الفهرس 
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Abstract
The quality of DUW is of considerable importance to patients and dental healthcare providers because they are exposed to water and aerosols generated from the DU during routine practice. Increasing numbers of people with diminished resistance to overt and opportunistic pathogens are seeking dental treatment; these people might be particularly susceptible to infection as a result of exposure to DUW. Fungal contamination of DUWLs output water is well documented and the contaminating fungi include environmental species of low pathogenicity as well as potentially pathogenic opportunistic spp.
Fungi genera include yeasts, commonly Candida and moulds such as Aspergillus and Penicillium. Fungi can cause invasive fungemia in immunocompromised patients as well as pulmonary infections. Some moulds can also produce mycotoxins which may cause serious liver diseases on chronic exposure.
This study aimed at assessing the mycological contamination of DUWLs and antifungal efficacy of H2O2 disinfectant.
This cross sectional study was carried out during a sixteen - month period, from the first of April 2017 till the end of July 2018. It included water samples randomly collected from 82 DUs located in nine governmental hospitals and three private clinics in Alexandria, Egypt as follows:
a) A total of 204 water samples from outlets of each of the high-speed handpiece, the air/water syringe and the cup filler waterlines (17 from each HCF).
b) A total of 24 water samples from DU reservoirs, 12 filled with tap water and 12 with distilled water (two from each HCF) to assess the water quality before entering the waterlines.
c) In addition, 24 tap water samples (two from each HCF) were collected from the tap adjacent to the DU to assess the water quality before entering the waterlines.
Water samples from outlets of the DUWLs were collected on two separate occasions at the beginning of the shift and at the end of the shift.
In addition, 49 water samples from outlets of the DUWLs (the high-speed handpiece, the air/water syringe and the cup filler) and water reservoirs that gave positive results for fungi were subjected to the application of H2O2disinfectant protocol.
All the collected samples were clearly labeled, transported and delivered in an ice box to the Microbiology Laboratory at the HIPH as soon as possible to be analyzed within 1-2 hours of collection.
The collected samples were subjected to enumeration of the fungi by MF. Isolation and identification of Candida spp., Aspergillusspp. and Penicilliumspp.were done by macroscopic and microscopic examination. All isolates of Candidaspp.were subjected to species identification using germ tube test, API Candida system test, and MALDI-TOF MS testing. Isolates of A.flavusas well as Candida spp. isolates were all tested for their biofilm forming ability as well as genes encoding biofilm formation.
The results of this study can be summarized as follows:
1. According to the Swedish standards for drinking water,total DUWLs acceptability percentages were95.8% of each of the high speed handpiece samples, 86.8% of the cup filler samples and 86.4 % of the air/water syringe samples, respectively.
2. Of the 204 samples from outlets of DUWLs, high speed handpiece samples showed the highest mean value of FCs (39.5 CFU/100ml), followed by air/water syringe (38.8 CFU/100ml), while the cup filler samples had the lowest mean value (17.2 CFU/100ml).Tap water samples, showed the highest FC mean value among all examined samples (380.42 CFU/100ml).
3. The more acceptability samples were revealed from DUs that used distilled water in their reservoir (91.5%)rather than those DUs using tap water in their reservoirs (36.5%).
4. The highest percentage of unacceptable samples was revealed from hospital A (29.4%), followed by hospitals G and I (23.5% each). On the other hand, all private dental clinics and hospital B had acceptable samples (100.0% each).
5. The mean values of FC varied significantly among the 7 dental specialty clinics (p˂0.042). The total mean value of FC was 133.62 CFUs /100ml. The highest acceptability was recorded from implant clinics (100.0%).
6. There was a decrease in FC mean values for the DUWL output water samples collected at the end of shift compared to samples collected at the beginning of the shift.
7. The DUWLs with age more than ten years gave a higher percentage of FC unacceptable samples than those less than five years of age (23.7% to 6.3 %, respectively).
8. Of the 204 examined samples, 186 samples were from DUs using sodium hypochlorite (at variable concentrations; 5.0%, 10.0%, 15.0% and 20.0%) while only 18 samples were from DUs using 2.0% % H2O2.
9. Most of the FC acceptable samples were from DUs that used H2O2 (2.0%) and 20.0% sodium hypochlorite (100.0%, and 96.9%, respectively). On the other hand, the lowest percentage of acceptable samples was from DUs that used 5.0% sodium hypochlorite (13.5%).
10. The percentage of FC acceptability increased (80.8%, 92.2%, 96.6%) with the increase in the concentration of sodium hypochlorite (10.0%, 15.0%, 20.0%, respectively).
11. Of the 204 examined samples, 111 were from DUs that used disinfectants weekly, while 93 water samples were from DUs that used disinfectants daily. The corresponding levels of acceptability were 87.4% and 92.5%, respectively.
12. Of the 204 DUWL output water samples, 76% yielded negative fungal cultures. A.flavus was prevalent in 8.3% of the examined water samples, followed by A.niger (5.9%), while Penicillium spp. and Candida spp. had a4.9% prevalence each.
13. Of the 49 examined water samples; 5 different spp.of fungi were isolated. The most frequent spp. were A.flavus (34.7%), followed byA.niger(24.5%) and Penicillium spp. (20.4%).On the other hand C.dubliniensisandC.albicanswere the least frequently isolated fungi (18.4%, 2.0%,respectively).
14. Hospital A had the highest rate of fungal contamination, since 22 mould isolates were yielded from its water samples. A.flavus was isolated from 64.7% and 11.8% of water samples from Hospitals A and B, respectively, and from only 5.9% of water samples from each of C, D, E, and F Hospitals.
15. Mould isolates were more prevalent in most Hospitals, while C.dubliniensis isolates were recovered only from Hospitals G, H and I hospitals. Only a single isolate of C.albicans was recovered in the present study, and it was isolated from hospital G. No fungal species were isolated from private clinics 1and 2.
16. The high percentage of A.flavus was isolated from minor operation clinics (30.4 %) followed by oral surgery clinics (15.2%), while C.dubliniensis isolates were recovered from periodontics clinics (42.9), fixed prosthodontics clinics (6.6%), and pediatric dentistry clinics (3.4%). Only a single isolate of C.albicans was recovered from periodontics clinics (7.1%).
17. The frequency of isolation of fungal species from DUWL output water samples varied according to the water source. It was found that A.flavus and C.dubliniensis were prevalent in 11.3% and 5.6%, respectively of samples from the high speed handpiece.
18. The single isolate of C.albicans was also recovered from the handpiece. A.flavus was also the most prevalent fungal spp. from the cup-filler (prevalent in 9% of its samples), followed by A.niger and Penicillium spp. (7. 5% each). The air/water syringe had 7.6% of its samples positive for each of A.niger and Penicillium spp., while A.flavus was prevalent in 4.5% of its samples.
19. At the beginning of the shift, all isolates were comprised of moulds (A.flavus, A.nigerandPenicillium spp. were prevalent in 15.7%, 10.8% and 9.8% of samples, respectively). On the other hand, at the end of the shift, all samples were free of moulds but 8.9% of samples were contaminated by Candida spp. (C.dubliniensisand C.albicans were isolated from 8.8% and 0.1% of samples, respectively).
20. The percentage of samples showing no fungal growth decreased significantly with the increase in the DUWLs age from <5 to 5-10 and more than10 years.
21. Before H2O2application, samples from high speeds handpiceses had the highest FC mean values (39.5 CFUs /100 ml), while those samples from cup-fillers had the lowest values (17.2 CFU/100 ml). After H2O2 application, there was a complete eradication of fungi from all examined water samples, yielding 100% acceptability. Candida spp. were completely eradicated by H2O2 application, while A.niger, Penicillium spp., and A.flavus had eradiation levels of 91.7%, 90% and 82.4%, respectively.
22. RBA was able to isolate all fungal species, while SDA had a lower isolation rate for moulds and yeasts (73.5%). A.flavusshowed significantly higher isolation rate on RBA medium than SDA (p= 0.044).There was a fair agreement between SDA and RBA as culture media for all the isolated fungi, except for A.niger, for which they had only a slight agreement. It was noted that only 66.7% of C.dubliniensis were recovered on SDA compared to (100.0%) RBA.
23. The CV method classified the 17 A.flavus biofilm producing isolates into 53% weak producers, 35.3% moderate producers and 11.7% strong producers. On the other hand, the XTT method classified the 17 biofilm producers into 76.5% weak producers, and 23.5% moderate producers.
24. The CV method classified the 10 biofilm producing Candida isolates into 40.0% weak producers, 50.0% moderate producers and 10.0 % strong producers. On the other hand, the XTT method classified the 10 biofilm producers into 40.0% weak producers, 60.0% moderate producers and 0.0% strong producers.
25. There was an upregulation of ALS3 and SAP5 gene expression (6.2% and 4.7%, respectively) in planktonic C.dubliniensis cells, which further increased in biofilm (8.2% and 5.3%, respectively). On the contrary, the expression of fluP gene was downregulated in planktonic cells (49.0%) with less downregulation in A.flavus biofilm (32.0%). Overall, these results denote higher gene expression of the 3 examined genes in biofilms compared to planktonic cells. The upregulation of ALS3 was higher than that of SAP5 in C.dubliniensis.
26. Of the 204 surveyed dentists, 116 (56.7%) were females and 88 (43.3%) were males. They were aged between 26 - 57 years, where 159 (78%) of them were vaccinated against HBV. Their years of experience ranged from 1- 18 years.
27. All of dentists enrolled in this study (100.0%) had knowledge about the causes of contamination of DUs and means to prevent them. In addition, the study has been strengthened by the observation of the dentists’ practices in dental clinics.
28. By observing the practices performed by the 204 dentists included in the present study, it was found that the majority of them did not perform the following practices: allowing water to flow out of the air/water syringes, allowing water to flow outside handpieces, covering light handles with disposable plastic sheets, covering head cushions with disposable plastic sheets, changing medical masks after each patient, wearing protective glasses, and disinfecting protective glasses after each patient (90.2%,88.2%, 89.7% 90.2%,88.2%,89.2%,88.7%, respectively).
29. Among the 82 examined DUs, the acceptability percentages regarding FCs were significantly higher than unacceptability percentages when the following practices were performed: medical masks were changed when exposed to any contamination, all water lines were flushed for 2 minutes at the beginning of the shift , patients used mouth wash before starting the treatment, water was allowed to flow out of the air /water syringes, hand hygiene was done before wearing gloves, and protective glasses were disinfected after each patient (85.0%-15.0%, 79.6%-20.4%, 77.3%-22.7%, 77.3%-22.7%, 75.9%- 24.1%, 71.4%-28.6%, respectively).