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Abstract
AIM OF THE WORK
Giving the continued and growing number of patients at risk for parasitic infections, being in endemic area and also due to similarity of musculoskeletal presentations of parasitic diseases to many rheumatic diseases, consequently the importance of parasitic infection as an underlying cause of rheumatic syndromes must be considered in patients undergoing evaluation for rheumatic Complaints. So this work was designed to estimate:
1- The prevalence rate of intestinal parasites among patients with unexplained rheumatic pain attending Rheumatology and Rehabilitation Outpatient Clinic in Suez Canal University Hospital.
2- The prevalence rate of parasitic rheumatism among infected patients.
RECOMMENDATIONS
Based on the practical work and results obtained from the present study, recommendations those worth mentioning are:
o When arthritis or arthralgia can not be defined, a parasitic infection has to be excluded.
o Parasitological diagnostic consideration should be given to this entity of patients with unexplained arthropathies particularly if they are living in an endemic parasitic country.
o Because most laboratories do not routinely test fecal specimens for Cryptosporidium species, Cyclospora and microsporidia, physicians must request specific testing for these organisms.
o Sanitary sewage disposal, hygienic habits and safe water supply should be stressed on and emphasized to raise the health standard for the Egyptian population.
o For microsporidian spores staining with modified trichrome stain, it is better to dry slides overnight at room temperature or for 2-3 hours on a slide warmer at 37 °C or in an incubator at 37 °C as morphological distortion may result if slides are dried too rapidly and to avoid washing off film during staining. Also, centrifugation at 1500 rpm for 10 min. will increase number of spores in the sediment.
o Role of HLA-B27 must be confirmed in all patients with parasitic rheumatism.
o As regards S. mansoni infection, more advanced techniques should be used in further studies as antigen and antibody detection in serum which are particularly useful in detecting infection in patients with light, chronic, or inactive infections, as well as molecular studies, DNA probing and gene sequencing since schistosomiasis is a chronic and prevalent disease in Egypt especially in rural areas.
DISCUSSION
Parasitic infections affect as much as 25% of the world’s population mostly prevalent in underdeveloped, agricultural and rural areas of tropical and subtropical regions (Abd El-Bagi et al., 2004 & Reeder and Palmer, 1994).
Many intestinal parasites can induce a variety of rheumatic manifestations leading to arthropathy which may follow parasitic infections after free interval (Bocanegra and Vasey, 1993 & El-Araby, 2003). These parasites may directly infiltrate musculoskeletal structures or induced an immune mediated mechanism with circulating immune complexes which are deposited in synovium in some cases (McGill, 1995 & McGill, 2003).
Diagnosis of parasitic rheumatism is based on the demonstration of infection with a pathogenic parasite, lack of response to anti-inflammatory agents; especially NSAD, and improvement following anti-parasitic therapy (Peng, 2002).
This work was designed to estimate the prevalence of intestinal parasites among 108 patients with unexplained rheumatic pain attending Rheumatology and Rehabilitation Outpatient Clinic in Suez Canal University Hospital by stool examination and then to identify the prevalence of parasitic rheumatism among infected patients.
In the present work, the overall prevalence of pathogenic intestinal parasites among patients with unexplained rheumatic pain was 46.3% (Tab. 1). The high prevalence of the intestinal parasites in the present work goes in agreement with several studies. In Egypt, a survey carried out by Sabry et al. (1990) in Menofiya Governorate, the prevalence rate was 62.3%. Geltman et al. (2003) and Caruana et al. (2006) found that the prevalence among African immigrants to the USA and Australia was 56% and 66% respectively.
Controversy, low prevalence rate (13.4%) was found in the study of Peruzzi et al. (2006) in Parma, Italy. Also, in Turkey, the overall prevalence of intestinal parasitic infection was 4.27%, among GIT symptomatic patients (Alver and Tore, 2006).
The great diversity in the prevalence of intestinal parasites may be related to the presence or absence of environmental risk factors such as seasonability of some parasites, residence, safety water and food supply, low socio-economic status and individual risk factors such as immune status of the patients (Dwivedi et al., 2007).
As regarding the response of infected patients with rheumatic pain to anti-parasitic treatment to fulfill the criteria of parasitic rheumatism according to Peng (2002), 16 patients (32%) gave good response with complete relief of rheumatic complaints and disappearance of the parasite from their stool. While, in 34 patients (68%) the rheumatic complaint persisted despite of full course treatment of the parasitic infection (Fig. 4). These data go in agreement with, Di Pietro et al. (1996) who reported that muscular and/or articular manifestations are not unusual in protozoal type infections. Also, McGill (2003) and Crum-Cianflone (2006) stated that a well-defined rheumatic syndrome occurs in a variety of parasitic diseases with distinct reported rates. Controversy, some authors considered this condition of parasitic rheumatism to be rare (Doury et al., 1977; 1990 & Sing et al., 2003).
This confusion may be resulted from that all of the reported cases were recorded in developed countries in which the prevalence of intestinal parasites especially in immunocompetent patients is low. In case of developing countries, the prevalence of parasitic rheumatism could be higher where safety food and water supply may be absent. On the other hand, patients those did not fulfill the criteria of parasitic rheumatism in the present study may be incidentally infected by these parasites with no relation with rheumatic pain. Although some authors proved that a genetic predisposition should be found with parasitic rheumatism which was occurring more in patients with HLA-B 27 positive (Hill Gaston and Lillicrap 2003), other authors denied this genetic predisposition (Hay et al., 1987; McGill, 2003 & Collings and Highton, 2004).
In the present study, single parasitic infection occurred in 60% of infected patients while, mixed infection occurred in 40% (32% double and 8% triple). In addition the majority of infections were due to protozoa (Tab. 3). The most frequent double co-infection occurred in 20% of both of Cryptosporidium and Cyclospora infected patients (Tab. 3). In coordinate with these data, Diaz et al. (2003) proved that 69% of parasitic infected patients had single infection versus 31% mixed infection. Also, Peruzzi et al. (2006) found that single parasitic infection was the dominant among their patients while mixed infection was the less common (113 patients versus 35). Moreover, in a study of Dwivedi et al. (2007) single infection was in 55.6%, double was in 38.9%, and triple in 5.6%.
In contrast, Ndamukong (2005) found that the prevalence of single and mixed infection was approximated in frequency among his patients (50.8% and 49.2% respectively).
The predominance of single parasitic infection in the present study may be because this study was done among immunocompetent patients where the chance of infection with more than one parasite is low. However, most of co-infections in the present study were between protozoa which are water and food borne, transmitted feco-orally, more common in low socio-economic states and rural areas (Garcia, 2001).
In the present study, the prevalence of intestinal parasites was 88% in females, while it was 12% in males among all of the infected patients (Tab. 4). In agreement with these results, Caruana et al. (2006) found that the rate of intestinal parasites was higher in females than males (11% versus 6%). Contrary, in Parma, Italy, Peruzzi et al. (2006) noticed that infected males were 84 while infected females were 64. Also, in Turkey, the prevalence among males was 59.6%, while among females was 40.4% (Alver and Tore 2006). Moreover, 70 % of males were affected versus 63 % of females in the study of Balcioglu et al. (2007).
On the other hand, other studies showed that there was no significant difference in the prevalence of intestinal parasites by sex (Omar et al., 1995; Geltman et al., 2003 & Champetier et al., 2005). The present study finding may be explained by high percentage of female patients with rheumatic pain among this study population.
As regards the age of the infected persons in the present study, high infection rate was found among adults (56%), followed by young age (28%) while, low infection rate was found among elderly (16%) as shown in Tab. 4. In agreement with these data, Peruzzi et al. (2006) proved that adults were more susceptible to infection compared to children (128 versus 20). In controversy to this result, in Gaza, parasitosis was generally declining with older age groups for both sexes (Shubair et al, 2000). Moreover, Geltman et al. (2003) found that patients ≤ 18 years were more likely than adults to have any parasite in their stool (62% versus 51%). Also, Al-Harthi and Jamjoom (2007) found that the infection rate was higher in ≤ 30 years of age.
The high percentage of intestinal parasitic infection among adult age group is due to high percentage of adults among the studied population (55.5%) this may be owed to the care of them in searching for the cause of this unexplained rheumatic pain in such age. whereas, in young age the problem is usually referred to a pediatrician who may manage the pain as a case of rheumatic arthritis for example while, in old age, the rheumatic pain is usually thought to be due to geriatric changes and osteoarthritis, moreover, parasites are often unmentioned in the differential diagnosis of rheumatic disease presentations (Peng, 2002).
Concerning the resident site, relatively higher percentage of the infected patients was living in rural Ismailia Governorate (56%), than those living in urban areas (44%), the difference was statistically non significant (Tab.4). In agreement with this result, in Egypt, El-Shazly et al. (2006a) reported that the prevalence was relatively high in rural than in urban areas in Dakahlia Governorate. While, Champetier et al. (2005) found that the prevalence was higher in rural areas (38.4%) compared to urban areas (30%) among school children in Haiti. Also, Dwivedi et al. (2007) proved that high prevalence rate was recorded among patients living in rural than urban areas (68.4% versus 44.4%). Such high prevalence in rural areas may be due to overcrowding, low level of sanitation and personal unhygienic conditions.
In the present study, most of the infected patients were in low socio-economic class (68%), the difference was statistically significant (Tab.4). In agreement with this result, Celiksoz et al. (2005) found that the highest prevalence of intestinal parasites was in low socio-economic class. In addition, several researches had demonstrated a firm relationship between parasitosis and lack of education and low socio-economic class (Gamboa et al 1998; Nematian et al., 2004; Balcioglu et al., 2007). This was not surprising since the low socioeconomic class is an important risk factor for intestinal parasitic infections where malnutrition with bad hygienic conditions and insufficient medical services were found (Tinuade et al. 2006).
In the present study, the majority of infected patients were GIT asymptomatic (60%) while, in 40% GIT symptoms were present (Tab.5). In agreement with the present study data, Okyay et al. (2004) found that most of the complaints of the study population were not significantly related with the prevalence of the intestinal parasitic infection among school children in Turkey.
Indeed, the development of acquired immune response to some parasitic infections depends on many factors such as age, intensity of infection, genetic background and duration of exposure which enable to develop the parasitic disease to sub-clinical or carrier status (Garcia 2001).
Concerning individual parasitic infection among the study population in the present study, Cryptosporidium was found in 22.2% (Tab. 2). In agreement with the obtained data, in Egypt, Benha, the total prevalence rate of cryptosporidiosis was 19.5% in a study carried on 1087 diarrheic patient of all age groups (Abdel-Maboud et al., 2000). Abdel-Messih et al. (2005) found that the prevalence rate was 17% in Nile River Delta of Egypt. Hussein (2006) reported that the prevalence rate of Cryptosporidium in diarrheic children attending Suez Canal University Hospital was 16.7% and 26.6% in immunocompetent and immunocompromized children respectively. In the study of Dwivedi et al. (2007) the prevalence rate of Cryptosporidium was 46 % among HIV infected individuals.
In the contrary, low prevalence rate was reported by Mikhail et al. (1989) who found that 9% of diarrheal patients from Aswan having Cryptosporidium. In Korea, Park et al. (2006) found that Cryptosporidium oocysts were detected in 1.5%. Also, Peruzzi et al. (2006) stated that cryptosporidiosis wasn’t found in Parma, Italy.
In the present study, infection with Cryptosporidium was more common in females than males (83.3% versus 16.7%) and in adults with a prevalence rate of 41.7% versus 33.3% and 25% for old and young age groups respectively (Fig. 5). Soliman, (1992) found that children (6-8 years) were the most affected among primary school children in a rural area of Alexandria. Helmy et al. (2006) found that females (24.5%) were more infected than males (20.4%) with cryptosporidiosis. In controversy, Park et al. (2006) found that the prevalence rate of Cryptosporidium in men was higher than in women with 1.9% and 1.2% respectively, while the majority of infected individuals were older than 50 years of age (94.6%).
In the present study, only 2 cryptosporidiosis patients (8.3%) were responding to anti-parasitic drugs well and both of them were young males, having oligo-articular pain, GIT symptoms and single infection (Tab. 6). On the other hand, the majority of cryptosporidiosis patients (91.7%) were not fulfilling the criteria of parasitic rheumatism (did not respond well to anti-parasitic drugs).
Many reports confirmed the result in the present study. Shephered et al. (1989) reported that cryptosporidiosis was a cause of reactive arthritis especially in children. Cron and Sherry (1995) reported that Reiter’s syndrome was associated with cryptosporidial gastroenteritis. Also, Sing et al. (2003) reported a case of 8 years immunocompetent old boy with cryptosporidiosis accompanied by arthritic symptoms involving several joints.
On the other hand, only four cases of Cryptosporidium reactive arthritis in adults were reported. Two were HIV patients (Hay et al., 1987). The other two were immunocompetent patients who had mono and oligo-articular joint affection. Similarly, HLA-B27 positive status has only been reported in one case, and was negative in the other three cases. Also, all of the patients were GIT symptomatic (Hay et al., 1987; Ozgul et al., 1999 & Collings and Highton, 2004).
Elevated ESR and CRP were found in 16.7% and 8.3% respectively of Cryptosporidium infected patients in this study (Fig. 5). In the study of Lee et al. (2005) all cases infected with Cryptosporidium had elevated ESR and some of them had elevated CRP. Also, Collings and Highton (2004) and Rees et al. (2004) found that the blood investigation of cryptosporidiosis reactive arthritis patients proved that ESR and CRP were elevated. This elevation in ESR and CRP was considered to reflect some types of inflammation existing inside the body (Lee et al., 2005).
In the present study, single infection with Cryptosporidum was reported in 50% of infected patients while mixed infection occurred in the other 50% (12 cases) (Tab.3). The most frequent association was with Cyclospora in 6 cases of double and 2 cases with triple infection (Fig. 3).
The present study data were in coordinate with Hussein (2006) who found that Cyclospora parasite was mostly associated with Cryptosporidium in 43.5% of Cyclospora infected children and Helmy et al. (2006) who found that 6 adult female cyclosporiasis patients out of 8 had Cryptosporidium infection. Meanwhile, Abaza et al. (1995) detected 16 cases having mixed infection with opportunistic parasites among which 11 cases were harboring G. lamblia and Cryptosporidium. Also, Abdel-Meseeh et al. (2005) found that Giardia was the dominant co-pathogen in 30% of Cryptosporidium infected children.
The present study finding may be explained by that, both Cryptosporidium and C. cayetanensis have the same source of infection and route of transmission. Moreover, the repeated occurrence of food and waterborne outbreaks of cryptosporidiosis and cyclosporiasis confirmed this association (Sterling and Ortega, 2006). While, no association was found between Cryptosporidium and G. lamblia in the present study and this may be related to factors as type of the study population, sample size and age of patients.
In the present study, Cyclospora cayetanensis were found in 14.8% of the studied population (Tab. 2). This finding goes in agreement with, El-Karamany et al. (2005), who stated that the prevalence rate of C. cayetanensis in Sharkia Governorate, Egypt was 17.4% among diarrheic patients living in El-Ekhewa Village. In Ismailia, the prevalence rate of C. cayetanensis among diarrheic children was 19.6% and 34.6% in immunocompetent and immunocompromized respectively (Hussein, 2006). World wide, Fryauff et al. (1999) reported that C. cayetanensis was the dominant adult pathogenic intestinal parasite in West Java, Indonesia, with 11.5% prevalence rate. Controversy, low prevalence rate was reported by Rizk et al (2001) among asymptomatic immunocompetent adults (2.8%) in Mansoura City. Such great variability in the prevalence could be due to environmental, seasonal, geographical (climate) factors.
In the present study, infection with C. cayetanensis was more common in females than males (75% versus 25%) and in adults than young ages (75% versus 25%). Meanwhile, no cases were detected among old age group (Fig. 6). These data were in coordinate with, Goncalves et al. (2005) who reported high percentage of cyclosporiasis in females (71.4%) in comparison to 43.3% in the healthy ones, while in males it was 57%. The adult age was the predominant infected age group in the study of Fryauff et al., (1999).
In contrast, studies developed in Gautemala by Bern et al. (1999) and Pratdesaba et al. (2001) showed that the prevalence of cyclosporiasis was five times higher among children than adults. However, in endemic countries the ages of patients may affect the epidemiology of Cyclospora infection suggesting that immunity became complete by adolescence so the prevalence was decanted in adults. On the other hand, continuous exposure to contaminated food and water may lead to presence of infection among adults (Karanja et al., 2007).
In the present study, 62.5% of patients infected with Cyclospora were asymptomatic and this finding is compatible with Eberhard et al. (1999) who found that most Cyclospora infections were asymptomatic.
In the present study, only 2 cyclosporiasis patients (12.5%) were fulfilling the criteria of parasitic rheumatism and both of them were young males, having oligo-articular pain, GIT symptoms and single infection (Tab.7). On the other hand, most of cyclosporiasis patients (91.7%) did not fulfill the criteria of parasitic rheumatism. In agreement of these results only two studies found a relation between C. cayetanensis infection and musculoskeletal manifestations such as Guillian Barrie syndromes (Richardson, et al., 1998) and Reiter’s syndromes (Conner, et al., 2001).
In the present study, Giardia lamblia was found in 11.1% of the studied population (Tab. 2). In agreement with the obtained data, in Egypt Alexandria, El-Sahn et al. (1997) found that the prevalence rate of giardiasis was 15.4%. However, 44% prevalence rate was found among GIT symptomic patients (Yakoob et al., 2005).
Worldwide, in Peru the prevalence rate was 10.57% in a study done by Cabrera et al. (2005) among adults. In Morroco, the prevalence rate of Giardia was 11.7% among individuals living in a farming population (El-Kettani et al., 2006). In a national survey done in Islamic Republic of Iran the prevalence rate of G. lamblia was 10.9% (Sayyari et al., 2005). Controversy, low prevalence rate of Giardia was recorded by Verie et al. (2003) in Northern Vietnam where it was 3%. In USA, the prevalence rate ranged from 5.7% to 6% in the studies of Garg et al. (2005) and Varkey et al. (2007) respectively.
Several factors could be contributed to the variation in the reported prevalence rates, viz.: sample size, diagnostic techniques, type of population, environmental variation and seasonal time of the study.
In the present study, the majority of giardiasis patients had elevated ESR and CRP (66.7%) as shown in Fig. 7. Also, the majority of giardiasis patients (8 cases out of 12) with a prevalence of 66.6% were responding well to anti-parasitic drugs (Tab. 8) while, the remaining 33.4% were not fulfilling the criteria of parasitic rheumatism (did not respond well). The eight cases with giardiasis who gave good response to treatment with relief of their rheumatic pain were females; 4 with young age, complained of mono-articular joint pain and 2 of them were GIT symptomatic, while the other 4 cases were adults, complained of poly-articular joint pain and 2 of them were GIT symptomatic.
In agreement with the present study data, Giardia was reported as a cause of reactive arthritis by Goobar (1977) who described musculoskeletal manifestations of giardiasis in a series of 66 infected children aged 2-15 years. Acute poly-arthiritis affection was the common feature. Also, Galland (1998) showed that there was positive association between giardiasis and arthralgia with a prevalence rate of 36%. Sometimes it was associated with HLA-B27 positive (Farthing, 1983 & LeBlanc and Birdi, 1999). Also, arthritis and arthralgia have been reported among a series of 10 children (5 to 11 years) and 70% of them were males with cysts of G. lamblia in feces seen by Meza-Ortiz (2001) in Mexico. All of the cases had GIT symptoms. Elevation on ESR was found in 60%, while CRP was negative in all cases.
A similar clinical picture was reported in adults. Shaw and Stevens (1987) reported an acute poly-arthiritis in female patient associated with giardiasis. Woo and Panayi (1984) and Barton et al. (1986) reported that 2 HLA-B27 positive adult patients developed asymmetrical oligo-arthritis following giardiasis. In contrast, 2 HLA-B27 negative adult patients with a slowly aggravating poly-arthritis were reported by Arman (1991) in Turkey. These patients had neither ESR elevation nor any other signs of inflammatory processes. However, (Layton et al., 1998) reported a mono-articular joint affection in HLA-B27 negative male patient infected with Giardia with elevated ESR and CRP. Recently, Carlson and Finger (2004) reported a HLA-B27 negative young woman had oligoarthritis after 2 weeks of being treated from infection with Giardia.
In the present study, B. hominis was found in 9.2% of the studied population (Tab. 2). In agreement with the obtained data, in Egypt, Khalifa (1999) identified 6% prevalence rate. In Ismailia the prevalence rate was 10.1% in school children (El-Shewy et al., 2002). However, high prevalence rate (22.4%) was reported by El-Shazly et al. (2006b). Worldwide, in Colombia the prevalence rate was 6.1% (Giraldo-Gomez et al., 2005).
On the other hand, low prevalence rate (0.04%) was reported by Degerli et al. (2005) in Turkey. While, in the study of Saksirisampant et al. (2006) in Central Thailand the prevalence rate was 0.19% among in primary schools children.
In the present study, most of the infected patients were living in urban areas (60%). These data were in coordinate with Doyle et al. (1990) who reported an urban-rural ratio as 3.4:1.0. In the contrary, O’Gorman et al. (1993) and El-Shewy et al. (2002) found that 31% and 29.7% of infected persons with B. hominis resided in urban areas whereas 69% and 70.3% were from rural areas respectively.
In spite of the high prevalence of blastocystosis in the present study no cases of parasitic rheumatism were found. These cases may be due to the non-pathogenic B. hominis human isolates which has long been described as a commensally organism (Udkow and Markel 1993; Shlim et al., 1995 & Tan 2004). In contrast, Lee et al. (1990) reported a case where the organism was found in synovial fluid aspirated from knee effusion of infectious arthritis patient. While in some cases, the organism may cause reactive arthritis (Lakhanpal et al., 1991 & Kruger et al. 1994).
In the present study, E. histolytica / dispar was found in 3.7% of the studied population (Tab. 2). In agreement with the obtained data, in Egypt El-Naggar et al. (2006) examined the stool of 180 apparently healthy children, and found the prevalence rate of E. histolytica / dispar as 3.3% and 3.7% by concentration sedimentation and Sheather’s floatation methods respectively. Worldwide, In Central America a survey done on 5 villages in Belize country by Aimpun and Hshieh (2004) found the prevalence rate of E. histolytica / dispar as 6%. While in Turkey, it was 2.3% among 34,883 patients (Yazar et al., 2005).
In contrast, El-Shazly et al. (2006b) reported that prevalence rate of E. histolytica / dispar was 19%. While, in Amazon Araujo and Fernandez (2005) found that the prevalence rate was 13.3% in city of Eirunepe.
In the present study, half the patients (2 cases) with E. histolytica / dispar infection gave good response to anti-parasitic treatment and were complaining of poly-articular joint pain. While, bad response occurred in the other half. The difference was statistically non significant.
In agreement with the current study data, E. histolytica infection has been associated with autoimmune phenomena (Salem et al., 1973) and development of symmetrical poly-arthritis very similar to rheumatoid and reactive arthritis (Zinneman, 1950). In some cases, organisms have been observed in synovial fluid, suggesting invasive infection (Burnstein and Liakos, 1983). Entamoeba dispar, which is morphologically identical to E. histolytica and was reported in asymptomatic patients (Horga and Naparst, 2006), may be found among non-responding patients in the present study.
In the present study, microsporidia was present in 2 cases (1.9%) as shown in Tab. 2; both cases complained of mono-articular joint pain. In Egypt, Abaza et al. (1995) found that the prevalence rate of microsporidia spores among immuno-compromised hosts was 2.3%, while El-Shazly et al. (2006b) reported a prevalence rate of 3.2% among immuno-competent patients attending Mansoura University hospitals. In contrast, no microsporidia spores were found in both malnourished and control children in the study of Rizk and Soliman (2001) in Mansoura, Dakahlia Governorate. Rheumatic manifestations of microsporidial infections are uncommon and have been described almost entirely among patients with AIDS (Kotler and Orenstien, 1998). However, several case reports described its association with infectious myositis (Ledford et al., 1985 & Cali et al., 2004). In the present study these cases didn’t respond well to treatment suggesting that it was incidental infection.
In the present study, S. mansoni was present in 2 cases (1.9%) as shown in Tab.2; both cases complained of mono-articular joint pain. In Egypt, El-Shazly et al. (2006a) found that the prevalence rate of S. mansoni in an urban and a rural area in Dakahlia Governorate were 0.5% and 1.6% respectively. High prevalence rate was reported by Nooman et al. (2000) where the prevalence of S. mansoni infection in rural Ismailia was 42.9%, peaked in the 20-30 years old age group and was higher in males than in females. Rheumatic manifestations are most common among patients with chronic schistosomiasis, (Kamel et al., 1989). Some of these manifestations appear to result from immune complex disease (Greenfield et al., 1986 & Bebars et al., 1992), whereas others result from direct infection (Bassiouni and Kamel, 1984 & Atkin et al., 1986). In the present study these cases didn’t respond well to treatment suggesting that it was incidental infection.
In the present work, A. lumbricoides was present in 2 cases (1.9%) as shown in Tab. 2; both cases complained of oligo-articular joint pain. In Egypt, El-Shazly et al. (2006a) found that the prevalence rate of A. lumbricoides in an urban and a rural area in Dakahlia Governorate was 0.1% of each. While, in a national survey done in Islamic Republic of Iran the prevalence rate of Ascaris was 1.5% (Sayyari et al., 2005). High prevalence rate (35.6%) was reported by Araujo and Fernandez (2005) among 413 individuals living in city of Eirunepe, Amazon. In 1978, Miehlke and Jentsch reported a case where Ascaris infection has been related to symmetrical polyarthritis. In the current study these cases didn’t respond well to treatment suggesting that it was incidental infection.
In the present work, S. stercoralis was present in 2 cases (1.9%) as shown in Tab. 2; both were female adults, living in rural areas, GIT symptomatic, with elevation of ESR, CRP, and blood eosinophils. Both cases occurred as single infection and complained of poly-articular joint pain. They gave good response to anti-parasitic treatment.
In Egypt, El-Shazly et al. (2006a) found that the prevalence rate of S. stercoralis in Dakahlia Governorate was 1.5%. While, high prevalence rate (11.1%) was reported by Salem et al. (1990) in Menoufia Governorate was.
In agreement with these results many reports involving patients with either oligo or poly-arthritis in association with intestinal Strongyloides infection were presented (Van Kuijk et al., 2003). Although Strongyloides larvae have been observed in a synovial biopsy specimen, suggesting an infectious type of arthritis (Alkoglu et al., 1984 & Patey et al., 1990), most authors considered this to be a reactive arthritis suggesting that arthritis may be mediated by immune complex formation (Bocanegra et al., 1981). On the other hand, Doury (1981) reported that arthritis is a less common feature of strongyloidiasis.
PARASITIC RHEUMATISM
In 1962, Bassiouni presented the first report about rheumatic diseases which may accompany parasitic infection, when he described the pattern of arthropathy which may develop secondary to infection with Schistosoma mansoni. Since that time many reports were introduced about rheumatic disease which may follow parasitic infections (El-Araby, 2003).
Many terms were introduced to describe the condition, e.g. reactive arthritis (Bocanegra et al., 1981), rheumatic syndromes associated with parasites (McGill, 1995), parasitic arthritis (Bocanegra, 1997), parasitic rheumatism (Doury, 1990; Peng, 2002). Arthropathy which may follow parasitic infections may fulfill the definition of reactive arthropathy, where the causative pathogen is the parasite, followed after free interval with a characteristic pattern of arthropathy as a reaction to the presence of the parasite (El-Araby, 2003).
In reactive arthritis, patients have a preceding infection during which they become naturally immunized against the etiological agent. During that process, usually in 1 to 3 weeks, antigens are transported to the synovial tissue. This may occur intracellularly within mononuclear or other phagocytosing cells (Salmi et al., 1995), as immune complexes (Lahesmaa-Rantala et al., 1987), or even in the form of free antigen (Lehtonen et al., 1994). The consequence is a CD4+ cell mediated reaction, manifested as acute arthritis (Burmester et al., 1995), immune complex mediated cytotoxicity may also participate (Lahesmaa-Rantala et al., 1987). For chronic antigen induced arthritis, a periodic or continuous supply of the antigen is required (Toivanen and Toivanen, 1999).
A variety of musculoskeletal syndromes have been described in association with numerous parasitic infections. Arthritis, enthesitis (inflammation at the site of insertion of ligaments and tendons onto bone), myositis and vasculitis were described in infected individuals resident in/or visitors to endemic areas (McGill, 1995).
Arthritis induced by parasitic infection is very polymorphic; symptoms are monoarticular, oligoarticular, or polyarticular, involving small, medium, and/or large joints. They can mimic the clinical picture of different inflammatory rheumatic diseases (Doury, 1990).
Many intestinal parasites can induce a variety of rheumatic syndromes as a result of infiltration of musculoskeletal structures by parasites or an immune mediated mechanism with circulating immune complexes and immunoglobulin deposits in synovium in some cases (Doury, 1990 & Bocanegra and Vasy, 1993).
Despite high prevalence of parasitic infections allover the world, the occurrence of parasitic rheumatism is uncommon which may be due to genetic predisposition related to the presence of histocompatibility antigen HLA-B27 (Doury, 1990). Difficulty to diagnose parasitic rheumatism, because of the wide range of clinical features, silent parasitic infection, or absence of the parasite at the time of arthropathy may also lead to its low prevalence (El-Araby, 2003).
The demonstration of infection with a pathogenic parasite, lack of response to anti-inflammatory agents; especially non steroidal anti-inflammatory drugs, and improvement following anti-parasitic therapy are the bases of diagnosis of parasitic rheumatism. Treatment consists of eradication of the parasite (Bocanegra and Vasey, 1993), which results in resolution of symptoms and immunological abnormalities (Bocanegra et al., 1981).
Peng (2002) proposed essential criteria for diagnosis of parasitic rheumatism which were inflammatory arthropathy, residence in an area of endemic parasitosis, absence of radiological changes, identification of a pathogenic parasite, inefficacy of anti-rheumatic drugs and efficacy of specific anti-parasitic treatment as well as supplemental criteria which were inflammatory synovial fluid, elevated erythrocyte sedimentation rate and peripheral eosinophilia.
Intestinal Parasites Commonly Causing Rheumatic Manifestations
I- Giardia lamblia:
The characteristic and distinctive morphological features of Giardia are well known and described initially in the latter part of the 1880s (Lambl, 1859). However, its role as a pathogenic organism was not recognized until the 1970s, after community outbreaks and after the appearance of the disease in travelers returning from endemic regions. Prior to that time, the organism was thought to be a harmless commensally organism of the intestine (Cash and Johnston, 2005).
Although, 6 spp. (G. duodenalis, G. agilis, G. muris, G. ardeae and G. psittaci) were identified, G. duodenalis (sometimes referred to as G. intestinalis or G. lamblia) is the only spp. found in human (Thompson, 2002). Indeed, G. duodenalis is clearly not a uniform spp. but a spp. complex comprising a variety of genetically and phenotypically distinct organisms (Monis and Thompson, 2003). Moreover, Giardia isolates recovered from human fall into one of the major genotypic assemblages A and B. Assemblage A consists of AI and AII subgroups which are zoonotic. Assemblage B comprises two subgroups III and IV which appears to be human-specific (Thompson, 2004).
Developmental Biology:
Giardia’s life cycle consists of three stages: cyst, excyzoite and trophozoite (Roxtrom-Lindquist et al., 2006). Infection usually starts with ingestion of cysts, followed by excystation and releasing of excyzoite in the upper part of the small intestine. The short-lived excyzoite is oval, has eight flagella and a metabolism intermediate between a trophozoite and a cyst (Fig. 1) (Bernander et al., 2001 & Roxtrom-Lindquist et al., 2006).
The excyzoite has 4 nuclei and undergoes cell division twice without DNA replication, generating 4 disease-causing trophozoites. This cellular amplification partly explains the low infectious dose of Giardia (10-100 cysts) (Katz and Taylor, 2001 & Leder and Weller, 2002). During the first cell division of the excyzoite, the four-adhesive disk fragments reassemble into new disks, enabling the newly formed trophozoites to attach quickly (Weiland et al., 2003). However, the heparin-sulphate binding protein α-1 giardin with lectin activity that is exposed on the outer surface of the excyzoite is one of the immunodominant proteins that help both trophozoite and exczoite in the attachment mechanism (Elmendrof et al., 2003; Palm et al., 2003 & Weiland et al., 2003).
Giardia growth in the small intestine is stimulated by bile, carbohydrates, and low oxygen tension (Procop, 2001). Excystation occurs within 5 minutes of exposure of the cyst to an environment with a pH between 1.3 and 2.7 (Petersen, 1972; Schultz, 1975). On the other hand, in response to the host signals in the lower part of the small intestine in the presence of neutral pH and secondary bile salts, trophozoites encyst and are excreted in feces as infective cysts (Lujan et al., 1997 & Leder and Weller, 2002).
The trophozoite form is tearDROP-shaped, measures 9-21 µm long X 5-15 µm wide, having a convex dorsal surface and a flat ventral surface that contains the ventral disk. It contains 4 pairs of flagella, two symmetric nuclei with prominent karyosomes produce the characteristic face like image that appears on stained preparations and a pair of distinctive median bodies (Thompson, 2004). Cyst form is smooth-walled and oval in shape, measuring 8-12 µm long X 7-10 µm wide and contains 4 nuclei (Petersen, 1972 & Schultz, 1975).
Pathophysiology:
Although Giardia is not invasive, its pathogenecity is not clearly understood. Highly variable giardiasis clinical pictures from asymptomatic to symptomatic are present (Thompson, 2004). A multi factorial pathophysiological mechanism was studied that is both parasite and host dependent. One of the fundamental changes is altered epithelial permeability that appears to result from a direct cytopathic effect induced by products of the parasite (Buret et al., 2002). In addition, peripheral membrane proteins, and in particular the tight junction-associated protein zonula occudin-1, which play an important role in the regulation of epithelial permeability are disrupted leading to digestive and absorptive changes that correlates with the loss of epithelial barrier function, this also may result from uptake of luminal antigens (Chin et al., 2002 & Scott et al., 2002). Interestingly, apoptosis and severity of the disease are determined by strain-dependent virulence factors of the parasite, as well as by the developmental, nutritional and immunological status of the host such as transient lactase deficiency, altered mucus secretion, brush border enzyme deficiency, and alteration in motility (Thompson, 2004).
Immune Response:
Immune response is important in eradicating the parasite from intestine during acute infection and also in development of protective immunity (Farthing et al., 1987). Experimental studies in animals had confirmed the presence of different types of immunity; natural and acquired (Roxtrom-Lindquist et al., 2006). Multiple Giardia antigens appear to be highly immunogenic like 170 kDa, 82-88 kDa surface antigens as well as Giardia heat shock antigen (Cook, 1995). Failure to produce antibodies against these antigens was found to be important factor leading to persistent infection (Char et al., 1992). An early B-cell independent phase occurs in the first two weeks after infection, followed by an antibody dependent immunity (Eckmann, 2003 & Li et al., 2004).
1) Natural immunity:
There are many factors that control the natural resistance to Giardia lamblia (Fig. 1) such as genetic factors, gastrointestinal factors e.g.: gastric acidity, mobility of the gastrointestinal tract, indigenous intestinal flora, mucous and bile salt secretion might influence the course of the infection (Roxtrom-Lindquist et al., 2006). Although proteases, lipases and bile salts kill most other microbes in the upper small intestine, it is tempting to speculate that Giardia cysteine-rich thick outer coat of variable surface proteins (VSPs), known to confer protease resistance, could also be protective against bile (Nash, 2002). Also, the membrane binding proteins of the cytoskeleton such as α-giardin together with VSPs coat stabilize the plasma membrane by supporting protein network (Benchimol et al., 2004).
As a part of the innate immune system in the small intestine, antimicrobial products are secreted by the intestinal epithelium and contribute to the maintenance of the mucosal barrier (Fig. 1) such as the antimicrobial peptides defense, lactoferrin and Penth cells secretion (Eckmann, 2003 & El-shewy and Aid, 2005). Also monocytes and macrophages are able to kill the trophozoite by an oxidative mechanism (Krause, 2004). HLA system has been enrolled in susceptibility to parasitic diseases. HLA-A2, HLA-B8 and HLA-B12 are detected with increased frequency in patients with giardiasis. Patients with the severest lesions had a higher frequency of HLA-B8, high serum C4 levels as well as significantly low levels of secretory IgA (Abaza et al., 1988).
2) Acquired immunity:
It includes; passive immunity through specific secretory IgA present in normal human milk and active immunity which includes humoral and cellular immune responses (Nayak et al., 1987). Giardia can down regulate the inflammatory response. Interferon- γ (IFN-γ) and interleukin-6 (IL-6) are needed for clearance and early control of acute infection (Ebert et al., 1999; Bienz et al., 2003).
Recent data show that mast cells can influence the development, magnitude and kinetics of acquired immune response by affecting denderitic cells, B cells and/or T cells (Galli et al., 2005).
Anti-Giardia IgM titers increase early in infection and then decline rapidly followed closely by sustained IgA antibody response (Goka et al., 1986 & Sharma and Mayhofer, 1988). Complement-induced lysis of trophozoites has been also demonstrated and can be mediated by the classical pathway with IgM from symptomatic giardiasis patients (Deguchi et al., 1987 & Heyworth and Pappo, 1990). A systemic IgG response directed against antigens that are present on the surface of G. lamblia trophozoites are found, which could participate in antibody-dependent cellular cytotoxicity (Smith, 1985). Although, CD8+ and CD4+ T-cells regulate the influx of intra-epithelial lymphocytes during human giardiasis its role is uncertain (Roxtrom-Lindquist et al., 2006).
Figure 1: G. lamblia interaction with human intestine (Roxtrom-Lindquist et al., 2006).
Epidemiology:
Giardia is the most prevalent protozoal infection of the human intestine worldwide causing 2.8 x 108 cases per annum (Lane and Lioyd, 2002). It is one of the most common causative agents of epidemic and endemic diarrheal illness throughout the world. Prevalence rates vary from 4-42% (Cash and Johnston, 2005). Some 4 to 16% of normal people in tropics harbor the parasite as carriers (Reeder, 1997). The prevalence is higher in populations with poor sanitation, close contact, water contamination and oral-anal sexual practices (DuPont and Backer, 1995).
In Egypt, the prevalence rate had been studied by different workers in different localities; while low prevalence rates (5 and 5.7%) had been reported in Sharkia and Menofiya governorates respectively (Mohammed et al. 1988 & Salem et al. 1990), high prevalence rates (19.4%, 20.3%, 21.7% , 24.7% and 27.59%.) had been recorded in Beniswif, Cairo, Behera, Mansoura and Ismailia governorates respectively (Boghdadi, 1986; Youssef et al. 1988; El-Serougi et al. 1990; Curtale et al., 1998 & Hussein, 2006).
Clinical picture:
After an incubation period of 1 to 2 weeks, symptoms of gastrointestinal distress may develop (Leder and Weller, 2002). Approximately 15% of patients infected with Giardia are asymptomatic, with cyst passage only. However, approximately 50% of patients infected with Giardia may present with a variety of symptoms including; acute diarrhea, nausea, vomiting, malaise, flatulence, and abdominal cramping, steatorrhea, and weight loss (Cash and Johnston, 2005). Chronic signs and symptoms of giardiasis may wax and wane over months if the condition is not treated (Leder and Weller, 2002).
Although, extra-intestinal manifestations of Giardia are rare (Cash and Johnston, 2005) Giardia was previously reported as a cause of reactive arthritis (Goobar, 1977 & Carlson et al., 2004). Typical cases involve the development in children of an acute polyarthritis, especially involving the knees, ankles, and hips, and sometimes associated with HLA-B27 (Farthing et al., 1983; LeBlanc and Birdi, 1999). Adults may also develop arthritis of the shoulders, elbows, and wrists, and some patients may develop symmetric rheumatoid like involvement affecting small joints of the hands and feet. Galland (1998) showed that there is positive association between giardiasis and; arthralgia, myalgia, and muscle weakness with prevalence rates of 36%, 34%, 46% respectively. Urticaria and erythema nodosom occasionally coexist (Farthing, 1983).
Diagnosis:
Giardia diagnosis by traditional microscopic methods following the application of fecal concentration techniques, especially zinc sulphate flotation and centrifugation, remains a reliable indicator of infection (Zajac et al., 2002). Cyst excretion occurs intermittently in both formed and loose stools, while trophozoites are almost only found in diarrhea (Fumess et al., 2000 & Katz and Taylor, 2001). Because Giardia is not invasive, eosinophilia, and peripheral or fecal leukocytosis do not occur (Leder and Weller, 2002). However, aspirations of duodenal contents and small bowel biopsy for demonstration of trophozoites also have been used in diagnosis, but this is more invasive (Garcia, 1998 & Cash and Johnston, 2005).
Immunodiagnosis using Antigen assays by ELISA or indirect immunofluorescent (IIF) antibody test (IFA) to detect antibodies to trophozoites or cysts had higher sensitivity and specificity compared with direct smear examination (Leder and Weller, 2002). Procedures have also been developed using ELISA and IIF to detect Giardia antigens and antibodies in feces, using polyclonal antibodies (Addis and Mathews, 1991; Cook, 1995 & Aldeen et al., 1998).
Giardia molecular techniques particularly PCR or by gene probes highly sensitive methods are developed but they are so sophisticated and money consuming to be commercially available (Mahbubani et al., 1992; Morgan, 2000 & McGlade et al., 2003).
Treatment:
Three classes of drugs are commonly used to treat giardiasis, namely the nitroimidazole derivatives, the arcidine dyes such as mepacrine and the nitrofurans as furazolidone (Roxtrom-Lindquist et al., 2006).
Although most experts recommend metronidazole and tinidazole as the drugs of choice because the brief treatment periods encourage good patient adherence, treatment failure occurs in as many as 20% of cases. Therefore treatment with a second-line drug as mepacrine or furazolidone may be necessary. The effectiveness of quinacrine is similar to that of nitroimidazole derivatives; however it is less tolerated because of its adverse effects. Furazolidone is the least effective antigiardial drug, but it is widely used. Additionally paromomycin has been suggested for pregnant women (Davidson, 1990; Nash, 2001 & Hokelek and Nissen, 2006).
II- Entamoeba histolytica:
Amoebic colitis and amoebic liver abscess were known to the ancients; Hippocrates recognized ”Dysenteries”, however more than 2000 years had been elapsed before amoebas were identified as a cause of dysentery when in 1875, Fedor Alexandrovich described amoebic trophozoites in the stool and colonic ulcerations of a farmer with a fatal case of dysentery (Stanley, 2003).
There are at least eight other amoebas that can be found in human and are generally accepted as non-pathogenic, but some of them are morphologically identical to E. histolytica as E. dispar that was reported in asymptomatic patients (Haque and Petri 2006 & Horga and Naparst, 2006).
Developmental Biology:
The parasite exists as either the amoeboid trophozoite stage or the infectious cyst. Human beings and perhaps some non-human primates are the only natural hosts (Stanley, 2003). Infection usually begins with ingestion of the mature cyst which is round, usually 15 μm in diameter, and is surrounded by a refractile wall that may include chitin. It contains 1-4 nuclei that are morphologically similar to the nuclei of the trophozoite, glycogen and chromatoid bodies (Beaver et al., 1984 & Ravdin, 1995). It survives the acid of the stomach, travels through the small intestine, and within the terminal ileum or colon, excysts to form trophozoite stage (Stanley, 2003).
Unlike, the inert cyst, E. histolytica trophozoite is highly motile, with pleomorhic shapes (diameter varying from 10-50 μm). The fuel for this constant motion comes from the anaerobic conversion of glucose and pyruvate to ethanol (Stanley, 2003).The trophozoite has a single nucleus with fine peripheral chromatine and a central karyosome. Ingested RBCs, bacteria and food particles may be present within the trophozoite (Beaver et al., 1984 & Stanley, 2003). Trophozoite produces lesions of amoebic colitis and invasion of the colonic mucosa which leads to dissemination of the organism to extra-colonic sites, predominantly the liver. Faced with an adverse colonic environment, the trophozoite changes to the cystic form, better adapted to survival (Markell et al., 1999; Horga and Naparst, 2006). Cysts passed in the feces can survive in moist environmental conditions for weeks to months. Ligation of a surface galactose-binding lectin on the surface of the parasite might be one of the triggering factors for encystation mechanism (Eichinger, 2001).
Pathophysiology:
Disease begins when E. histolytica trophozoites adhere to colonic epithelial cells, and leukocytes through a surface protein called the galactose/N-acetylegalactosamine specific lectin, a complex of disulphide-linked 170 kDa subunits and 35/31 kDa subunits and an associated 150 kDa (Mann, 2002). When human cells are touched by amoebic trophozoites, they become immobile within minutes, lose their cytoplasmic granules and structures, and eventually their nucleus cytolysis is undertaken by amoebapores (Leippe, 1997). The trophozoite can induce apoptosis but is independent TNF- α receptor 1. Amoebapores could be necessary both for the lytic and for the apotosis pathways (Stanley, 2003). Subsequently, alternation in intestinal cell permeability causes a leak of ions (i.e. Na+, K+, Ca+) from target cell cytoplasm (Leroy et al., 2000). A number of hemolysins, encoded by plasmid (ribosomal deoxyribonucleic acid) and are cytotoxic to the intestinal mucosal cells, have been described in E. histolytica. An extra-cellular cysteine kinase causes proteolytic destruction of tissues, producing flask-shaped ulcers (Horga and Naparst, 2006). The E. histolytica cysteine proteinases are directly implicated in invasion and inflammation of the gut (Stanley, 2003).
The pathogenic strains can evade the complement-mediated lyses in the blood stream. Trophozoites ascend the portal veins to produce liver abscesses filled with acellular proteinaceous debris. Triangular areas of hepatic necrosis also may occur due to ischemia caused by portal venous obstruction. The trophozoites of E. histolytica may be present along the periphery of these hepatic lesions (Horga and Naparst, 2006).
Immune Response:
The intestinal epithelial cells sense the E. histolytica infection and produce IL-1B, IL-8, and cyclo-oxegenase (COX)-2 mediators which attract neutrophiles and macrophages to the site of amoebic invasion (Stenson et al., 2001). The activation of the intestinal epithelial cells takes place via the transcription nuclear factor (NF-kB). E. histolytica trophozoite can lyse neutrophiles which might release mediators that lead to colonic epithelial cells and hepatocyte damage and this explains why so few neutrophils are seen in direct contact with amoebic trophozoites in biopsy (Seydel et al., 1998). Moreover, E. histolytica cystein proteases cleave and inactivate the anaphylatoxins C3a and C5a, as well as human IgA and IgG (Stanley, 2003).
Cell-mediated immunity is important in limiting the disease and preventing recurrences. Antigen-specific blastogenic responses occur, leading to production of lymphokines, including IFN-d, which activate killing of E. histolytica trophozoites by macrophages. Incubation of CD8+ lymphocytes with E. histolytica antigens in vitro elicits cytotoxic T-cell activity against the trophozoites (Horga and Naparst, 2006).
Epidemiology:
E. histolytica is the third leading prarasite cause of death worldwide in humans (WHO 1997 & Haque et al., 2003). Amoebiasis affects about 50 million persons each year, resulting in approximately 100,000 deaths annually (Horga and Naparst, 2006). It is more prevalent in areas with high levels of poverty, illiteracy, overcrowding, inadequate water supplies and poor sanitation. In that areas direct feco-oral transmission occurs frequently and thus the endemic character of the disease is maintained (Mohamed et al., 1988). In Egypt, the prevalence rate of E. histolytica and E. dispare in rural area of Menofiya was 21.4% and 24.2% respectively (Abd-Alla et al., 2000). The Amoebic colitis prevalence among diarrheic patients in Cairo was 38% (Abd-Alla and Ravidin, 2002).
An even higher incidence of amoebiasis (up to 50-80%) has been reported in certain tropical countries due to bad housing conditions and poor personal hygiene (Walsh, 1986; Li and Staneley, 1996 & Reed, 2001).
Clinical picture:
The incubation period varies from 2 days to 4 months. About, 4-10% of asymptomatic E. histolytica infected individuals develop disease over a year (Haque et al., 2001).
Intestinal amoebiasis:
Acute amoebic colitis has a gradual onset presenting with 1 to 2 week history of abdominal pain, diarrhea, and tenesmus. Fever is noted in only a minority of patients. Lower quadrant abdominal tenderness may be noted. Fulminant amoebic colitis is a rare complication of amoebic dysentery. It presents with a rapid onset of sever bloody diarrhea, sever abdominal pain, and high fever. Children younger than 2 years are at increased risk and intestinal perforation is common (Reed, 1992 & Ravdin, 1995).
Chronic amoebic colitis occurs if symptoms of acute amoebic colitis persist for more than a month or if there are relatively long intervals of mild symptoms that are usually accompanied by formed stool or constipation with periodic reactivation of the acute state (Haider and Rasul, 1975). Amoeboma is a localized chronic infection of the cecum or ascending colon. It presents as a right lower quadrant abdominal mass, accompanied by symptomatic dysentery. It may result from repeated invasion of the colon by E. hitolytica complicated with pyogenic infection (Reed, 1992).
Extra-intestinal amoebiasis:
The liver is the most frequent extra-intestinal site of amoebic affections which present as acute hepatitis and amoebic liver abscess (Markell et al., 1999) then, lung, brain or splenic abscess, pericarditis, peritonitis or empyema, skin or genitourinary ulcers (Horga and Naparst, 2006). However, Chronic E. histolytica infection has been associated with autoimmune phenomena, including the appearance of antibodies to colonic epithelial cells (Salem et al., 1973) and development of symmetrical polyarthritis very similar to rheumatoid and reactive arthritis (Zinneman, 1950; Rappaport et al., 1951; Kasliwal, 1970; Peng, 2002). In some cases, organisms have been observed in synovial fluid, suggesting invasive infection (Burnstein and Liakos, 1983 & Peng, 2002).
Some non-pathogenic amoebae have been implicated in arthritic syndromes. A reactive arthritis like presentation has been associated with E. hartmani (Schimer et al., 1998). Another case report, showed a United States serviceman heavily infected with Endolimax nana presented by diarrhea and a symmetrical polyarthritis resembling rheumatoid arthritis, metronidazole rapidly reversed all abnormalities (Burnstein and Liakos, 1983).
Diagnosis:
Microscopic identification of cysts and trophozoites in the stool is the common method for diagnosing E. histolytica (Walsh, 1986). Iodine solution was used as staining reagent to demonstrate the number of nuclei in cysts and the character of the peripheral chromatin and stored glycogen (Hegazi et al., 1975). Concentration techniques used for detection of E. histolytica are zinc sulphate centrifugal flotation and formalin ether (or formalin ethyl acetate) sedimentation (El-Naggar et al., 2006). Confirmation by permanently stained fecal smear is essential for accurate diagnosis of E. histolytica and for spp. differentiation (Garcia et al., 1979). The most commonly used permanent stain is the trichrome stain and its modifications (Aldeen and Hale, 1992).
Culture of stool samples takes between one and several weeks and requires special laboratory facilities making it impractical for use (Strachan et al., 1988).
Rectosigmoidoscopy and colonoscopy are invasive methods and may carry the risk of perforation (Horga and Naparst, 2006). Liver function testes are normal but may show minimal elevation of alkaline phosphatase (Reed, 2001).
Antibody detection is most useful in patients with extra-intestinal amoebiasis when organisms are not generally found on stool examination. It can also distinguish between infection with E. histolytica and E. dispar, since patients infected with E. dispar have no detectable serum anti-amoebic antibody titers (Abd-Alla et al., 1992). Circulating antibodies have been detected by virtually all known serological testes as the latex agglutination, IFA, indirect haemaglutination, immunobloting, radioimmunoassay, counter immunoelectrophoresis, and ELISA. The later test is the most sensitive and specific (Robert et al., 1990; Martinz and Espinosa, 1998). However, antigen detection may be useful as an adjunct to microscopic diagnosis in detecting E. hitolytica and to distinguish between pathogenic and nonpathogenic infections (Haque et al., 1998).
The application of PCR technique in direct diagnosis of amoebiasis had been used in epidemiologic typing of infection (Acuna-Soto et al., 1993). However, Al-Hindi et al. (2005) recommended using PCR for diagnosis of stool specimens from patients with E. histolytica/dispar and that treatment should be prescribed for only patients positive for E. histolytica.
Treatment:
Amoebicidal drugs comprise two groups: Luminal amoebicide, to destroy cysts in intestinal lumen (it includes; iodoquenol, paraomomycin, and diloxanide furoate), and tissue amoebicides, to destroy trophozoites present in tissues (it includes; metronidazole, tinidazole, and omedazole). Recommended drugs for treatment of symptomatic intestinal disease and for hepatic abscess are metronidazole and tinidazole. Since these drugs may not eliminate the intraluminal cysts immediately, this therapy is followed by treatment with iodoquinol, paromomycin, or diloxanide furoate (Horga and Naparst, 2006).
III- Cryptosporidium:
In 1907, Tyzzer was the first person to describe life cycle stages of the protozoan parasite Cryptosporidium and proposed a new genus. This genus remained relatively obscure for nearly half a century until new spp. were identified from different animals (Fayer, 2004). Although, 8 spp. of Cryptosporidium (C. parvum, C.hominis, C. baileyi, C. felis, C. canis C. mealagridis, C.muris, and C. suiss) cause human infection only two spp. (C. hominis and C. parvum) are the most common (Xiao et al., 2004).
While, Cryptosporidium possesses mitochondria-like organelle (Tetley et al., 1998) as found in classical coccidian, molecular data suggest that it may be more related to gregarines (Carreno et al., 1999; Hijjawi et al., 2002).
Developmental Biology:
The Cryptosporidium parasite exists in 8 forms within the life cycle that is complex (Fig. 2). The life cycle begins upon ingestion or inhalation of the oocysts which contain 4 sporozoites. Excystation occurs in the gastrointestinal tract and sporozoites are liberated. Excystation is possibly triggered by a combination of environmental conditions such as pH, bile salts, carbon dioxide and temperature (Fayer and Leek, 1984 & Keusch, et al., 1995). The free sporozoites attach to epithelial cells and at this point are sometimes referred to as trophozoites. The trophozoite appears as round or oval uninucleate intracellular forms, 2.7 x 2.7 μm in diameter. It represents a transitional stage from sporozoite and merozoites to meronts (Thompson et al., 2005).
The trophozoites then undergo asexual reproduction to form merozoites. As a result of asexual multiplication of the trophozoite, meront development occurs and there are two different types of meront (meront I and meront II), which give rise to two morphologically different types of merozoites (Hijjiawi et al., 2001). Merozoites released from type I meronts are actively motile, circular to oval in shape and small in size (1.2 x 1μm). Merozoites released from type II meronts are either broadly spindle-shaped with pointed ends measuring 3.5 x 2 μm in size, or rounded to pleomorphic measuring 1.6 x1.5 μm. Then the merozoites released from type II meronts, develop into the sexual stages by transforming into microgamonts (5.6 μm x 3.4 μm in diameter containing microgametes) and macrogamonts (4x4μm and having a large peripheral nucleus). Following fertilization these give rise to zygotes (5 x 7μm) which undergo further asexual development, producing oocysts containing four sporozooites (Thompson et al., 2005).
Two types of oocysts are recognized thick and thin walled oocysts. The thin walled oocysts are important in internal auto-infection. Consequently, oocysts are excreted from the gut in infective form, with potential for direct fecal-oral transmission (O’Donoghue, 1995 & Fayer, 2004).
Pathophysiology:
Location of the Cryptosporidium parasite in the intestine is intracellular but extra-cytoplasmic. Apical complex proteins (e.g. circumsporozoite-like antigen, GP 900 and GP40) have been recognized to facilitate the attachment and invasion process of the sporozoite (Tzipori and Ward, 2002; Thompson et al., 2005). Parasite migration across the apical surface of enterocytes is facilitated by actin polymerization-dependent motility. Then it attaches to the host actin filaments and actin-binding proteins where the cytoskeleton is recognized with the aid of actin polymerization factors (Sibley, 2004).
The invasion and colonization of the epithelial surface displace the microvillus brush border and induce loss of surface epithelial cells which leads to loss of membrane-bound digestive enzymes and impair nutrient and electrolytes transport. Villous shortening and fusions as well as crypt hyperplasia also occur (Argenzio et al., 1990; Buret et al., 2003). Moreover, Cryptosporidium disrupt epithelial tight junction that can lead to increase epithelial cell permeability (Buret et al., 2003). Additionally, enterotoxin has been proposed to cause chloride secretion resulting in secretory diarrhea (Guarino et al., 1995 & Arafa et al., 2000). However, distribution of infection may determine the intensity of clinical signs as infection of the proximal small intestine frequently leads to watery diarrhea, while infections localized to pyloric region, the distal ileum and large intestine are frequently asymptomatic (Thompson et al., 2005).
Immune response:
Both humoral and cell-mediated immunity may be required to eliminate the infection caused by Cryptosporidium. Cell mediated immunity is believed to be the primary method, through CD4+ T cell and IFN-γ which are critical in parasite elimination (Theodos, 1998 & Riggs, 2002). Also, peripheral lymphocytes are less important than CD4+ intra-epithelial lymphocytes (IELs) which increase early in infection and CD8+ IELs which increase over the recovery phase (Riggs, 2002). The humoral immune response is considered less important but there is elevation in serum IgM and mucosal IgA antibodies (Okhuysen et al., 1998). Moreover, passive immunity transferred to infants from their mothers either across the placenta (Ungar et al., 1987) or through breast feeding appears to provide partial protection against sever clinical cryptosporidiosis (Pramoalsinsap et al., 1993).
Epidemiology:
Cryptosporidium is now considered to be one of the most common enteropathogens that causes human diarrhea. The disease is distributed worldwide with a prevalence rate ranging from 3-20% (Laureiro et al., 1986 & Fayer, 2004). It is the most common detected enteric pathogen in AIDS patients (Chen et al., 1992) and is considered as an important cause of death in healthy children in developing countries (Molbac et al., 1993 & Thompson et al., 2005).
Seroprevalence studies using antibody assays suggest that 25-35% of the population in developed countries had cryptosporidiosis some time in their life (Kourtis, 2006). In developing countries, serologic and stool studies have documented high rates of infection in Latin America, Africa, the Middle East, and South Asia. In patients with AIDS, the rate of cryptosporidiosis is higher in developing countries, ranging from 12-48% of patients with diarrhea (Clinton White and Eisen, 2006).
In Egypt, Mikhail et al. (1989) found that 9% of diarrheal patients in Aswan were due to Cryptosporidium. Also, Handousa et al. (1991) reported that 7.7% of patients with gastroenteritis were having cryptosporidiosis. However, in rural area in Alexandria, the rate of infection was 13.5% among primary school children (Soliman, 1992). While in Benha, it was 19.5% among diarrheic patients of all age groups (Abdel-Maboud et al., 2000). In cancer patients, Hassan et al. (2002) found that the incidence was 23.8% while, it was 37.7% and 91% in children and adult immunodeficient patients respectively. Abdel-Messih et al. (2005) found that 17% of children were infected with Cryptosporidium in Nile River Delta of Egypt. In Fayoum, El-Mohamady et al. (2006) found that 15% of children less than 60 months old were infected. In Ismailia, Hussein (2006) reported that the prevalence of Cryptosporidium in diarrheic children attending Suez Canal University Hospital was 16.7% and 26.8% in immunocompetent and immunocompromized children respectively.
Clinical pictures:
Infection with Cryptosporidium may or may not cause symptoms. The main symptoms are related to the gastrointestinal tract, but respiratory symptoms may also develop (Kourtis, 2006). After an incubation period of 5-10 days (range 2-28) the patient develops a self limiting watery diarrhea, which may be associated with abdominal cramps and a low-grade fever. Diarrhea contains mucous but rarely blood or leukocytes (Thompson et al., 2005). In patients who are immunocompetent, the duration of diarrhea ranges from 2-26 days (mean of 10). Diarrhea can persist longer in individuals who are immunocompromized. Oocyst shedding can continue for up to 2 weeks after clinical improvement (Clinton White and Eisen, 2006). Biliary tract involvement is recorded. Nonspecific respiratory symptoms, including shortness of breath, wheezing, cough, hoarseness, and croup, may be a manifestation of infection in the respiratory tree (Kourtis, 2006).
Reactive arthritis that affects the hands, knees, ankles, and feet have been described with Cryptosporidium infection (Hay et al., 1987; Shephered et al., 1989; Thompson et al., 2005 & Kourtis, 2006). Moreover, Cron and Sherry (1995) reported that Reiter’s syndrome (triad of arthritis, conjunctivitis and urethritis) was associated with cryptosporidial gastroenteritis.
Diagnosis:
Stool specimens as well as other secretions like sputum or bile can be examined to detect oocysts. Specimens are submitted as fresh materials or in formalin 10% or sodium acetoacetic acid formalin (SAF) preservatives (Garcia et al., 1987). Recently, the most used sensitive concentration method is the Sheather’s sugar floatation that provides the best results (Kvac et al. 2003). However, multiple stool samples (at least three) should be tested before a negative diagnostic interpretation is reported, because shedding may be intermittent (CDC, 2002).
The diagnosis of cryptosporidiosis was improved with the discovery of the acid-fast property of Cryptosporidium oocysts. There are several staining techniques based on the original Ziehl Neelsen staining with some modifications (Garcia et al. 1983; Martinz and Belda neto, 2001). In these acid-fast staining, the oocysts of Cryptosporidium are revealed as intense pink-reddish forms, 4-6 µm, against a green or blue background depending on the dye used in the counterstaining step. Fluorescent staining with arcidine orang causes both oocysts and yeasts to fluoresce, while with fluorochrome Truant auramine-rhodamine stain, only oocsts fluoresce (Ma and Soove, 1983). Hot Safranin staining can also be used (Mehta, 2002).
A variety of tests such as Enzyme Immuno Assay (EIA) and IFA (both direct and indirect methods) can detect Cryptosporidium, although they are not particularly specific when required to distinguish between C. parvum and non-C. parvum (Graczyk et al., 1996; Mehta, 2002). However, ELISA is a highly sensitive and specific technique, and is useful for screening large numbers of specimens in a short time period. Also, it does not rely on skills in microscopy (Mehta, 2002; El-Sibaei et al. 2003). Michel et al. (2000) showed that co-agglutination test for detecting cryptosporidial antigen in stool, serum and water is clearly superior to ELISA especially in large-scale epidemiological surveys.
Recently, molecular techniques were developed to detect C. parvum in stool specimen and identified two genotypes (C. hominis which is affecting human only and C. parvum which is zoonotic) by using different types of PCR (Mehta, 2002; Thompson et al., 2005).
Treatment:
Pharmaceutical treatment of cryptosporidiosis is not satisfactory. Many agents have been tried with variable and limited success (Kourtis, 2006). The majority of immunocompetent individuals suffer a short self-limiting course that requires supportive care with fluid replacement, nutrition and occasionally anti-diarrhoeal medication (Clinton White and Eisen, 2006). Recently, nitazoxanide (Alinia) is approved as an oral suspension to treat children with diarrhea caused by Cryptosporidium. Significant reduction in the duration of diarrhea and oocyst shedding were recorded, its dose in adults is 500 mg BID for 3 days while pediatric dose is 100 mg (1-4 years old) and 200 mg (4-12 years old) BID for 3 days (Rossignol et al., 2001). Also, bovine transfer factor and bovine colostrum from animals were under trails as a treatment, as well as orally administered immunoglobulin preparations (Kourtis, 2006).
IV- Cyclospora cayetanensis:
In the early to mid 1990s, Cyclospora infection came to worldwide attention (Ortega et al., 1998). Since the advent of AIDS epidemic, C. cayetanensis has been increasingly recognized as an enteric pathogen (Tolan and Schroeder, 2007).
Of the 19 known Cyclospora spp. that infect animals (primates, other mammals and reptiles), C. cayetanensis infection is the only spp. affecting humans (Lainson, 2005) and no animal reservoir for C. cayetanensis is known or suspected (Eberhard et al., 2000 & Shoff and Behrman, 2006).
Developmental Biology:
Cyclospora has an obligate intracellular life cycle within the epithelium of the gastrointestinal tract where both asexual (merogony or schizogony) and sexual (gametogony) reproduction occur. The infection is initiated when a person ingests sporulated oocysts in contaminated food or water which are spherical in shape and between 7.5 and 10 μm in diameter with a 50 nm thick wall with outer fibrillar (63 nm) coat (Abou El-Naga, 1999). In the presence of bile and trypsin, usually in the jejunum oocyst excysts releasing sporozoites thus invade the intestinal epithelial cells. Merogony results in the generation of many merozoites, each of which is capable of infecting a new host cell and initiating another generation of merogony (Ortega et al., 1997). Then the type I meronts with 8-12 merozoites are developed and released, presumably by cell rupture, to invade other epithelial cells and repeat the process (Goodgame, 1996 & Sterling and Ortega, 1999).
After several cycles of type I meronts, type II meronts develop with each cell containing 4 merozoites. After invading epithelial cells, some of these form single macrogametes and others divide multiple times to form microgametes. When released, a microgamete fertilizes a macrogamete, which develops into a zygote. The zygote, in turn, develops into an oocyst with an environmentally resistant wall. The oocyst passes into the environment in feces, as a non-sporulated non-infectious oocyst consequently; human-to-human transmission does not occur (Sterling and Ortega, 1999 & Mansfield and Gajadhar, 2004).
In the environment, the oocyst sporulates, becoming infectious for humans. During sporulation, the sporont divides into 2 sporocysts, each containing 2 sporozoites. Time course of sporulation is 7-12 days in the environment at ambient temperature. The preferred temperature is 26-30°C. Contamination of food or drinking water leads to human ingestion and infection. The infectious inoculum is small but has not been precisely quantitated (Eberhard et al., 2000; Shoff and Behrman, 2006).
Pathophysiology:
Grossly, moderate to severe erythema of the distal duodenum is observed in patients with Cyclospora infection. Distal duodenal histopathological findings include acute and chronic inflammation, reactive hyperemia with vascular dilatation and villous capillary congestion, parasitophorous vacuoles that contain both asexual and sexual forms, crypt hyperplasia, epithelial disarray, and partial villous atrophy. Electron micrographs have demonstrated intracellular particles similar to sporozoites (Mansfield and Gajadhar, 2004; Shoff and Behrman, 2006). Abnormal findings on lactulose or mannitol studies or studies of both have demonstrated intestinal barrier disruption. Abnormal findings on D-xylose studies have demonstrated malabsorption (Long et al., 1990).
Immune response:
The nature of the immune response to Cyclospora is not clear, and only few observations can currently be made. Patients’ sera have demonstrated Cyclospora-specific antibodies (Shoff and Behrman, 2006). Acquired immunity may play a role in susceptibility to Cyclospora infection. It is likely that previous exposure confers some resistance against challenge infection. During a wet season, Cyclospora was the main protozoan cause of GIT illness and diarrhea among adult foreign residents of West Java, Indonesia. However, in this area asymptomatic infection was common and the parasite rarely associated with illness in indigenous population (Fryauff et al., 1999). On the other hand, a variety of autoimmune sequel such as Guillian-barre syndrome and immune complex such as Reiter’s syndrome have been associated with cyclosporiasis (Richardson, et al., 1998 & Conner, et al., 2001).
Epidemiology:
The true prevalence of this organism is not yet known. The majority of cases occur in tropical areas where the infection rates as high as 20% have been observed with its peak in children aged 2-4 years (Ortega et al., 1993).
C. cayetanensis causes an estimated 16,264 cases of food borne illness in the United States each year out of the estimated 76 million cases of food borne illness overall (Shoff and Behrman, 2006).
It was reported for the first time in Egypt at 1995 in AIDS patient from Alexandria (Awadalla et al., 1995). However, the extent of infection and its effect in human health is still under studying, the prevalence reaches about 9% in children in Cairo and El-Menofiya Governorate (Nassef et al., 1998). Subsequently, Abou El-Naga (1999) detected cyclospora infection in 4% of immunocompromized patients. In contrast, El-Nazer et al. (1998) in Assiut University hospital, found Cyclospora oocyst among 25% of non diarrheic immunocompromised patients. However, no cases were detected among immunocompetent and immunocompromised primary school children in Port Said city (Saad, 2001). On the other hand, C. cayetanensis oocysts were detected in 5.6% malnourished children and in 2.8% of control group in the study of Rizk and Soliman (2001) in Mansoura, Dakahlia Governorate. In Ismailia, the prevalence of C. cayetanensis among diarrheic children attending Suez Canal University Hospital was 19.6% and 34.6% in immunocompetent and immunocompromised children respectively (Hussein, 2006).
Moreover, oocysts can survive in water for 2 months at 4°C, for 7 days at 37°C and they are resistant to chlorine disinfection. On the other hand, heating (60°C for 60 min.) and freezing (-18°C) prevents sporulation. Desiccation for 15 min. ruptures the oocyst wall. Pesticides at recommended levels do not affect sporulation. So, washing contaminated vegetables does not completely remove all of the oocysts (Shoff and Behrman, 2006). In addition several outbreaks of cyclosporiasis have been linked to contaminated water, fresh produce and food (Mansfield and Gajadhar, 2004) which is common in spring and early summer seasons (Tolan and Schroeder, 2007).
Clinical pictures:
After exposure, in non-immune individuals, the incubation period is usually 1-11 days (mean of 7). The onset of illness may be abrupt in as many as 30% of cases. It may be preceded by flu-like illness. The patient develops watery diarrhea (may be explosive) with a median of 6 stools per day, but in some patients there is no diarrhea (Mansfield and Gajadhar, 2004). Other symptoms include; anorexia, weight loss, fatigue, abdominal cramps, vomiting, flatulence, abdominal bloating, less frequently joint pain and low grad fever (Loony, 1998). After a few days, acute symptoms subside and then may recur (61% of cases) in a waxing-waning pattern. Alternatively, a patient may experience persistent symptoms. The illness usually lasts 6-7 weeks but has been reported to persist for several months. The duration can be several months to a year in patients with HIV (Shoff and Behrman, 2006).
Extra-intestinal cyclosporiasis was reported such as acalculous cholecystitis and Guillian-barre syndrome (Richardson, et al., 1998 & Zar et al., 2001). Moreover, Reiter’s syndrome was reported following protracted symptoms of Cyclospora cayetanensis (Conner, et al., 2001).
Diagnosis:
Laboratory confirmation of cyclosporiasis in symptomatic and/or asymptomatic patients is defined as detection of Cyclospora oocysts in stool samples by microscopic examination or in intestinal fluid, or small bowl biopsy specimens, or demonstration of sporulation, or DNA by PCR in stool, duodenum/jejunal aspirates or small bowel biopsy specimens (CDC, 1997).
Direct smears are usually unsuccessful as the organisms excreted in stool are few and with intermittent excretions. However, the concentration methods help in diagnosis not only by concentration but also by cleaning the oocysts in feces. Then the stool sample could be examined as wet mount by phase contrast and/or bright field microscope first by using X400 and then by X1000. It is recommended to use an ocular micrometer to measure the size (7.5-10 μm), which is valuable in exclusion of Cryptosporidium (4-6 μm) and other artifacts (Ratman et al., 1985 & Ortega et al., 1998).
Oocysts can be concentrated by differential centrifugation. However, the most employing methods are by FEA or sucrose floatation in Sheather’s solution (Ortega et al., 1998). Also, potassium hydroxide concentration has been described by Markell et al. (1999).
Cyclospora is sensitive to acid-fast and safranin staining. The acid-fast stains show great variability because many organisms remained as unstained glassy, wrinkled spheres and the others show faint pink to deep red (Garcia, 2001). By using modified safranin stain, which gives superior results, the oocysts stained reddish orange (Baxby et al., 1994). Auto-fluorescence is one of the confirmatory methods used in diagnosis of Cyclospora oocysts (Berlin et al., 1994). This feature enhanced their detection at least two folds over the direct wet mount (Clark, 1995 & Berlin et al., 1998).
Routine light microscopy does not permit accurate visualization of the sporozoites within each sporocyst but, the induction of sporulation and excystation experimentally were able to identify the contents of the oocyst (Smith et al., 1988). This is not essential for diagnosis but it could be used for more confirmation (Smith et al., 1997).
Electron microscopy (EM) is the most assured way for diagnosis of Cyclospora in stool specimens. Oocysts have a bilayered thick wall. Each oocyst contains two ovoidal sporocysts. Stieda and substieda bodies are present. There are two sporozoites in each sporocyst (Ortega et al., 1998).
Although, Shoff and Behrman (2006) considered that, until now no reliable immunological techniques are available to detect Cyclospora, Wang et al. (2002) proved that ELISA used in specific antibody detection was more simple, practical, specific and sensitive in diagnosis of Cyclospora infection.
Several molecular techniques had been described for the detection of Cyclospora oocysts as nested PCR that was unable to differentiate Cyclospora spp. and Eimeria spp. (Relman et al., 1996), an oligonucleatid-ligation that can differentiate between Cyclospora and Eimeria (Jinneman et al., 1998), intervening transcribed spacer region which can be used to identify genotyping of C. cayetanensis (Adam et al., 2000), quantitative real time PCR that can evaluate the intensity of Cyclospora infection (Varma et al., 2003). Recently single-nucleotide polymorphisms (SNP-PCR) and restriction fragment length polymorphism (RFLP-PCR) were developed to distinguish C. cayetanensis from other Cyclospora spp. and Eimeria (Chu et al., 2004 & Li et al., 2007).
Treatment:
The only effective treatment for eradicating Cyclospora infection is the Co-trimoxazole. The adult dose is Trimethoprim 160 mg and Sulphamethoxazole 800 mg (TMP-SMX) twice daily for 7-10 days (Mansfield and Gajadhar, 2004). The children dose is TMP (5 mg/kg.d) plus SMX (25 mg/kg.d) three times daily (Medico et al., 1997).
V-Blastocystis hominis:
The first accurate description of Blastocystis hominis organism was in 1911, and named Blastocystis entercola (Alexieff, 1911). This name was changed to B. hominis and classified as yeast genera by Brumpt (1912). Then, B. hominis was reclassified as protozoan belonging to class Blastocystis with new order Blastocystida (Zierdt, 1978 & Zierdt, 1991).
Developmental biology:
Four commonly described forms are present, namely the vacuolar (otherwise known as central body), granular, amoeboid, and cyst forms. The appearance of the organism is largely dependent upon environmental conditions (Zierdt, 1991 & Tan, 2004).
The vacuolar form is the typical cell form of Blastocystis which varies greatly in size, with diameters ranging between 2 µm and 20 µm. Otherwise it is known as central body form because it has a large central vacuole surrounded by a thin band of peripheral cytoplasm which contains other organelles (Khalifa, 1999). The function of the vacuole is for storage purposes (Boreham and Stenzel, 1993). Other functions, such as cell division during reproduction (Zierdt, 1991) and the deposition of apoptotic bodies have been proposed (Puthia et al., 2006).
The granular form is somewhat morphologically identical to the vacuolar forms except that distinct granules were observed in the central vacuole and/or cytoplasm (Dunn et al., 1989). Within the central vacuole, these granules appear in different forms too. Three types were suggested; metabolic, lipid, and reproductive granules (Tan and Zierdt, 1973).
An amoeboid form is rather rare in occurrence, only in patients with acute diarrheal syndrome. It is irregular in shape and has pseudopodia of slow movement, central nuclei and no central vacuole (Lanuza et al., 1997 & Tan et al., 2001).
The cyst form is smaller in size and has a thick multilayered cyst wall. It lacks a central vacuole but few nuclei, multiple vacuoles and food storage deposits were observed. It is the most resistant form of this parasite and is able to survive in harsh conditions because of its thick multilayered cyst wall. It could survive well at room temperature for up to 19 days, indicating its strong resistance (Zaman et al., 1995 & Moe et al., 1996).
Infection occurs in human when the fecal cyst form is being ingested. After ingestion, the cyst develops into other forms which may in turn re-develop into cyst forms. Through human feces, the cyst forms enter the external environment and are being transmitted to human and animals via the fecal-oral route (Tan, 2004). Five modes of reproduction were observed in B. hominis, namely, binary fission, endodyogeny, plasmotomy, budding and schizogeny (Zhang et al., 2007).
Pathophysiology:
B. hominis is able to produce a protease that breaks up IgA antibody which is produced and secreted into the gastrointestinal lumen. By breaking up this antibody, it allows the persistence of B. hominis in the human gut (Puthia et al., 2005). Puthia et al. (2006) proposed that, in response to the proteases secreted by Blastocystis, self-destruction of the host cells (apoptosis) will eventually occur.
Blastocystis has the ability to alter the arrangement of F-actin in intestinal epithelial cells. Actin filaments are important in stabilizing tight junctions, they in turn stabilize the barrier which is a layer of cells between the intestinal epithelial cells and the intestinal content. The parasite causes the actin filaments to rearrange, and so compromising barrier function. This has been suggested to contribute to the diarrheal symptoms sometimes observed in Blastocystis patients (Tan, 2004 & Puthia et al., 2006). Impaired intestinal cell permeability was proved by Zuckerman et al. (1994).
The histopathological changes of intestinal mucosa vary from enteritis (Gallagher and Venglarcik, 1985), terminal ileitis (Tsang et al., 1989) and ulcerative colitis (Jeddy and Farrington, 1991). Toxic and allergic reaction may play a role in its pathogenesis (Garavelli et al., 1991a). In colonic ulceration, B. hominis was found in superficial ulcers and infiltrating to the superficial lamina propria. In addition, inflammation with chronic inflammatory cells and eosinophils was present in the caecum, transverse colon and rectum of the patient (Al-Tawil et al., 1994).
Epidemiology:
Blastocystosis has a world wide distribution. It is endemic in tropical and subtropical countries and more common in developing countries compared to industrialized nations (Garavelli et al., 1988 & Sohail and Fischer, 2005).
In the United States, the prevalence rate of B. hominis is about 2.6%, while in less developed countries the prevalence of 30% might reach (Hoeprich et al., 1995). In South America, prevalence ranges from 16.8% to 34.1% (Sheehan et al., 1986 & Devera et al., 1997). In Europe, prevalence rate of B. hominis infection was from 4% to 16.5% among diarrheic patients that is higher in adults than children and slightly higher in females than males (Carbajal et al., 1997 & Svenungsson et al., 2000).
In Asia, prevalence rate of B. hominis infection was 8% among food handlers, 12.8% among preschool children and 60% among school children (Tanriverdi et al., 1988; Amin, 1997 & Pegelow et al., 1997). Prevalence rate in Saudia Arabia and Kuwait was 17.5% and 2% respectively (Qadri et al., 1989 & Zaki et al., 1991). In Australia, the prevalence rate of B. hominis was 6% (Hellard et al., 2000). However, in Africa, in Kenyan Rift Valley, the prevalence rate of B. hominis was 3% (Joyce et al., 1996). While in Democratic Sahara Republic the prevalence among children was 22% (Paricio et al., 1998).
In Egypt, the prevalence rate of B. hominis was 8.5% in symptomatic and 4% in asymptomatic food handlers in Qualyobia governorate (Sadek et al., 1997). Among immunocompromized children in Mansoura Governorate, B. hominis prevalence rate was 9% (Azab, 1998). In Alexandria, the prevalence of B. hominis in immunocompetent children was 8.4% (Ibrahim et al., 1998) while in diarrheic patients was 6% (Khalifa, 1999). El-Shewy et al. (2002) detected a prevalence rate of 10.1% among school children in Ismailia city.
Clinical pictures:
Not all Blastocystis infections display symptoms, and even when symptoms are present they are not specific to Blastocystis infection. Common symptoms attributed to B. hominis infection are diarrhea, abdominal cramps, nausea, and in more acute cases, profuse watery diarrhea and fever. Other possible associated symptoms include fatigue, anorexia, flatulence, headache, chills and other gastrointestinal effects (O’Gorman et al., 1993; Tan, 2004). Ertug et al. (2007) found a correlation between the presence of B. hominis and lower anthropometric indices in children.
Symptoms are usually associated with parasite concentration of >5 per high power field (Udkow and Markell, 1993) and the severity of symptoms increases with large number of the organism in feces (Garcia and Bruckner, 1997). The mean duration of symptoms is around 11 days (Senay and MacPherson, 1990). However, symptoms last longer and more sever in immunocompromised patients (Hoeprich et al., 1995).
B. hominis may be associated with a variety of diseases, including tropical pulmonary eosinophilia (Enzenauer et al., 1990), diabetes (Scaglione et al., 1990), leukaemia (Garavelli et al., 1991b). Lee et al. (1990) reported a case where the organism was found in synovial fluid aspirated from knee effusion of a rheumatoid arthritis patient who developed B. hominis acute diarrhea, which was associated with increased inflammation and effusion of the left knee, suggesting invasive infection (infectious arthritis). While in some cases, the organism may cause reactive arthritis (Lakhanpal et al., 1991). Kruger et al. (1994) reported a case of 46 years old female patient who developed a chronic diarrhea and oligoarthritis some days after returning from a trip to Senegal.
Diagnosis:
Diagnosis of B. hominis in man is based on identification of the parasite in the fecal sample. As there is irregular shedding pattern of the organisms in feces, so it is recommended that more than one sample should be examined before exclusion of infection (Vennila et al., 1999). Usually the parasite is identified in the vacuolated form. Cystic stage is not so easy to identify, because of its relatively small size (Zaman, 1998). However the recognition of cyst is greatly facilitated if the fecal specimen is first concentrated using Ficoll column technique (Zaman, 1996).
Wet mount preparation and trichrome stained smears are recommended for stool examination in blastocystosis (Stenzel and Boreham, 1996). In fresh saline smears, organisms were identified as refractile particles, rounded to ovoid (5-20 µm) with well defined wall, central body and a thin layer of cytoplasm containing number of nuclei (Khalifa, 1999). Other permanent stains can be used as iron hematoxylin (Guimaraes and Sogayar, 1993), safranin-methylene blue and modified Ziehl-Neelsen stains (Khalifa, 1999). Culture of clinical specimens is beneficial when microscopic diagnosis is uncertain (Boreham and Stenzel, 1993).
Fluorescent stains also have been used to detect B. hominis organisms. These include; truant-auramine stain, which cause fluorescence of both wall and central structure but not nuclei and auramine carbol fuchsin, which causes fluorescence of both central body and nuclei against red background (Khalifa, 1999). Transmission electron microscopy and scanning electron microscopy can be used for further morphological studies of the parasite (Zaman et al., 1995 & Zaman et al., 1998). Although both are not required for routine diagnosis, they are of great importance in research (Stenzel and Boreham, 1996).
Moreover, invasive techniques have occasionally been used in diagnosis, but generally are not required (Stenzel and Boreham, 1996). These techniques may include; duodenal aspirate using entero test (Narkewicz et al., 1989), colonic scrapings during colonoscopy (Dellers et al., 1992), touch cytology (Debongnie et al., 1994), endoscopy and sigmoidoscopy (Zukerman et al., 1994).
Enzyme Linked Immuno-Sorbent Assay can detect IgG antibodies against B. hominis (Hussain et al., 1997). Although ELISA was used to confirm infection, to screen populations and to determine rise and fall in circulating antibodies overtime, however it is now important for diagnosis of infection especially with growing numbers of patients (Zierdt et al., 1995). Also a strong antibody response consistent with B. hominis infection can be demonstrated using IFA (Garavelli et al., 1995).
Molecular identifications of B. hominis using PCR, electrophoretic karyotyping, DNA sequencing, and RFLP analysis have been developed. However, these are expensive methods and might not be a feasible method in hospitals and clinics (Yoshikawa et al., 1996; Ho et al., 2001 & Yoshikawa et al., 2004).
Treatment:
In immunocompetent patients, blastocytosis tends to be self-limiting (Kain et al., 1987). However treatment is necessary for patients with persistent symptoms (Hoeprich et al., 1995). Six protozoal drugs were found to be effective against B. hominis. In order of effectiviness, they are: emitine, metronidazole, furazolidone, TMP-SMX, chlorohydroxy-iodoquinolone, chloroquine and diiodohydroxyquinoline. However, metronidazole is the most widely used at 250 mg three times daily. TMP-SMX could be used to cure infections resistant to metronidazole (Zierdt, 1991). Moreover, oral administration of oil of Mediterranean oregano Oreganum vulgare resulted in complete disappearance of B. hominis in 8 of 14 cases (Force et al., 2000).
VI- Schistosoma mansoni:
In 1851, Theodor Bilharz, described a parasitic infection that was later termed schistosomiasis. Currently, 200 million people in 74 countries have this disease; 120 million of them have symptoms, and 20 million have severe illness (Chitsulo et al., 2000). Five spp. of Schistosoma are known to infect human. where infection with; S.mansoni, S. japonicum, S. mekongi and S. intercalatum is associated with chronic hepatic and intestinal fibrosis and infection with S. haematobium results in fibrosis and calcification of the urinary tract (Ross et al., 2002).
Developmental biology:
All Schistosoma infections follow direct contact with fresh water that harbors free swimming larval stage known as cercariae (Laughlin, 1984). The cercaria penetrates the skin or mucous membranes with the assistance of lytic proteinase (Curwen and Wilson, 2003). The process is quite rapid, cercariae changes its appearance to become schistosomula which is tail less and worm like in appearance (Rifaat, 1976).
After penetration, the trilaminate tegument of the cercariae is replaced by the seven layer membrane of the schistosomulum and adult worm which consist of two closely opposed lipid bilayers (Hockley et al., 1975). Schistosomulae are transported predominantly via the blood vessels, and a small proportion via lymphatics, traveling passively with flow of blood via the right side of the heart to lodge in lung capillaries (Miller and Wilson, 1978). In the lung, the schistosomulae undergo morphological changes and become more elongated (Wilson and Barnes, 1987). They find their way into the microcirculation to the heart, lungs and eventually to the small vessels of the liver (Laughlin, 1984).
In the liver, schistosomulae become mature adult unisexual worms. Four to six weeks after cercarial penetration, mating occurs and egg lying starts (Laughlin, 1984). The broad male blocks the blood flow, while the female extends forwards to lay its eggs receding backwards in doing so. Egg lying starts 30-40 days from the date of infection (Pellegrino et al., 1962). Eggs that reach the lumen of the intestine are passed out in the stool. Hatching takes place in fresh water and the liberated miracidium locates its appropriate snail Biomphalaria alexandrina (Laughlin, 1984).
Inside the snail, the miracidium develops into a mother sporocyst and two weeks later into daughter sporocyst (Rifaat, 1976). The second generation of sporocysts migrates to the digestive glands of the snail, where they develop cercariae (Diconza and Hasen, 1972). Shedding of cercariae takes place in water in bright sunlight. Cercariae emerge from the snail, about 4 weeks after initial penetration by the miracidium. Without successful host contact, cercariae die within 24- 48 hours (Schmidt and Roberts, 1981).
Pathophysiology:
Not all Schistosoma eggs are excreted from the body, and up to 50% can embolize to other body areas, leading to a host immune reaction and granuloma formation. Granulomas begin to form with maturation of miracidium at 6 days and become localaized within 2 weeks. The most common sites for S. mansoni are the liver and intestine. Other less commonly affected sites include lungs, central nervous system and kidneys (Colley et al., 1986 & Nissen and Walker, 2005).
A chronic inflammatory response to Schistosoma eggs, is mediated by both cellular and humoral mechanisms, this immune reaction is the actual root of pathology in hepatosplenic disease rather than a direct pathogenicity (Stickland and Ramirez, 2000 & Schafer and Hale, 2001).
Immune response:
After infection, the schistosomula expresses antigens on their surface that evoke a host immune response which provides some degree of resistance to re-infection. As the worms mature they become less antigenic and more resistant to the immune system-mediated killing mechanisms, because the worms are able to incorporate host antigen onto their surface, thus preventing the host from recognizing these parasites as foreign (Capron, 1992 & Dunne et al., 1992).
In infected individuals, there is a marked increase in the levels of IgE and IgG4 against various parasitic antigens (Langley et al., 1994). Individuals exposed to infection develop an age acquired resistance (Etard et al., 1995). These antibodies have also been associated with resistance to re-infection. In acute schistosomiais, the levels of IgA and IgM correlate with the intensity of infection (Rabello et al., 1995).
The immune response to schistosomiasis is highly regulated through Th1 and Th2 responses with local cytokine production. Granulomatae are notable for the presence of eosinophils and lead to the development of widespread collagen deposition and scar tissue (Auriault et al., 1996 & Pearce and MacDonald 2002).
Human skin appears to be a critical site in which the initial events of the host and parasite interaction occur and where the immune response is commenced. Induction and modulation of granuloma formation is under the control of clones of CD4+ and CD8+ T cells. Cytokines produced in response to the parasite, such as IL-7 in the skin and IFN-α in the liver, also seem to influence the development of schistosomal immunity (Capron, 1992 & Chikunguwo et al., 1993).
Clinical picture:
Disease syndromes associated with S. mansoni infection are related to the stage of infection, previous host exposure, worm burden, and host response. Syndromes include cercarial dermatitis, acute schistosomiais (Katayama fever), and chronic fibro-obstructive disease which is related to tissue changes resulting from egg deposition (Garcia, 2001).
Cercarial dermatitis follows skin penetration by cercaria, leading to petechial hemorrhages with edema and pruritus with subsequent maculopapular rash which may become vesicular. However, cercarial dermatitis is a constant feature of human infection with avian schistosomes, with cercarial death occurring in the subcutaneous tissues and immediate hypersensitivity reaction occurring at invasion sites (Pearce and MacDonald 2002).
Acute schistosomiasis is associated with heavy primary infections and the initiation of egg production. Characteristic features include high fever, hepatosplenomegaly, lymphadenopathy, eosinophilia, and dysentery (Garcia, 2001).
Egg deposition and granuloma formation affect many organs. The most commonly affected site with S. mansoni is the liver, leading to hepatosplenic disease with the complication of hepatosplenomegaly, portal hypertension, and oesophageal varices (Garcia,2001), less commonly affected sites include lungs, central nervous system and kidneys (Nissen and Walker, 2005).
Rheumatic manifestations are most common among patients with chronic schistosomiasis, resembling classical reactive arthritis or other sero-negative spondyloarthropathies with varying combinations of peripheral enthesitis, large joint asymmetric oligoarthritis, and sacroileitis. However, high percentage of rheumatoid arthritis-like presentations involving the metacarpophalengeal and proximal interphalengeal joints, wrists, ankles, and knees were reported (Kamel et al., 1989; Fachartz et al., 1993; Rolland et al., 1998). Some of these manifestations appear to result from immune complex disease (Greenfield et al., 1986; Bebars et al., 1992), whereas others result from direct infection (Bassiouni and Kamel, 1984; Atkin et al., 1986).
Epidemiology:
The estimated mortality owing to S. mansoni in sub-Saharan Africa is 280,000 per year (Pearce and MacDonald 2002). Schistosomiasis is one of the 10 tropical diseases especially targeted for control by WHO (Morel, 2000). Despite major advances in control, schistosomiasis continues to spread to new geographical areas (Patz et al., 2000).
In Egypt, The prevalence rate of S. mansoni infection in rural Ismailia was 42.9%. Prevalence and intensity peaked in the 20-30 years old age group and was higher in males than in females (Nooman et al., 2000). Another study in 9 rural governorates in Egypt revealed a prevalence of 17.5-42.9%. Morbidity occurred in >60% of cases due to irreversible fibrosis and portal hypertension (El-khoby et al., 2000). On the other hand, a study in Alexandria revealed that prevalence of S. mansoni accounted for 20.5% (Zaki et al., 2003). While in Giza governorate, the prevalence in a village at the Nile bank south to Cairo was 25.1% (Sayed et al., 2004). Recently, El-Shazly et al. (2006a) found that the prevalence rates of S. mansoni in an urban and a rural area in Dakahlia Governorate were 0.5% and 1.6% respectively.
Diagnosis:
Eggs may be found in feces as early as 5 weeks after infection. Patients with a low worm burden or old (chronic) infections may have very few eggs in their feces, and infection may not be confirmed. For that reason multiple stool examinations should be performed (Garcia, 2001). Engeles et al. (1997) suggested that examination of few samples collected on different days was more effective than examination of more slides from one sample. Occasionally S. mansoni eggs are detected in urine (Garcia, 2001).
Direct microscopic examination of stool smears is not very sensitive but may be useful for screening purposes. Concentration techniques can be used to detect eggs in stool. Recommended methods include FEA technique and Kato-Katz thick smear which is simple and sensitive quantitation technique (Ebrahim et al., 1998).
In chronic infections in which the worm burden is light, hatching test can be performed. Rectal biopsy specimens have been particularly useful in detecting eggs in patients with light, chronic, or inactive infections. The biopsy tissue can be crushed between two glass slides. The technique is more effective than histological examination and allows assessment of the spp. and viability of the eggs (Garcia, 2001).
A large number of serological tests have been used in the diagnosis of schistosomiasis (Tsang and Wilkins, 1991). The most frequently used tests are the circumoval precipitin test, the cercaria hullen reaction, the indirect fluorescent-antibody test, the indirect hemagglutination test, and ELISA (Rossi et al., 1991; Tsang and Wilkins, 1991). ELISA has also been used to detect circulating schistosome antigens in the serum and urine of infected patients and it may be the preferred method of diagnosis (Barsoum et al., 1991; Hassan et al., 1992; Polman et al., 1995).Detection of circulating anodic antigen and circulating cathodic antigen in serum and urine for the diagnosis of active schistosomal infections appears promising as a complimentary diagnostic tool with fecal/urine microscopy and antibody detection (Stickland and Ramirez, 2000).
Treatment:
Praziquantel (a pyrazinoquinolone) has become the main anti-schistosomal agent because it is effective against many parasites and is well tolerated orally with a single dose of 40 mg/kg. Other oral compounds available are oxamniquine (a nitroquinolone) and metrifonate (an organophosphorous cholinesterase inhibitor), but these have limited parasite specificity (Guiniady et al., 1994; Abd El-Wahab, 1996 & Stickland and Ramirez 2000). Recently, myrrh extract (mirazid) was under trail as a novel anti-schistosomal drug which showed a remarkable effect against S. mansoni mature and immature stages also high efficacy rate among schistosomiasis patients without any side effects (Massoud et al., 2000; Sheir et al., 2001 & Massoud et al., 2004).
VII- Strongyloides stercoralis:
Infection with Strongyloides stercoralis was first reported in the year 1876 in French soldiers working in Vietnam (Siddique and Berk, 2001). Then its life cycle, hyperinfection and disseminated infection were described by Fulleborn in 1926 (Schwartz and Tarlow, 2005 & Vadlamudi et al., 2006).
Developmental biology:
Strongyloidiasis is typically acquired when the infective (filariform) larvae penetrate the skin during contact with contaminated soil. This is facilitated by a potent histolytic protease that is secreted by the organism, although ingestion of filariform larvae (fecal-oral route) may also result in infection (Dees et al., 1990). The larvae migrate into the pulmonary circulation via the lymphatic system and venules. Larvae are swallowed, and reach the gastrointestinal system, where they are embedded in its folds (Woodring et al., 1994). Besides this migratory pathway, a direct route from skin to duodenum can also be taken (Keiser and Nutman, 2004).
Larvae moult twice and become parasitic adult female worms with 2 x 0.05 mm in diameter (Mahmoud, 1996) which anchor themselves with their mouths to the mucosa of the small intestine or burrow their anterior ends into the submucosa. The parasitic female worm produces eggs by means of parthenogenesis, yielding non infective (rhabditiform) larvae. These larvae either can be passed out of the host’s body in the feces or, under certain conditions such as constipation, can cause autoinfection (Schmidt and Roberts, 1989). Rhabditiform larvae that are passed into the stool can become either filariform larvae or free-living adult males and females (1 x 0.06 mm) capable of producing rhabditiform larvae (i.e. free-living cycle). The latter, in turn, can either develop into infective (filariform) larvae (i.e. able to penetrate human skin and initiate the parasitic cycle) or repeat the free-living cycle (Schmidt and Roberts, 1989 & Vadlamudi et al., 2006).
Autoinfection involves premature transformation of non infective larvae (rhabditiform, 0.25 X 0.015 mm) into infective larvae (filariform, 0.5 X 0.015 mm), which can penetrate the intestinal mucosa (internal autoinfection) or the skin of the perineal area (external autoinfection), thus establishing a developmental (parasitic) cycle within the host. Infection can be maintained by repeated migratory cycles for the remainder of the person’s life (Schmidt and Roberts, 1989).
Pathophysiology:
At the portal of entry, the larvae cause petechial hemorrhages, which are accompanied by intense pruritus, congestion, and edema. Once in the pulmonary capillaries, the larvae produce hemorrhages, which form the avenue of spread into the alveoli. An inflammatory response associated with eosinophilic infiltration follows, and the sequence of events that occurs in the lungs results in pneumonitis. In the intestine, S. stercoralis can produce an inflammatory reaction and induce a malabsorption syndrome (O’Brien, 1975). Also Sullivan et al. (1992) found that strongyloidiasis was associated with hypoalbuminemia in the infected children.
The two most severe forms of strongyloidiasis are hyperinfection syndrome and disseminated strongyloidiasis. Hyperinfection syndrome represents an acceleration of the normal life cycle of S. stercoralis, leading to excessive worm burden without the spread of larvae outside the usual migration pattern (e.g. GIT, lungs) (Keiser and Nutman, 2004).
Disseminated strongyloidiasis involves widespread dissemination of larvae to extra-intestinal organs (e.g. CNS, heart, urinary tract, endocrine organs), which are not ordinarily part of the parasite life cycle (Harcourt-Webster et al., 1992). In these severe forms of strongyloidiasis, the migration of the larvae from the gastrointestinal tract into the circulation and various organs may result in bacteremia and, occasionally, meningitis with enteric microorganisms. The enteric microorganisms either are carried by the filariform larvae or enter the circulation via the intestinal ulcers (Link and Orenstein, 1999).
Immune response:
There are two broad types of defense mechanisms involved against larvae, the first one is against the filariform larvae and the second is against the host adopted larvae that cause autoinfection (Vadlamudi et al., 2006). Both the humoral and cellular immune responses are required in Strongyloides infection (Keiser and Nutman, 2004). Indeed, Herbert et al. (2002) proved that B-cells play no part in the defense against primary infection but play an important role in subsequent challenge infections. Increased circulating IgG, IgM and specific serum IgA levels were reported (McRury et al., 1986; Douce et al., 1987 & Genta et al., 1987). In addition, IgG4 is the most prominent parasite-specific response in chronic, uncomplicated S. stercoralis infections and that IgG2 and IgG4 were more elevated in immunocompetent than immunosupressed patients (Genta and Lillibridge 1989). On the other hand, peripheral blood eosinophils, intestinal mast cells and goblet cells, IgE, Th2 cells and cytokines such as IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 are shown to be the mediators of immunity against S. stercoralis (Vadlamudi et al., 2006). In severe strongyloidiasis, no significant change in the T-cell numbers were noted compared with asymptomatic or mildly symptomatic patients suggesting the presence of an inhibitory serum factor to the function of T cell (Trajman et al., 1997).
Epidemiology:
Strongyloides stercoralis has a worldwide distribution, especially in tropical and subtropical countries, affecting probably 100 million humans worldwide that is found mainly in institutionalized persons, immigrants, or veterans (Mahmoud, 1996 & Polenakovik and Polenakovik, 2006). The mortality from disseminated infection could be up to 87% (Schwartz and Tarlow 2005).
In the United States, strongyloidiasis is the most important nematode infection in humans with a tendency towards chronic persistent infection, autoinfection, hyperinfection and disseminated infection involving other organs may occur (Concha et al., 2005).
Sub-Saharan Africa, South and Southeast Asia, Central and South America, and Eastern Europe are considered endemic areas of Strongyloides infection with a prevalence rate reaching 60% (Carpenter Rose and Richard, 2006).
In Egypt, Salem et al. (1990) found that the prevalence rate of S. stercoralis in Menofiya Governorate was 11.1% by using IFA. While, El-Shazly et al. (2006a) found that the prevalence of S. stercoralis in an urban and a rural area in Dakahlia governorate was 1.5% of each using direct stool examination. In immunocompromised patients the prevalence rates were 3.6% and 5.3% by using stool culture technique as reported by Khalil et al. (1991) and Azab et al. (1992) respectively.
Clinical pictures:
The vast majority of patients with strongyloidiasis are asymptomatic. Patients who become symptomatic do so shortly after exposure, or they develop late symptoms. As larvae enter the body, initial manifestations (larva currens) include a transient pruritic, erythematous and serpiginous eruption (Von Kuster and Genta, 1988). Migration of the larvae through the lungs produces a pneumonitis that resembles Löffler syndrome. Symptoms include a productive cough, at times with blood-streaked sputum, dyspnea, and fever. Strongyloidiasis can also produce a clinical syndrome that mimics either asthma or pneumonia (Woodring et al., 1994).
The intestinal phase of the disease presents with epigastric abdominal cramping, indigestion, anorexia, vomiting, and diarrhea which is profuse, watery, and mucoid. Periods of alternation between diarrhea and constipation may occur (Grove, 1989). Malabsorption of fat and vitamin B-12 has been reported (O’Brien, 1975). Invasion of the CNS may produce symptoms of meningitis, including headache, nausea, vomiting, nuchal rigidity, confusion, focal seizures and, in extreme cases, coma (Harcourt-Webster et al., 1992).
Arthritis is a less common feature of strongyloidiasis (Doury, 1981). Many reports involving patients with either oligo- or poly-arthritis in association with intestinal Strongyloides infection were presented (Van Kuijk et al., 2003). It usually involves the lower extremity large joints but sometimes the sacroiliac or upper extremity large joints as well (Jaramillo et al., 1978; Masseau et al., 2005 & Richter et al., 2006). Although Strongyloides larvae have been observed in a synovial biopsy specimen, suggesting an infectious type of arthritis (Alkoglu et al., 1984; Patey et al., 1990), most authors consider this to be a reactive arthritis (Van Kuijk et al., 2003). Bocanegra et al. (1981) found evidence of abnormal humoral immunity to the parasites, immune complexes in serum, and immunoglobulin deposits in the synovia suggesting that arthritis may be mediated by immune complex formation. Specific anti-parasitic treatment resulted in resolution of the symptoms and immunologic abnormalities (Van Kuijk et al., 2003).
Diagnosis:
The most common diagnostic stage of S. stercoralis is the rhabditiform larva, but the filariform larva, adult females and even eggs can be seen in some patients (Cunni et al., 1977). The chance of diagnosis using one direct smear of stool sample has been reported to be between 30-60% (Koga et al., 1990). Pelletier (1984) suggested the screening of large bulk of feces by examining multiple slides for a period of up to 9 hours per specimen. While Nielsen and Mojon (1987) proved that the examination of 7 stool samples on 7 consecutive days can improve the diagnostic value of stool examination. On the other hand, Assefa et al. (1991) and Soroczan (2002) recommend Baermann’s technique for detecting S. stercoralis larvae.
However, Koga et al. (1992) and Massoud et al. (2006) recommended that agar-plate stool culture is superior to filter paper method and gave 100% positivity, even in cases were negative by coprological methods either direct smear and/or sedimentation technique.
Small intestinal biopsy may be used as the parasite was demonstrated in less than 50% of open duodenal and jejunal biopsies as well as to evaluate the mucosal morphology and function during the disease (Goldsmid and Davis, 1978). Moreover, larvae and adult worms can be detected in the sputum and in bronchoalveolar lavage, ascetic fluid, pancreatic aspirate and CSF (Berry et al., 1983). Koga et al. (1990) reported that small intestinal intubation, endoscopy with aspiration or the string test, are the most sensitive procedures in diagnosis.
Several immunodiagnostic assays have been developed, including skin testing with larval extracts, IIF using killed larvae, filarial complement fixation testing, indirect haemagglutination testing, radioallergosorbent testing for specific IgE, and ELISA for IgG antibodies. However, results may not help to distinguish acute from prior infection, making these tests less helpful in endemic areas (Salem et al., 1990 & Tolon and Muiz, 2006).
Treatment:
Thiabendazole is still the recommended therapy for strongyloidiasis with about 70 to 90% eradication rate (Mahmoud, 1996). Albendazole and ivermectin, which have success rates of 60 to 90% but fewer side effects, are considered alternatives (Hagelsjkaer, 1994 & Mahmoud, 1996). Recently, Massoud et al. (2006) proved that strongyloidiasis patients were cured with mirazid double course for one month, only few patients showed resistance, they were later cured by either repeating the course of mirazid or combining it with mebendazole.
VIII-Ascaris lumbricoides:
Ascaris lumbricoides causes the most common helminthic human infection in the world. Ascariasis is found most commonly in countries with poor sanitary conditions (Khuroo, 1996).
Developmental biology:
Infection begins via ingestion of water or food (raw vegetables or fruits) contaminated by embryonated (infective) eggs which hatch and release small larvae that penetrate the intestinal wall. Larvae migrate to the pulmonary bed via the portal veins, during that time they may cause pulmonary symptoms. After migrating up the respiratory tract and being swallowed, they mature, copulate, and lay eggs in the intestine. Adult worms may live in the gut for 6-24 months, where they can cause partial or complete bowel obstruction in large numbers, or they can migrate into the appendix, hepatobiliary system, or pancreatic ducts. The entire developmental process from egg ingestion to egg passage from the adult female takes from 8 to 12 weeks (Gilles, 1984 & Freedman, 1992).
Pathophysiology:
When larvae break out the lung tissue and into the alveoli, there may be some damage to the bronchial epithelium. With re-infection and subsequent larval migrations, there may be intense tissue reactions, even with small number of larvae (Bradley and Jackson 2004). Changes caused by Ascaris are attributed to direct tissue damage, host immune response, obstruction of an orifice or the lumen of gastrointestinal tract by an aggregation of worms, and nutritional deficiencies due to presence of adult worm (Tietze and Tietze, 1991).
Immune response:
The human immune response against Ascaris parasites is characterized by prominent humoral and cellular responses that are directed primarily against the larval stages of infection. Ascaris infection stimulates the secretion of all antibody isotypes, although high circulating levels of total IgE and parasite-specific IgE are the most characterized features. In endemic regions the celluler response to A. lumbricoides infection is characterized by a polarized Th2 response with the prominent production of both IL-4 and IL-5 (Cooper, 2002). Age prevalence and age intensity relationships for A. lumbricoides are typically convex, peaking in children or adolescent (Elkins et al., 1988). This could be explained by age-specific variation in exposure (Bundy and Medley, 1992), although this is consistent with a slow increase in acquired resistance with age (Cooper, 2002).
Epidemiology:
It is estimated that more than 1.4 billion people are infected with A. lumbricoides, representing 25% of the world’s population. A number of features account for its high prevalence including ubiquitous distribution, the durability of eggs under a variety of environmental conditions, the high number of eggs produced per parasite, and poor socioeconomic conditions that facilitate its spread, yet prior infection does not confer protective immunity (Seltzer, 1999).
Although ascariasis occurs at all ages, it is most common in children 2 to 10 years old and prevalence decrease over the age of 15 years. Infections tend to cluster in families (Haswell-Elkins et al., 1989). The highest prevalence of ascariasis occurs in tropical countries where warm, wet climates provide environmental conditions that favor year round transmission of infection. The prevalence is also great in areas where suboptimal sanitation practices lead to increase contamination of soil and water (Warren and Mahmoud, 1977).
Ova can survive in the environment for long periods and prefer warm, shady, moist conditions under which they can survive for up to 10 years. The eggs are resistant to usual methods of chemical water purification but are removed by filtration or by boiling. Developing larva will be destroyed by sunlight and desiccation. There is no significant animal reservoir (Khuroo, 1996). The majority of people with ascariasis live in Asia (73%), Africa (12%), and South America (8%), where some populations have infection rates as high as 95% (Sarinas and Chitkara, 1997; Reeder, 1998).
In the United States, the prevalence of infection decreased dramatically after the introduction of modern sanitation and waste treatment in the early 1900s (Jones, 1983). It is estimated that the current prevalence of A. lumbricoides in stool samples is approximately 2% in the United States, but it may be more than 30% in children between the ages of 1 to 5 years, particularly in rural areas (Jones, 1981; Tietze and Tietze, 1991). It is also seen in travelers from endemic areas (Sarinas and Chitkara, 1997).
In Egypt, the prevalence rate of A. lumbricoides among solid waste scavengers (Zabaline) was 26% (Miller et al., 1982). In Alexandria, the prevalence among primary governmental schools in the Eastern zone was 10.2% with increased risk of under weight and stunted growth in infected children than those without infection (El-Sahn et al., 1997). While, El-Shazly et al. (2006a) found that the prevalence of A. lumbricoides in an urban and a rural area in Dakahlia governorate was 0.1% of each.
Clinical picture:
The majority of infections with A. lumbricoides are asymptomatic. Clinical disease is largely restricted to individuals with high worm load (Khuroo, 1996). When symptoms occur, they relate either to the larval migration stage or to the adult worm intestinal stage. During larvae migration Ascaris pneumonitis may develope and is accompanied by an allergic reaction consisting of dyspnea, a dry or productive cough, wheezing or coarse rales, fever (39.5 to 40 °C), transient eosinophilia, and a chest X-ray suggestive of viral pneumonia. This picture is frequently called Löffler syndrome. In addition to eosinophilia, Charcot-Leyden crystals, the sputum may contain larvae. Asthma and urticaria may continue during the intestinal phase of ascariasis (Proffitt and Walton, 1962; Garcia, 2001). Transplacental migration of larvae has also occasionally been reported (Chu et al., 1972).
The presence of the adult worms in the intestine usually causes no difficulties unless the worm burden is very heavy, however, because of the tendency of adult worms to migrate even a single worm can cause serious sequelae. Fever, general anesthesia, or sub-therapeutic doses of anti-helminthics may provoke the worms to migrate. This migration may result in intestinal blockage; entry into the bile duct; pancreatic duct, or other small spaces; or entry into the liver or peritoneal cavity. They can also migrate out of the anus or come out the mouth or nose. Other body sites, such as kidney, appendix, or pleural cavity, have been involved (Garcia, 2001).
Heavy infections are also associated with impaired absorption of dietary proteins, lactose, and vitamin A and C. It has also been proposed that heavy infections may be associated with impaired cognitive development in school children (Hlaing, 1993 & Oberhelman et al., 1998).
Ascaris infection has been related to two cases of vasculitis (Grcevska and Polenakovic, 1993) and a case of symmetrical polyarthritis (Miehlke and Jentsch, 1978).
Diagnosis:
In the larval migration phase of the infection, diagnosis can be made by finding the larvae in sputum or in gastric washings (Proffitt and Walton, 1962). During the intestinal phase, the diagnosis can be made by finding the eggs (unfertilized or fertilized) or adult worms in the stool. The eggs are most easily seen on a direct wet smear or a wet preparation of the concentration sediment (Tietze and Tietze, 1991).
Intestinal disease can often be diagnosed from radiographic studies of the gastrointestinal tract, in which the worms can be visualized. Abdominal radiography may reveal adult worms (Reeder, 1998), obstructing Ascaris lesions cause cylindrical filling defects on contrast computed tomography scans, cholangiopancreatography by endoscopy or magnetic resonance imaging, or magnetic resonance cholangiopancreatography may detect adult worms in bile or pancreatic ducts, and also ultrasonography which may detect worms in gallbladder (Sandouk et al., 1997 & Misra and Dwivedi, 2000).
Antibodies to Ascaris often cross react with antigens from other helminthes (McSharry et al., 1999). On the other hand, Malafiej and Spiewak (2001) reported that serological investigations exhibits higher sensitivity than the traditional methods especially in the lung phase of the disease, the larval stage or in cases of infection with an individual parasite, when the feces samples do not contain eggs.
Treatment:
Benzimidazoles are effective for the treatment of intestinal ascariasis. The most commonly recommended agents are albendazole (the adult dose is 400 mg/day as a single dose, while pediatric dose is 200 mg/day as a single dose, dose can be repeated after 3 weeks if not cured), and mebendazole (adult dose is 100 mg BID for 3 days). Ivermectin (150-200 mcg/kg as a single dose) and pyrantel pamoate (11 mg/kg/dose as a single dose; not to exceed 1 g) are alternatives (Liu and Weller, 1996 & Urbani and Albonico, 2003). A new anti-helminthic agent from China, tribendimidine, has recently been show to be as efficacious as albendazole (Xiao et al., 2005).
Intestinal parasites less commonly causing rheumatic manifestations
Microsporidia:
Organisms of the order Microsporidia encompass hundreds of spp. of obligatorily intracellular, spore-forming protozoa which cause an ever-growing number of clinical syndromes, ranging from asymptomatic infection to diarrhea and malabsorption to disseminated infection. In Egypt, Abaza et al. (1995) found that the prevalence of microsporidia oocysts among immuno-compromised hosts was 2.3%, while none were found in both malnourished and control children in the study of Rizk and Soliman (2001) in Mansoura, Dakahlia governorate. Rheumatic manifestations of microsporidial infections are uncommon and have been described almost entirely among patients with AIDS but several case reports described its association with infectious myositis (Ledford et al., 1985 & Kotler and Orenstien, 1998). Cali et al. (2004) reported a case of Brachiola algrae infecting skeletal muscles in a 56 years old woman who was being treated for rheumatoid arthritis with immunosuppressive drugs. Diagnosis requires identification of characteristic spores in stool specimens or other affected body fluids or tissues. Serological assays have not been of great value. Treatment, when indicated, is albendazole (400 mg twice a day) which appears to be effective against most microsporidial spp. (Kotler et al., 1990).
Isospora belli:
Isospora belli causes a self-limited diarrheal illness in immunocompetent hosts. In individuals with immunocompromization, it may cause chronic life-threatening diarrhea and dehydration (Whiteside, 1984). Most infections are acquired in tropics or subtropics, while it is much less common in temperate zones (De Paula et al., 1989). In Egypt, Khalil et al. (1991) and Abaza et al. (1995) did not report any case of isosporiasis in the immunocompromised hosts. On the other hand, Rizk and Soliman et al. (2001) found that I. belli oocysts were detected in 2.8% of malnourished children in Mansoura, Dakahlia governorate. Isospora belli has also been associated with a reactive arthritis in an AIDS patient (Gonzalez et al., 1994).
Ancylostoma duodenale:
Ancylostoma duodenale is the predominant spp. in the Mediterranean region, northern regions of India and China, and North Africa. In Egypt, El-Shazly et al. (2006a) compared the prevalence of gastrointestinal helminthes in an urban and a rural area in Dakahlia governorate; in Mansoura city (as an urban area) no A. duodenale was recorded while, in Gogar village (as a rural area) the prevalence was 0.1%. A. duodenale infection causes iron deficiency anemia and protein malnutrition (Hotez and Pritchard, 1995). Bissonnette and Beaudet (1983) reported one case of an asymmetrical lower extremity oligoarthritis which was related to A. duodenale infection.
Taenia saginata:
Both of T. solium and T. saginata infections are cosmopolitan (Botero, 1993; Schantz, 1996). In Egypt, El-Shazly et al. (2006a) showed that the prevalence rate of T. saginata in Mansoura city was 0.1%. In only one case report, T. saginata has been associated with a symmetrical arthritis of wrists, metacarpophalengeal joints, and interphalengeal joints (Bocanegra et al., 1981).
INTRODUCTION AND RATIONALE
Parasitic infections affect as much as 25% of world’s population (Reeder and Palmer, 1994). They are mostly prevalent in underdeveloped agricultural and rural areas of tropical and subtropical regions, causing reduced worker productivity and wastage of economic resources (Abd El-Bagi et al., 2004).
Infected patients may remain asymptomatic or develop a variety of clinical manifestations, ranging from local symptoms to a multisystem and sometimes lethal disease. The latter is more common in immunocompromised and debilitated patients (Masseau et al., 2005).
Underlying infectious etiologies have long been thought in rheumatic diseases (Saag and Bennett, 1987). Parasitic infections can induce a variety of rheumatic syndromes as a result of infiltration of musculo-skeletal structures by parasites or an immune mediated mechanism (Bocanegra and Vasey, 1993 & McGill, 2003).
Rheumatic syndromes, including inflammatory arthritis, inflammatory myositis, and vasculitis have been described among multiple different parasitic infections of all parasitic divisions including protozoa, nematode, and platyhelminthes. Individual parasitic divisions are often associated with particular rheumatic syndromes (Peng, 2002). Rheumatic pain is the main clinical manifestation of rheumatic syndromes and is defined as; pain involving articular (related to joints) and/or extra-articular structures (related to tendons and muscles) (Kelley et al., 1989).
Evidence for underlying parasitic infection should be sought in patients presenting with unexplained or atypical rheumatic syndromes (Peng, 2002). Diagnosis of parasitic rheumatism is based on the demonstration of infection with a pathogenic parasite, lack of response to anti-inflammatory agents; especially non steroidal anti-inflammatory drugs (NSAD), and improvement following anti-parasitic therapy. Treatment consists of eradication of the parasite (Bocanegra and Vasey, 1993).
Protozoan-type infections were usually described with muscular and/or articular manifestations and directly correlated to the presence of the organism (Di Pietro et al., 1996). Entamoeba histolytica was reported in association with infectious knee arthritis (Than-Saw et al., 1993) and reactive arthritis (Afsar et al., 1995). Moreover, some non-pathogenic amoebae which include other Entamoeba species (spp.) have been implicated in arthritic syndromes (Schimer et al., 1998). Blastocystis hominis is another pathogenic protozoan which was associated with reactive arthritis (Kruger et al., 1994). In some cases, it has been observed in synovial fluid, suggesting invasive infection (Lee et al., 1990).
Giardia lamblia has been frequently associated with a reactive arthritis like syndrome that involves infection of the bowel but not of extraintestinal sites (Goobar, 1977). Galland (1998) showed that there was positive association between giardiasis and arthralgia with a prevalence rate of 36%.
Cryptosporidium is the most common enteric infection in immunocompromised patients, and has been associated with reactive arthritis (Shepherd et al., 1989). Moreover, Reiter’s syndrome was reported following protracted symptoms of Cyclospora cayetanensis (Conner, et al., 2001). Isospora belli has also been associated with reactive arthritis (Gonzalez et al., 1994). Rheumatic manifestations of microsporidial infections are uncommon and have been described almost entirely among patients with AIDS but several case reports described its association with infectious myositis (Ledford et al., 1985 & Kotler and Orenstien, 1998).
Nematodes are the second group of intestinal parasites commonly associated with rheumatic manifestations (Peng, 2002). Strongyloides stercoralis can be associated with reactive arthritis (Van Kuijk et al., 2003 & Masseau et al., 2005). It also has been directly observed in joints of some affected patients (Akoglu et al., 1984). Ascaris infection has been related to a case of symmetrical polyarthritis (Miehlke and Jentsch, 1978) and two cases of vasculitis (Grcevska and Polenakovic, 1993). Only one case of an asymmetrical lower extremity oligoarthritis was related to Ancylostoma duodenale infection (Bissonnette and Beaudet, 1983).
Rheumatic manifestations are described typically among patients with chronic schistosomiasis. The most common syndromes resemble classical reactive arthritis, seronegative spondyloarthropathies, sacroileitis, and rheumatoid arthritis like presentations (Kamel et al., 1989 & Rolland et al., 1998). Taenia saginata has been associated with rheumatoid arthritis like presentations in only one case report (Bocanegra et al., 1981).
RESULTS
This study was designed to estimate the prevalence of intestinal parasites among patients with unexplained rheumatic pain and to identify the prevalence of parasitic rheumatism among infected patients. One hundred and eight patients were recruited in the study according to the inclusion criteria. The patients were conducted from Rheumatology and Rehabilitation Outpatient Clinic in Suez Canal University Hospital in the period from October 2005 to June 2006.
1- Prevalence of intestinal parasitic infections:
In the present work, microscopic examination of direct, concentrated and stained fecal smears revealed an overall prevalence of pathogenic intestinal parasitic infections among patients with unexplained rheumatic pain as 46.3% (Tab. 1).
The prevalence of different pathogenic intestinal parasites detected among the studied patients (Tab. 2) was in a descending order; Cryptosporidium (22.2%), C. cayetanensis (14.8%), G. lamblia (11.1%), B. hominis (9.2%), E. histolytica / dispar (3.7%). Microsporidia, S. mansoni, A. lumbricoides and S. stercoralis were 1.9% for each. Mixed infections occurred in 20 cases (40%). Sixteen cases (32%) had double infection while only 4 cases (8%) had triple infection (Tab. 3). The most frequent association was Cryptosporidium and C. cayetanensis (8 cases) (Fig. 3).
As regards to the response of infected patients with unexplained rheumatic pain to anti-parasitic treatment, fulfilling the criteria of parasitic rheumatism, 32% (16 patients) gave good response with complete relief of rheumatic complaints and disappearance of the parasite from their stool (good response). While, in 68% the rheumatic complaints persisted (didn’t respond well) despite of full course treatment of parasitic infection (Fig. 4).
Table 1: The overall prevalence of pathogenic intestinal parasites detected among patients with rheumatic pain.
No. %
Infected 50 46.3
Non-infected 58 53.7
Total 108 100
Table 2: Prevalence of different pathogenic intestinal parasites detected among patients with rheumatic pain.
Parasites Infected Non- Infected
No. % No. %
Cryptosporidium 24 22.2 84 77.8
C. cayetanensis 16 14.8 92 85.2
G. lamblia 12 11.1 96 88.9
B. hominis 10 9.2 98 90.8
E. histolytica / dispar 4 3.7 104 96.3
Microsporidia 2 1.9 106 98.1
S. mansoni 2 1.9 106 98.1
S. stercoralis 2 1.9 106 98.1
A. lumbricoides 2 1.9 106 98.1
Chi2=65.52 p-value < 0.0001 (Statistically highly significant).
Table 3: Prevalence of single and mixed intestinal parasites detected among infected patients.
Intestinal parasites detected Infected group
Single 30
(60%) Mixed (20)
40% Total
50
(100%)
Double 16
(32%) Triple 4
(8%)
Cryptosporidium 12 (24%) 10 (20%) 2 (4%) 24 (48%)
C. cayetanensis 2 (4%) 10 (20%) 4 (8%) 16 (32%)
G. lamblia 8 (16%) 2 (4%) 2 (4%) 12 (24%)
B. hominis 2 (4%) 6 (12%) 2 (4%) 10 (20%)
E. histolytica / dispar 2 (4%) 2 (4%) 0 4 (8%)
Microsporidia 0 2 (4%) 0 2 (4%)
S. mansoni 0 0 2 (4%) 2 (4%)
A. lumbricoides 2 (4%) 0 0 2 (4%)
S. stercoralis 2 (4%) 0 0 2 (4%)
Chi2 = 32.064 & P-value < 0.009 (Statically Significant)
Figure 3: Distribution of the prevalence of single and mixed parasitic infections in relation to each parasite.
Figure 4: The response of infected patients to anti-parasitic drugs, fulfilling the criteria of parasitic rheumatism.
2. Sociodemographic data of the studied patients (Tab.4):
In the present study 10 men (9.3%) and 98 women (90.7%) were passed baseline assessments. The percentage of males among the infected patients was 12% while, females were 88% and in non-infected patients males and females were 7% and 93% respectively. The difference was statistically non significant (p-value > 0.05).
The patients were divided into three age groups; young age (<18 years) with 24.1%, adult age (18-49 years) with 55.5%, and old age (≥50 years) with 20.4%. As regards the age of the infected patients in the present study, high infection rate was found among adults (56%) while, low infection rate was found among elderly people (16%). The difference was statistically non significant (p-value > 0.05).
Concerning the residence site, the majority of patients in the study population were living in rural areas (63%), while the minority was living in urban areas (37%). As regards to the infected patients, relatively higher percentage was living in rural areas (56%), than those living in urban areas (44%). Also the majority of non-infected patients were living in rural areas (69%). The difference was statistically non significant (p-value > 0.05).
High percentage of the studied population was found in the low socio-economic class (50%). The majority of infected patients were in low socio-economic class (68%) compared to non infected (34.5%). Most of non-infected patients were in medium class (51.7%). The difference was statistically significant (p-value < 0.002).
Table 4: Comparison between sociodemographic data of infected and non- infected patients.
Infected Non-infected Total Chi2
P-value
No. % No. % No %
Sex male
Female 6
44 12
88 4
54 7
93 10
98 9.3
90.7 FE
0.281
Total 50 100 58 100 108 100%
Age Young
Adult
Old 14
28
8 28
56
16 12
32
14 20.7
55.2
24.1 26
60
22 24.1
55.5
20.4 1.47
0.479
Total 50 100 58 100 108 100%
Residence Urban
Rural 22
28 44
56 18
40 31
69 40
68 37
63
1.94
0.164
Total 50 100 58 100 108 100%
Socio-economic state High
Medium
Low 2
14
34 4
28
68 8
30
20 13.8
51.7
34.5 10
44
54 9.3
40.7
50
12.52
0.002
Significant
Total 50 100 58 100 108 100%
3. Clinical and laboratory data of the studied patients (Tab.5):
Gastrointestinal symptoms were present in 27.8% of patients in the studied population. Among the infected patients, 40% were GIT symptomatic, while 60% were GIT asymptomatic. In non-infected patients, 17.2% were GIT symptomatic and 82.8% were GIT asymptomatic. This difference was statistically significant (p-value <0.008).
All patients in this study had only joint pain (arthralgia) and no one had extra-articular pain. In comparison between the patterns of joint affection poly-articular (> 4 joints) constituted the highest percentage (44%) of infected patients followed by mono-articular (only one joint) with 32% then, oligo-articular pain (2-4 joints) constituted the lowest percentage (24%). In non-infected patients oligo-articular constituted the highest percentage (41.4%) while; mono-articular constituted the lowest percentage (24.1%). This difference was statistically non significant (p-value > 0.05).
Erythrocyte sedimentation rate was elevated in 20.4% of the studied population. Among the infected patients 28% had elevated ESR compared to 13.8% in non infected patients. CRP was detected in 16.7% from all studied population. Among the infected patients 24% had elevated CRP compared to 10.3% in non-infected patients. Eosinophilia was found only in infected patients with 1.9% from all of the studied population. This difference was statistically non significant (p-value > 0.05).
Table 5: Comparison between clinical and laboratory data of infected and non-infected patients.
Clinical & lab. Data Infected Non-infected Total Chi2
P-value
No. % No. % No. %
GIT symptoms Present 20
30 40
60 10
48 17.2
82.8 30
78 27.8
72.2
6.93
<0.008
Significant
Absent
Total 50 100 58 100 108 100
*Pattern of joint affection Mono-articular 16
12
22 32
24
44 14
24
20 24.1
41.4
34.5 30
36
42 27.8
33.3
38.9
3.66
0.161
Oligo-articular
Poly-articular
Total 50 100 58 100 108 100
Blood investigations ESR High 14
36 28
72 8
50 13.8
86.2 22
86 20.4
79.6
3.34
0.068
Normal
Total 50 100 58 100 108 100
CRP High 12
38 24
76 6
52 10.3
89.7 18
90 16.7
83.3
3.60
0.058
Normal
Total 50 100 58 100 108 100
Eosinophilia Present 2
48 4
96 0
58 0
100 2
106 1.9
98.1
FE
0.212
Absent
Total 50 46.3 58 53.7 108 100
* By history and confirmed by examination.
I- Cryptosporidium
As regards to the demographic data and socio-economic classes of Cryptosporidium infected patients (Fig. 5); the majority of patients were females (83.3%). Most of the patients were adults (41.7%), while 25% and 33.3% were in young and old age groups respectively. Cryptosporidium infected patients living in rural areas were more than urban areas with 58.3% and 41.7% respectively. Patients in low socio-economic classes constituted the majority (50%), followed by the medium class (41.7%) and 8.3% were found in high class. Blood investigations revealed elevation of ESR and CRP in 16.7% and 8.3% respectively but no blood eosinophilia was detected.
Figure 5: The sociodemographic data and blood investigation results of Cryptosporidium infected patients.
On the other hand, the clinical characters of Cryptosporidium infected patients (Tab. 6) proved that; the majority of patients (91.7%) were not fulfilling the criteria of parasitic rheumatism and most of them (41.7%) had poly-articular joint pain. Only 2 patients responded to anti-parasitic drugs (8.3%) and both of them had oligo-articular pain. The differences were statistically significant (p-value <0.038).
Cryptosporidiosis GIT symptomatic patients were 41.7% and equal percentages of them were complaining of mono-articular and poly-articular pain (16.7%). While GIT asymptomatic patients were 58.3% from which high percentage (25%) were complaining of poly-articular pain. The difference was statistically non significant (p-value > 0.05).
Single and Mixed parasitic infections occurred with equal percentages (50%). The majority of patients with single infection had poly-articular (25%) while the minority had mono-articular (8.3%). The majority of patients with mixed infection had mono-articular (25%) while the minority had oligo-articular (8.3%). The differences were statistically non significant (p-value > 0.05).
Table 6: The relation between clinical characters (response to anti-parasitic treatment, GIT symptoms, type of infection) of Cryptosporidium infected patients and joint affection pattern.
Clinical characters of cryptosporidiosis patients Mono-articular Oligo-articular Poly-articular Total Chi2
p-value
No % No % No % No %
Parasitic Rheumatism* Responding 0 0 2 8.3 0 0 2* 8.3 6.55
< 0.038
Sig.
Non-responding 8 33.3 4 16.7 10 41.7 22 91.7
Total 8 33.3 6 25 10 41.7 24 100
GIT symptoms Symptomatic 4 16.7 2 8.3 4 16.7 10 41.7 0.411
0.814
Non sig.
Asymptomatic 4 16.6 4 16.7 6 25 14 58.3
Total 8 33.3 6 25 10 41.7 24 100
Type of infection Single 2 8.3 4 16.7 6 25 12 50 0.800
0.670
Non sig.
Mixed 6 25 2 8.3 4 16.7 12 50
Total 8 33.3 6 25 10 41.7 24 100
* The two cases who gave good response to treatment were young males, with GIT symptoms, and complained of oligo-articular joint pain.
Photo 1: Cryptosporidium oocyst in stool stained with modified acid-fast staining (X1000).
II- Cyclospora cayetanensis
As regards to the demographic data and socio-economic classes of Cyclospora infected patients (Fig. 6); the majority of patients were females (75%). Most of the patients were adults (75%), while 25% were young. Meanwhile, no cases were detected among old age group. Cyclospora infected patients were living in rural areas and urban areas with equal percentages (50%). Patients in medium socio-economic classes constituted 62.6%, followed by the low class (37.5%) and none were found in high class. The difference was statistically significant. Blood investigation gave normal results.
On the other hand, the clinical characters of Cyclospora infected patients (Tab.7) proved that; the majority of patients (87.5%) were not fulfilling the criteria of parasitic rheumatism and equal percentages from them (37.5%) were complaining of mono-articular and poly-articular joint pain. Only 12.5% were responding to the anti-parasitic drugs and all of them were complaining of oligo-articular pain. The differences were statistically significant (p-value < 0.032).
Cyclosporiasis GIT symptomatic patients were 37.5% and most of them were complaining of mono-articular pain (25%). On the other hand, GIT asymptomatic patients were 62.5% and high percentage of them (37.5%) were complaining of poly-articular. The differences were statistically significant (p-value < 0.049).
Single parasitic infection occurred only in 12.5% and all of them were complaining of oligo-articular pain. Mixed infections occurred in the majority of patients with 87.5% from which equal percentages were complaining of mono-articular and poly-articular joint pain (37.5%). The differences were statistically significant (p-value < 0.032).
Table 7: The relation between cinical characters (response to anti-parasitic treatment, GIT symptoms, type of infection) of Cyclospora infected patients and joint affection pattern.
Clinical characters of cyclosporiasis patients Mono-articular Oligo-articular Poly-articular Total Chi2
p-value
No % No % No % No %
Parasitic Rheumatism* Responding 0 0 2 12.5 0 0 2* 12.5 6.86
< 0.032
Sig.
Non-responding 6 37.5 2 12.5 6 37.5 14 87.5
Total 6 37.5 4 25 6 37.5 16 100
GIT symptoms Symptomatic 4 25 2 12.5 0 0 6 37.5 6.04
< 0.049
Sig.
Asymptomatic 2 12.5 2 12.5 6 37.5 8 62.5
Total 6 37.5 4 25 6 37.5 14 100
Type of infection Single 0 0 2 12.5 0 0 2 12.5 6.86
< 0.032
Sig.
Mixed 6 37.5 2 12.5 6 37.5 14 87.5
Total 6 37.5 4 25 6 37.5 16 100
* The two cases who gave good response to treatment were young males, with GIT symptoms, and complained of oligo-articular joint pain.
Photo 2: Cyclospora cayetanensis oocyst in stool with modified acid-fast staining (X1000).
III- Giardia lamblia
As regards to the demographic data and socio-economic classes of giardiasis patients (Fig. 7); all the patients were females. The majority of them were adults (66.7%), while 33.3% were young. Meanwhile, no cases were detected among old age group. Most of Giardia infected patients were living in rural areas (83.3%), while only 16.7% were living in urban areas. Patients in low socio-economic classes constituted 66.7%, followed by the medium class (33.3%) and none were found in high class. As regards blood investigations, elevated ESR and CRP were found in 66.7% of cases with G. lamblia infection but no blood eosinophilia was detected.
On the other hand, the clinical characters of Giardia infected patients (Tab.8) proved that; the majority of patients (66.6%) were fulfilling the criteria of parasitic rheumatism from which equal percentages (33.3%) had mono-articular and poly-articular joint pain. In the remaining 33.4% the complaint persisted from which equal percentages were complaining of oligo-articular and poly-articular pain (16.7%). The differences were statistically significant (p-value < 0.05).
Gastrointestinal symptomatic giardiasis patients were 33.4% with equal percentages complaining of mono-articular and poly-articular pain (16.7%). On the other hand, GIT asymptomatic patients were 66.7% with equal percentages were complaining of mono-articular and oligo-articular pain (16.7%) while, 33.3% had poly-articular joint pain. The difference was statistically non significant (p-value > 0.05).
Single parasitic infection occurred in 66.6% from which equal percentages complaining of mono-articular and poly-articular pain (33.3%). Mixed infection occurred in 33.4% with equal percentages were complaining of oligo-articular and poly-articular pain (16.7%). The difference was statistically significant (p-value < 0.05).
Table 8: The relation between cinical characters (response to anti-parasitic treatment, GIT symptoms, type of infection) of Giardia infected patients and joint affection pattern.
Clinical characters of Giardiasis patients Mono-articular Oligo-articular Poly-articular Total Chi2
p-value
No % No % No % No %
Parasitic Rheumatism Responding 4 33.3 0 0 4 33.3 8* 66.6 6.00
< 0.05
Sig.
Non-responding 0 0 2 16.7 2 16.7 4 33.4
Total 4 33.3 2 16.7 6 50 12 100
GIT symptoms Symptomatic 2 16.7 0 0 2 16.7 4 33.4 1.5
0.472
Non- sig.
Asymptomatic 2 16.6 2 16.7 4 33.3 8 66.6
Total 4 33.3 2 16.7 6 50 12 100
Type of infection Single 4 33.3 0 0 4 33.3 8 66.6 6.00
< 0.05
Sig.
Mixed 0 0 2 16.7 2 16.7 4 33.4
Total 4 33.3 2 16.7 6 50 12 100
* The eight cases with giardiasis who responded to treatment with relief of their rheumatic pain were females; 4 with young age, complained of mono-articular joint pain and 2 of them were GIT symptomatic, while the other 4 cases were adults, complained of poly-articular joint pain and 2 of them were GIT symptomatic.
Photo 3: Giardia lamblia cyst in stool stained with trichrome stain (X1000).
IV- Blastocystis hominis
All cases infected with B. hominis were females and the majority of them were adults (80%), while 20% were young age. Most of the infected patients were living in urban areas (60%), while 40% were living in rural areas. High percentage of B. hominis infected patients were found in low socio-economic class (60%), followed by the medium class (40%) and none were found in the high class. Blood investigations were normal (Fig. 8).
Figure 8: The sociodemographic data and blood investigation results of Blastocystis hominis infected patients.
As regards to the clinical data (Tab. 9), none of the B. hominis infected patients gave good response to B. hominis infection treatment as the rheumatic pain persisted in all cases.
Gastrointestinal symptoms were found in only 20% of B. hominis infected patients while, 80% were GIT asymptomatic. All of symptomatic patients had mono-articular joint pain while, high percentage of asymptomatic patients (60%) had poly-articular pain followed by oligo-articular joint pain (20%). The difference was statistically significant (p-value < 0.007).
The majority of B. hominis patients had mixed parasitic infections (80%) while, single parasitic infection occurred only in 20% of patients. All of the patients with single infection had mono-articular joint pain while, most of patients with mixed infection were presented with poly-articular joint pain (60%). The difference was statistically significant (p-value < 0.007).
Table 9: The relation between GIT symptoms, type of infection in B. hominis infected patients and joint affection pattern
Clinical characters of B. hominis patients Mono-articular Oligo-articular Poly-articular Total Chi2
P-vaue
No % No % No % No %
GIT symptoms Symptomatic 2 20 0 0 0 0 2 20 10
< 0.007
Significant
Asymptomatic 0 0 2 20 6 60 8 80
Total 2 20 2 20 6 60 10 100
Type of infection Single 2 20 0 0 0 0 2 20 10
< 0.007
Significant
Mixed 0 0 2 20 6 60 4 80
Total 2 20 2 20 6 60 12 100
Photo 4: Blastocystis hominis vacuolar form in stool stained with trichrome stain (X1000).
V- Entamoeba histolytica / dispar:
All cases with Entamoeba histolytica / dispar infection were females. Equal percentages were found among adult and old age groups (50%), while none were found among young age group. As regards the residence, equal percentages were found living in urban and rural areas (50%). Also equal percentages were found in low and medium socio-economic classes (50%) and none were found in the high class. Blood investigations were normal (Fig. 9).
Figure 9: The sociodemographic data and blood investigation results of E. histolytica / dispar patients.
On the other hand, the clinical characters of Entamoeba histolytica / dispar infected patients (Tab.10) proved that; 50% of the patients were fulfilling the criteria of parasitic rheumatism and complaining of poly-articular joint pain. While bad response occurred in the other 50% who were complaining of mono-articular joint pain. The difference was statistically non significant (p-value > 0.05).
GIT symptoms were found in 50% of Entamoeba histolytica / dispar cases who were complaining of poly-articular joint pain, while the other 50% were GIT asymptomatic and were complaining of mono-articular joint pain. The difference was statistically non significant (p-value > 0.05).
Single infection occurred in 50% of cases with Entamoeba histolytica / dispar who were complaining of poly-articular joint pain, while mixed infection occurred in the other 50% of cases who were complaining of mono-articular joint pain. The difference was statistically non significant (p-value > 0.05).
Table 10: The relation between cinical characters (response to anti-parasitic treatment, GIT symptoms, type of infection) in E. histolytica / dispar patients and joint affection pattern.
Clinical characters of amebiasis patients Mono-articular Oligo-articular Poly-articular Total FE
p-value
No % No % No % No %
Parasitic Rheumatism Responding 0 0 0 0 2 50 2* 50 Non-sig.
Non-responding 2 50 0 0 0 0 2 50
Total 2 50 0 0 2 50 4 100
GIT symptoms Symptomatic 0 0 0 0 2 50 2 50 Non- sig.
Asymptomatic 2 50 0 0 0 0 2 50
Total 2 50 0 0 2 50 4 100
Type of infection Single 0 0 0 0 2 50 2 50 Non-sig.
Mixed 2 50 0 0 0 0 2 50
Total 2 50 0 0 2 50 4 100
P-value of Fisher Exact test = 0.167 (Not significant)
* The two cases who gave good response to treatment were adult females, with GIT symptoms, and complained of poly-articular joint pain.
Photo 5: Entamoeba histolytica / dispar cyst in stool stained with trichrome stain (X1000).
VI - Microsporidia
Both cases with microsporidia infection were females, adults, living in urban areas with medium socio-economic class. Both cases were asymptomatic and blood investigation showed normal results. Both cases were complaining of mono-articular joint pain and gave bad response to treatment with persistence of the rheumatic pain. Both cases occurred as mixed infection.
Photo 6: microsporidian spores in stool stained with modified trichrome stain, with belt-like strip (X1000).
VII- Schistosoma mansoni
As regards to the demographic data and socio-economic classes of S. mansoni patients; both cases were males, adults, living in rural areas with low socio-economic class. As regards to the clinical data; both cases were symptomatic and blood investigation showed normal results. Both cases were complaining of mono-articular joint pain and gave bad response to treatment with persistence of the rheumatic pain. Both cases occurred as mixed infection.
Photo 7: Schistosoma mansoni egg in stool by wet mount preparation (X100).
VIII- Ascaris lumbricoides
As regards to the demographic data and socio-economic classes of A. lumbricoides patients; both cases were males, young age, living in urban areas with low socio-economic class. As regards to the clinical data; both cases were asymptomatic and blood investigation showed normal results. Both cases were complaining of oligo-articular joint pain and gave bad response to treatment with persistence of the rheumatic pain. Both cases occurred as single infection.
Photo 8: Ascaris lumbricoides fertile egg in stool by wet mount preparation (X100).
IX- Strongyloides stercoralis
As regards to the demographic data and socio-economic classes of S. stercoralis patients; both cases were females, adults, living in rural areas with low socio-economic class. As regards to the clinical data; they were GIT symptomatic and blood investigation results showed elevation of ESR, CRP, and blood eosinophils. Both cases occurred as single infection. Both cases were complaining of poly-articular joint pain and gave good response to anti-parasitic treatment with relieve of rheumatic pain.
Photo 9: Strongyloides stercoralis rhabditiform larva in stool by wet mount preparation (X100).
SUBJECTS AND METHODS
1- SUBJECTS
This study was designed to estimate the prevalence rate of intestinal parasites among patients with unexplained rheumatic pain, as well as the prevalence rate of parasitic rheumatism among infected patients, and was conducted on 108 patients attending Rheumatology and Rehabilitation Outpatient Clinic in Suez Canal University Hospital and fulfilling these inclusion criteria.
Inclusion Criteria:
o Age groups are above 3 years old.
o Both sexes.
o Acceptance to share in the study.
o Complaining of unexplained rheumatic pain.
o Residence in an area of endemic parasitosis.
o Inefficacy of anti-rheumatic drugs.
Criteria used for diagnosis of parasitic rheumatism according to Peng (2002):
Essential criteria
• Inflammatory arthropathy.
• Residence in an area of endemic parasitosis.
• Absence of radiologic changes.
• Identification of parasite.
• Inefficacy of anti-rheumatic drugs.
• Efficacy of specific anti-parasitic treatment.
Supplemental criteria
• Inflammatory synovial fluid.
• Elevated erythrocyte sedimentation rate.
• Peripheral eosinophilia.
All patients were subjected to the following:
a) History taking covering the following:
o Full demographic data, including: Age, sex, residence, any special habits of food handling, and socioeconomic state.
The socioeconomic status of the patient was assessed using a scoring system. The maximum score was 20, and the socioeconomic status was classified as follows: score<10 = low, score 10-15 = medium and score 16-20 = high (Fahmy and El-Sherbini, 1983 & Abou El-Fotouh et al., 1996).
o Present complaint, including:
- Articular and extra-articular complaints (site and duration of pain).
- Symptoms of intestinal parasitic infections (diarrhea, dysentery, abdominal discomfort, and dyspepsia).
o Past history of previous parasitic infections.
b) General examination and local examination for the articular system by the doctor on duty.
c) Rheumatologic investigations: including;
o Blood tests:
• Complete Blood Count (CBC),
• Erythrocyte Sedimentation Rate (ESR),
• C-Reactive protein (CRP),
• Rheumatoid factor (RF) and
• Anti-nuclear antibody (ANA).
o Radiological:
• Plain X-ray for the affected joints was performed.
d) Stool samples were collected and examined.
e) Treatment by suitable anti-parasitic drugs was prescribed to infected patients, according to stool examination followed by stool re-examination 2 weeks after finishing treatment to be sure of parasite elimination and to detect cases with drug resistance.
Drugs used in treatment of infected cases included; metronidazole (Flagyl) 750 mg tds. for 5-10 days (adult dose) and 50 mg/kg/day tds. for 10 days (pediatric dose) for cases infected with G. lamblia, E. histolytica / dispar and B. hominis. Praziquantel (Distocid) 40 mg/kg single dose was used for cases with bilharziasis. Albendazole (Zental) 400 mg/day 3-7 days (adult dose) and 200 mg/kg 3-7 days (pediatric dose) was used for treatment of cases with S. stercoralis, A. lumbricoides and microsporidia. Paromomycin (Humatin) was used for treatment of cases with Cryptosporidium in a dose of 25 mg/kg daily for 5 days. While, C. cayetanensis infection was treated by TMP-SMX (Sutrim), the adult dose is TMP 160 mg and SMX 800 mg twice daily for 7-10 days and the pediatric dose is TMP (5 mg/kg.d) plus SMX (25 mg/kg/d) tds.
2- MATERIALS
Materials used in stool sample collection:
- Dry clean covered plastic cups.
- Wooden sticks.
- Plastic vials with screw lid for stool preservation (30 ml).
Materials used for wet mount preparation:
- Microscopic slide, 1” x 3”.
- Microscopic slide cover, 22x 22 mm.
- Wooden applicator.
- Saline solution: sodium chloride (NaCl) with 0.9% concentration.
Materials used for iodine staining (Garcia, 2001):
-Lugol’s iodine, prepared as:
*Potassium iodine 10 g
*Iodine crystals 5 g
*Distilled water (D.W.) 100 ml
Iodine crystals were ground with potassium iodine using a hammer and mortar in the presence of 5 DROPs of D.W. until iodine dissolved completely, and then 95ml of D.W. were added.
Material used for formalin ethyl acetate (FEA) sedimentation:
- Conical-bottom, 15 ml polyethylene centrifuge tubes.
- Ethyl acetate.
- 10% formalin.
- Distilled water.
- Lab-Top hanging bucket centrifuge.
- Miscellaneous: glass funnels, gauze, wooden applicators and phenol-containing jar for stool disposal.
Materials used for stool preservation:
- Formalin 10% solution (Garcia, 2001):
* Formaldehyde 100 ml
* D.W. 900 ml
- Schaudinn’s fixative (Garcia, 2001):
* Mercuric chloride; saturated aqueous 600 ml
* Ethyl alcohol (95%) 300 ml
The fixative was prepared in stock solution. Just before use, 5 ml of glacial acetic acid were added to 100 ml of stock solution.
- Polyvinyl alcohol (PVA) preservative (Garcia, 2001):
*PVA powder 10 g
* Ethyl alcohol (95%) 62.5 ml
*Mercuric chloride; saturated aqueous 125 ml
*Glacial acetic acid 10 ml
*Glycerin 3 ml
The liquid ingredients were mixed in a 500 ml beaker. Then PVA powder was added without stirring. The beaker was covered with a large Petri dish and the solution was allowed to soak overnight. The solution was heated slowly to 75ºC and stirred until a homogenous slightly milky solution was obtained. For its use; part of stool sample was mixed in PVA at a ratio of 1:3.
Materials used for different stains:
- Coupling jars.
- Water filled plastic bottles for washing.
- Slide rack, slide warmer, slides and covers.
- Forceps.
- Stop watch.
- Laboratory test tubes 15ml.
- Rods.
- Adhesive labels.
- Paper towels.
- Wooden applicator.
- Canada balsam.
Materials used for Modified Acid-Fast staining (Garcia, 2001):
- Absolute methyl alcohol.
- Kinyoun Carbol Fuchsin:
Basic fuchsin 4 g
Ethyl alcohol (95%) 20 ml
Phenol liquid 8 ml
Distilled water 100 ml
Ethyl alcohol was added slowly to basic fuchsin dye in 500 ml Erlenmeyer flask, with continuous stirring to dissolve the powder. Phenol was mixed well with distilled water and this mixture was added slowly to the dye.
- Acid alcohol:
Sulfuric acid (conc.) 10 ml
Absolute ethyl alcohol 90 ml
- Malachite green:
Malachite green powder 3 g
Distilled water 100 ml
Materials used for trichrome staining (Garcia, 2001):
- Trichrome stain:
Chromotrope 2R 0.6 g
Light green SF 0.3 g
Phosphotungstic acid 0.7 g
Glacial acetic acid 1 ml
Distilled water 100 ml
The glacial acetic acid was added to the dry components and allowed to stand for 15-30 min. to ripen, and then distilled water was added. The resultant staining solution was kept in a glass or plastic bottle.
-Iodine alcohol:
A stock solution is made by adding iodine crystals to 70% alcohol (1 to 2 g / 100 ml), then for use; the stock solution is diluted with 70% alcohol until a strong tea color (port wine) is obtained.
- Acid alcohol:
Ethyl alcohol (90%) 99.5 ml
Glacial acetic acid 0.5 ml
- Ethyl alcohol (70% and 100%)
- Xylene.
Materials used for modified trichrome stain (Weber, et al. 1992):
- Modified trichrome stain:
Chromotrope 2R 6 g
Fast green 0.15 g
Phosphotungstic acid 0.7 g
Glacial acetic acid 3 ml
Distilled water 100 ml
The glacial acetic acid was added to the dry components and allowed to stand for 15-30 min. to ripen, and then distilled water was added. The resultant staining solution was kept in a glass or plastic bottle.
- Automatic micropipette, 10 micron and its tips.
- Absolute methyl alcohol.
- Acid alcohol:
Ethyl alcohol (90%) 999.5 ml
Glacial acetic acid 4.5 ml
- Ethyl alcohol: 100% absolute and 95%.
- Xylene.
Materials used for stool culture (Garcia, 2001):
- Pyrex Borosilicate glass tubes with screw cap (16 x 100).
- Filter paper, cut as 10cm x 0.5cm strips.
- Wooden disposable applicator.
- Distilled water (3ml / tube).
- Rack for test tubes.
Materials used for Modified Kato-Katz thick smear (Cheesbrough, 1998):
- Nylon mesh screen.
- Template.
- Wooden or plastic applicator.
- Cellophane tape; soaked in a solution consisting of:
Glycerin 100 ml
Distilled water 100 ml
Malachite green 3% 1 ml
Cellophane tape should remain in this solution for 24 hours before use.
- Phenol containing jar for stool disposal.
- Glass slides.
3- METHODS
Specimen collection and handling:
One stool specimen was collected from each patient for three successive days in a clean and dry plastic container. Patients were instructed to avoid contaminating the specimen with urine or tap water. Each stool sample was subjected to the following:
1. Direct smear examination and iodine staining.
2. The sample was fixed in Schaudinn’s fixative and stained with trichrome stain on the day of collection or preserved in PVA for later staining.
3. Part of the fresh stool sample was preserved in formalin 10% (1-3 parts).
Direct stool examination (Baroody, 1946 &Garcia, 2001):
A small piece of stool was emulsified in a DROP of saline on a slide and covered with a glass cover (22x22 mm) to form a thin fairly transparent film free of air bubbles. Another film was made using a DROP of Lugol’s iodine mixed with a small piece of stool.
Both films were examined using low and high powers of the light microscope. For more confirmation of the result of the direct smear, the same samples were examined using FEA sedimentation.
Formalin ethyl acetate (FEA) Sedimentation (Garcia, 2001):
- About 4 grams of fresh stool was mixed thoroughly with 10 ml of 10% formalin in a tube and lift to stand for at least 30 min. for fixation.
- A sufficient amount of the mixture was strained in wet gauze not more than 2 layers thickness.
- Saline solution was added to the top of the tube and centrifuged at 1500 rpm.
- The supernatant fluid was decanted and the sediment was re-suspended in formalin 10%, up to half the tube only.
- Four to five ml of ethyl acetate was added and the tube was closed by stopper, and then shacked vigorously for at least 30 sec.
- After 15 sec. the stopper was carefully removed.
- The solution was centrifuged at 1500 rpm for 10 min.
- After centrifugation 4 layers were formed (sediment was in the bottom, followed by layer of formalin, then plug of fecal debris and finally layer of ethyl acetate at the top).
- The plug of debris was freed by ringing it with applicator stick and all of the supernatant fluid was decanted.
- The sediment was spread on a slide and examined.
Modified Acid-Fast staining (Garcia, 2001):
- A thin smear of one DROP from each concentrated sample was heat fixed on a slide warmer adjusted at 60 ºC for 5-10 min. until dry.
- The slide was fixed in absolute methyl alcohol for 30-60 sec.
- Then it was stained with Kinyoun carbol fuchsin for 1 min. and was rinsed briefly with distilled water and drained.
- It was de-stained with acid/alcohol for 2 min. and was rinsed briefly with distilled water and drained.
- The slide was counterstained with malachite green for 2 min. and then rinsed briefly with distilled water and drained.
- Then the slide was dried by putting it on a slide warmer at 60 ºC for about 5 min.
- The slide was mounted with cover slip using Canada balsam.
- The slide was examined for at least 10 min. or 200-300 fields by x100 objective.
Trichrome staining (Garcia, 2001):
*Fecal smears for staining from fresh stools were prepared as follows:
1. Fresh fecal material was smeared onto cover slips 22x22 mm. The amount of fecal material smeared was so thin that newsprint could be read through the smear.
2. Immediately (without drying) the slips were placed face downwards in Schaudinn’s for fixation for 30 min.
*Polyvinyl alcohol preserved samples were processed as follows:
1. The preserved specimen was thoroughly mixed and strained through gauze to remove large particulate matter.
2. After sedimentation, a portion of the fecal matter was removed with applicator stick and placed on absorbent paper to remove excess PVA.
3. The material was then streaked onto slides, and the slides were allowed to dry overnight.
*The prepared cover slips and slides were processed as follows:
- Ethyl alcohol 70% for 5 min. (this step is done only with Schaudinn’s fixed smears).
- Iodine alcohol (70%) for 1 min. for fresh specimens or for 5-10 min. for PVA air-dried smears.
- Ethyl alcohol 70% for 5 min.
- Ethyl alcohol 70% for 3 min.
- Trichrome stain for 10 min.
- Acid alcohol for 1-3 sec.
- Absolute ethyl alcohol several dips.
- Absolute ethyl alcohol (2 changes) for 3 min. each.
- Xylene (2 changes) for 5-10 min. each.
- Mounting with Canada balsam.
- Smears were allowed to dry overnight or after 1 hour at 37 ºC.
- Smears were examined microscopically with the X100 objective.
Modified trichrome staining (Weber, et al. 1992):
A thin smear of 10 µl of 10% formalin preserved stool sample (un-concentrated) was made and allowed to air dry then exposed to the following steps:
- Fixation in absolute methyl alcohol for 5 min. then allowed to air dry.
- Staining in modified trichrome stain for 90 min.
- Rinsing in acid/alcohol for no more than 10 sec.
- Dipping in ethyl alcohol (95%) several times.
- Dehydrating in ethyl alcohol (95%) for 5 min.
- Dehydrating in absolute alcohol for 10 min.
- Dehydrating in xylene for 10 min.
- Mounting with a cover slip, using Canada balsam.
- Slides were examined under oil emersion (X1000), for at least 100 fields (about 10 min. per slide).
Stool culture ”Harada Mori filter paper strip culture” (Garcia, 2001):
Immediately after collection, a small piece of stool (1-2 g) was smeared onto the middle of 1x10 cm filter paper, the tip of which was tapered and submerged in a 3 ml distilled water-filled glass tube with crock-screw cap, so that the water level did not reach the site of sample smearing. The tubes were tightly capped, labeled and incubated at 25-28 ºC in an upright position for 10 days. After this period, the tubes were centrifuged at 2500 rpm for 2 min. and a direct wet smear from the sediment was examined microscopically for S. stercoralis hatched larvae, which migrate from the stool smear through the filter paper to the water.
Modified Kato-Katz thick smears technique (Cheesbrough, 1998):
- Feces were pressed through the nylon mesh screen to remove large particles.
- A portion of the sieved sample was then transferred to the hole of a template on a slide.
- After filling the hole, the template was removed and the remaining sample (about 10 mg) was covered with a piece of cellophane soaked in glycerol (glycerol clears the fecal material from around the eggs).
- The slide was turned face down on a paper to spread the sample.
- Slides were labeled and examined after 24 hours, using the low power (X100) of the light microscope.
DATA MANAGEMENT AND STATISTICAL ANALYSIS
Data collected throughout history, basic clinical examination, laboratory investigations, imaging results and outcome measures were coded, entered and analyzed using Microsoft Excel software. Data were then imported into (SPSS 10.0) software for analysis. In qualitative data, Chi square and Fisher Exact tests were used to test difference for significance. P-value (2 tailed) was set at <0.05 for significant results and <0.01 for highly significant results.
SUMMARY AND CONCLUSION
Parasitic infections affect as much as 25% of world’s population. They are mostly prevalent in underdeveloped agricultural and rural areas of tropical and subtropical regions.
Infected patients may remain asymptomatic or develop a variety of clinical manifestations. Rheumatic syndromes can occur as a result of infiltration of musculo-skeletal structures by parasites or an immune mediated mechanism.
Many intestinal parasites can induce a variety of rheumatic manifestations. However, arthropathy which may follow parasitic infections may fulfill the definition of reactive arthropathy.
Underlying parasitic infection should be sought in patients presenting with unexplained or atypical rheumatic syndromes. Diagnosis of parasitic rheumatism is based on the demonstration of infection with a pathogenic parasite, lack of response to anti-inflammatory agents especially NSAD, and improvement following anti-parasitic therapy. Treatment consists of eradication of the parasite.
So, this work was designed to estimate the prevalence of intestinal parasites among patients with unexplained rheumatic pain, as pain is considered as a major and usual symptom in most of rheumatic diseases, and also to identify the prevalence of parasitic rheumatism among infected patients. It was conducted on 108 patients attending Rheumatology and Rehabilitation Outpatient Clinic in Suez Canal University Hospital. Stool examination were done including wet mount preparation, FEA sedimentation, filter paper stool culture, modified Kato-Katz thick smear, in addition to staining with modified acid-fast staining, trichrome stain, and modified trichrome stain.
This study showed the following results:
- The overall prevalence of pathogenic intestinal parasitic infection among patients with unexplained rheumatic pain was 46.3% (50 cases).
- The prevalence of different intestinal parasites detected among the study patients was in a descending order; Cryptosporidium (22.2%), C. cayetanensis (14.8%), G. lamblia (11.1%), B. hominis (9.2%), E. histolytica (3.7%). Microsporidia, S. mansoni, A. lumbricoides and S. stercoralis were 1.9% for each. Non-pathogenic E. coli were present in 14 cases (13%) other than the fifty infected patients. Single infection occurred in 30 cases (60%) and mixed infection occurred in 20 cases (40%) from which 16 cases (32%) had double infection while, only 4 cases (8%) had triple infection. The most frequent association was Cryptosporidium and C. cayetanensis (8 cases).
- Among the infected patient group, the prevalence of intestinal parasites was higher in females than in males (88% versus 12%), and in adults (56%) versus 28% and 16% in young and old age groups respectively. The majority of infected patients were living in rural areas (56%) and most of them were in the low socio-economic class (68%).
- Gastro-intestinal symptoms were identified among 40 % of infected patients, while 60 % were GIT asymptomatic.
- The response of infected patients with unexplained rheumatic pain to anti-parasitic treatment was measured according to the criteria of parasitic rheumatism which include; essential criteria which are inflammatory arthropathy, residence in an area of endemic parasitosis, absence of radiological changes, identification of a pathogenic parasite, inefficacy of anti-rheumatic drugs and efficacy of specific anti-parasitic treatment as well as supplemental criteria which are inflammatory synovial fluid, elevated erythrocyte sedimentation rate and peripheral eosinophilia. Sixteen patients (32%) gave good response with complete relief of rheumatic complaints and disappearance of the parasite from their stool; 8 cases had G. lamblia and the other 8 cases had Cryptosporidium, C. cayetanensis, E. histolytica / dispar and S. stercoralis (2 cases for each). While, in 68% the rheumatic complaints persisted despite of full course treatment of parasitic infection.
الملخص العربي
مقدمة:
تصيب الطفيليات المعدية ٢٥٪ تقريبا من تعداد سكان العالم و خاصة في الدول النامية مسببة تقليل نسبة الإنتاج و ضياع الموارد الاقتصادية. حيث أن معدل الإصابة بالأمراض الطفيلية قد يصل إلى ٩٠٪ من تعداد السكان في بعض الأماكن الاستوائية.
الطفيليات المعدية ممكن أن تسبب العديد من الأعراض الروماتيزمية نتيجة لاختراق التكوين العضلي الهيكلي أو عن طريق التأثير على الجهاز المناعي. و يوجد الكثير من الطفيليات المعوية التي يمكن أن تسبب العديد من الأعراض الروماتيزمية. و ينبغي التفكير في أن الطفيليات هي المسببة لهذه الآلام خاصة الغير معروف سببها و التي لا تنطبق عليها خصائص الأمراض الروماتيزمية المعروفة.
و يعتمد تشخيص الحالات الناتجة عن أمراض طفيلية على وجود طفيلي معدي و عدم استجابة المريض للأدوية المضادة للالتهاب و التحسن بعد علاج العدوى الطفيلية بينما يعتمد العلاج على التخلص من الطفيلي.
الهدف من البحث:
هدفت هذه الدراسة الوصفية إلى البحث عن معدل انتشار الطفيليات المعوية بين المرضى المصابين بآلام روماتيزمية غير معروفة السبب و معدل إنتشار الروماتيزم الطفيلي بالمرضى المصابين بالطفيليات المعوية و قد أجريت هذه الدراسة على المرضى المترددين على العيادة الخارجية للروماتيزم و التأهيل في مستشفى جامعة قناة السويس و عددهم 108مريض المنطبقة عليهم الصفات المطلوبة للبحث.
خطوات البحث:
تم أخذ التاريخ المرضي و الفحص الإكلينيكي و كذلك ملء إستمارةالإستبيان الخاصة بالحالة الإقتصادية و المعيشية لكل مريض كما تم فحص ثلاث عينات براز لكل حالة على مدى ثلاثة أيام متتالية و بفارق أسبوعين للحالات السلبية بالطرق الآتية :
٭ بالطريقة المباشرة (اللطخة)
٭ بطريقة التركيز (فورمالين إثيل أثيتات بالترسيب)
٭ تم فحص الطبقة الراسبة بكل من الصبغات التالية :
- الأسد فاست المعدلة
- الترايكروم
- الترايكروم المعدلة
٭ تم عمل شرائح بطريقة الكاتو للمرضى المشتبه في اصابتهم بالبلهارسيا المعوية.
٭ تم عمل مزرعة براز للبحث عن دودة الاسترونجلويدس ستيركوراليس.
تم وصف العلاج اللازم بالجرعات المناسبة للحالات الإيجابية و إعادة فحص البراز بعد إسبوعين من إنهاء العلاج للتاكد من إستجابة المرضى للعلاج لتحقيق معايير الروماتيزم الطفيلي.
و قد تم عمل تحليل البراز بمعمل الطفيليات بكلية الطب بجامعة قناة السويس. و تم إدخال البيانات و النتائج للكمبيوتر و ترتيبها في جداول و أشكال بيانية و إجراء الإحصاء التحليلية.
نتائج البحث:
و قد أثبت البحث أن نسبة الإصابة بالطفيليات المعوية المعدية بين هؤلاء المرضى هي 46.3٪ معظمهم من الإناث (88٪) و البالغين (56٪), كما أن معظمهم يقطن المناطق الريفية (56٪) و ذو مستوى معيشي منخفض (68٪). اربعون بالمئة من المرضى المصابين بالطفيليات المعوية كانوا يعانون من أعراض خاصة بالجهاز الهضمي.
و قد تم كشف العديد من الطفيليات المعوية و إحتلت الإصابة بطفيلي الكريبتوسبوريديام المكانة الأولى بنسبة 22.2٪ من إجمالي المرضى، تليها السيكلوسبورا كايتانينسيس بنسبة 14.8٪، ثم الإنتاميبا كولاي بنسبة 13٪، ثم البلاستوسيستيس هومينيس بنسبة 9.2٪، ثم الإنتاميبا هستوليتيكا بنسبة 3.7٪، بينما كانت نسبة الإصابة 1.9٪ لكل من الطفيليات الآتية: الميكروسبوريديام و الشستوسوما مانسوني و الأسكاريس لامبريكويدس و الإسطوانية الروثية. و كانت الإصابة بطفيلي معوي واحد في 60٪ من الحالات، بينما في 40٪ كانت الإصابة بأكثر من طفيلي أكثرهم من المصابين بطفيلي الكريبتوسبوريديام و السيكلوسبورا كايتانينسيس.
و قد استجاب 32٪ (16 حالة) من المرضى المصابين بالطفيليات المعوية للعلاج و تحسنت الآلام الروماتيزمية عندهم و كان معظمهم من المصابين بطفيلي الجيارديا لامبليا (8 حالات)، بينما في 68٪ من هؤلاء المرضى استمرت الآلام الروماتيزمية.
و نستخلص من هذه الرسالة أنه يجب أن تؤخذ الطفيليات المعدية في الاعتبار كسبب من أسباب الأعراض الروماتيزمية عند تقييم حالة المرضى الذين يشكون من آلام روماتيزمية غير معروفة السبب و خاصة مع استمرار و زيادة عدد المرضى المعرضين للإصابة بالطفيليات المعدية ووجودنا في منطقة تتوطن فيها الأمراض الطفيلية و عدم وجود وعي طبي كافي و كذلك للتشابه بين الأعراض العضلية و الهيكلية المصاحبة للأمراض الطفيلية و العديد من الأمراض الروماتيزمية.
المناقشة:
تم مناقشة النتائج في ضوء الدراسات السابقة.
الخاتمة:
و اختتمت الرسالة بموجز قصير من التوصيات.
المراجع:
انتهت الرسالة بذكر المراجع التي وردت في متن الرسالة.