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HIV/AIDS patients are at high risk of infection with toxin producing strains of C. difficile because of frequent hospitalization, exposure to antibiotics and antibiotic prophylaxis for opportunistic infections. There is very little data in the Indian scenario on the prevalence of C.difficle infection in Indian population.
Methods:The prevalence of Clostridium difficile infections in HIV-positive patients with regard to the presence of its toxin and growth in culture was investigated. Enzyme immunoassay (EIA, Premier toxins A and B; Meridian Diagnostic Inc.) was used for the detection of toxin from 237 fresh stool samples collected from HIV positive diarrheal patients. Culture was performed oncycloserine-cefoxitin-fructose agar and brain heart infusion agar.
Results: Clostridium difficilewas found in 12/237(5.06%, 95%CI=2.64 to 8.68%) of HIV positive diarrheal patients (nine patients were positive by EIA and three by culture).The presence of C.difficile in ART treated patients 7/66 (10.6%) was significantly higher (P<0.016) as compared to ART naÃ¯ve patients 05/171 (3%).Out of 12, seven patients were on antiretroviral therapy for average of 34.42 months with mean CD4+ T cells count of 186±98.81 and five patients were antiretroviral naÃ¯ve with mean CD4+ T count of 181±68.70.All of these 12 patients were on antibiotics for previous two months and four patients out of 12 were hospitalized in previous 30 days.
Conclusions:The prevalence of Clostridium difficile infections in HIV/AIDS patients is variable according to population selected and site of study. The relatively lower prevalence of C.Difficilein our study population as compared to previous studies could be attributed to higher use of metronidazole antibiotic use in our study participants.
Key words: Clostridium difficile, HIV/AIDS, Antibiotic associated diarrhea.
Patients with HIV/AIDS are at high risk for various opportunistic infections during their lifetime. Among them, diarrhea is significant cause of morbidity as observed in majority of the studies1 and is an independent marker of poor prognosis2.Recent reports indicate that diarrhea occurs in 40-80% of HIV infected patients untreated with highly active antiretroviral therapy (HAART) in developed countries and in about 90% of such patients in developing countries.3,4HIV/AIDS patients are at high risk of infection with toxin producing strains of C. difficile because of frequent hospitalization and exposure to antibiotics. According to previous studies, prevalence of C. difficile associated diarrhea in HIV positive patients varies from 34-10%.5,6while its prevalence among HIV negative patient is varies from 4%- 12.1%as reported by various authors.6,7 However, there is very limited data from India regarding C.difficileassociated diarrhea(CdAD) in HIV/AIDS patients.
2. MATERIAL AND METHODS
We conducted a hospital-based, cross-sectional study to estimate the prevalence of C. difficile diarrhea and determine associated risk factors in HIV infected individuals attending the ART clinic at AIIMS.A total of 237 fresh stool samples were collected from 267 screened HIV positive diarrheal patients (30 patients excluded, one HIV-2 positive and 29 patients unwilling to participate).HIV-1 positive patients older than 18 years of age with diarrhea, defined as more than three episodes of loose watery stools per day,were recruited for this study.Patients who were unwilling to participate in the study were excluded. C.difficile was isolated using stool culture on cycloserine-cefoxitin-fructose agar (CCFA) and brain-heart-infusion agar (BHIA) and identification of the organisms was done by standard Gram stain and biochemical methods as described in previous studies. This was also augmented by ELISA (Premier toxins A and B; Meridian Diagnostic) to detect C. difficile toxins A & B in stool samples. A patient was considered to have tested positive for C.difficile if the cultures grew C.difficile orif the ELISA for stool cytotoxin assay. CD4 count and viral load was also done in all patients to assess the HIV status of the study participants. Stool was also tested for Salmonella, Shigella and entero-parasites, other pathogens were not tested due to limited time and funding.
This study was the part of a PhD thesis and study population was a sample of convenience based on the number of diarrheal patients visiting the ART clinic during the study period.Primary analysis was comparison of C.difficilepositive diarrheal patientswithC.difficile negative diarrheal patients. The data are presented as proportions (with 95% confidence interval [CI]) for qualitative variables and mean (standard deviation [SD]) for quantitative variables. Bivariate analysis of the quantitative variables was done using t-test, that of qualitative variables using chi-square test and that of ordinal variables using Mann-Whitney's test. Information and stool samples were collected from patients after obtaining informed consent and the study protocol was approved by the Institutional Ethical Committee of the All India Institute of Medical Sciences, New Delhi. Information and stool samples were collected from patients after obtaining informed consent and the study protocol was approved by the Institutional Ethical Committee of the All India Institute of Medical Sciences, New Delhi.
A total of 237 fresh stool samples were analyzed for C. difficile from HIV positive diarrheal patients. The maximum number, 220/237(92.82%) of suspected cases were recruited from outpatients departmentand 17/237(7.17%) of the study patients were hospitalized. Age range of patients was 14 to 84 years.A majority of the patients were men (75.5%). The clinical characteristics of study patients of both groups are detailed in Table 1. Mean CD4+ T cells count were lower in C.difficile positive patients (183.69 ± 79.83) as compared to C.difficile negative (234. 51 + 188.94) patients, but this difference was not statistically significant. The HIV viral load in C.difficile positive group (219963 ± 260803) was higher compared to C.difficile negative group (156272 ± 206221); however this was not statistically significant. There was also no significant difference in the duration of diarrhea, antibiotic use patterns between patients with C. difficile and those without.
Of the 237patients, 12 tested positive for C. difficile infection, an estimated prevalence of 5.0% (95% CI: 2.6 to 8.7). Of the 12 detected to have C. Difficile infection, 9 tested positive for the toxin but had negative cultures, 3 tested negative for cytotoxin but tested positive for culture but none tested positive for both..Seven of the twelve patients were on antiretroviral therapy for average of 34.42 months with mean CD4+ T count of 186±98.81 and five patients were antiretroviral naÃ¯ve with mean CD4+ T count of 181±68.70. All of these 12 patients were on antibiotics before the onset of diarrhea. Five of these had tuberculosis as opportunistic infection and one had CMV retinitis.We also observed a statistically significant [05/171(3%) vs. 7/66(10.6%); P<0.01] difference in the prevalence of C. difficile in ART naÃ¯ve and ART treated patient respectively.
Other pathogenic organism isolated from the study group included Isospora belli in 27/237(11.39%) and Shigella in one.No Salmonella sp. was isolated from any stool samples in our study.The mean duration of illness was 7days (ranges1-60days) and mean frequency of stool was five episodes per day.A total 195/237 (82.27%) patients were on multiple antibiotics.Out of 195 patients who were on antibiotics, 12(6.15 %) patients had CdAD. Most of the patient experienced diarrhea during treatment or within 15days of starting antibiotics.Details of antibiotic data are summarized in Table 2.
The prevalence of C.difficile was 5.06% in our study population, which is lower than 58.8% reported by Wongwanichet.al.11and10% by Uppal et.al6.However, there is conflicting report of prevalence of C.difficile diarrhea in HIV positive pre HAART and post HAART era. Anastasi et.al.12reported that C.difficile is still the most common infectious cause of diarrhea in HIV patients. Although Tacconelli et.al.13found that the incidence of CdAD in HIV patients has decreased in the HAART era in western countries, the incidence is still higher than patients without HIV infection.
In our study, we observed statistically significant difference in the prevalence of C. difficile in ART naÃ¯ve and ART treated patient respectively. ART treated patients are more prone to C.difficile infection and this discrepancy in positivity in either group may be due to use of ATT, and prophylactic antibiotics for opportunistic infection and in addition CD4+ T count<200cu/mm indicating immunological failure in 5 out of 7 leads them into more risk in ART treated group. This risk was absent in ART naÃ¯ve subjects except low CD4 cell count.
In the present study, the positivity of C. difficile was higher statistically significant (P= 0.001) in the older age group, and in male gender. This is supported by report by other authors.14 Thiscould be due to low immunity in elderly and more risk of exposure in male patients may be responsible.
Prior use of antibiotics is a key risk factor for C.difficile infection. We found that patients affected with CdAD were taking more antibiotics for longer period of time though statistically there was no significant difference. Common antibiotics associated with CdAD are clindamycin, penicillin, and cephalosporin; however, antibiotics associated with diarrhea in our study (Quinolones, Antitubercular medications, Co-trimoxazole) are not traditionally associated with a high risk for CdAD as reported by other authors.15 It may be due to an association with tuberculosis or more intensive empiric use of these antibiotics in a sicker group of patients.
In our study, we observed that patients with more advanced HIV disease were more frequently infected with C.difficile as described here as low CD4+ T cells count though statistically not significant (p>0.5) and more opportunistic infections amongC.difficilepositive patients.This is consistent with previous studies.16 This is consistent with the hypothesis that anadvanced HIV infection leads to imunocompromised host, havemore frequent hospitalization andmore antibiotic use, all which are risk factors for C.difficile infections.
For the diagnosis of C .difficile infections, standard methods include stool culture and ELISA for C. difficile toxins (A & B) detection in stool was done. Stool culture is standard method, but requires longer time and cannot differentiate toxigenic from non-toxigenic strain, where toxin detection is rapid and specific (toxin B) method for diagnosis of C. difficile infection.17 Stool culture positivity in the absence of toxin detection as revealed in our study may be due to presence of non-toxigenic strain or less production of toxin as reported by other authors.18
Prior use of antibiotics (metronidazole) may be responsible for culture negativity in toxin positive cases. MostlyC. difficile toxin EIAs were used for diagnosis of CdAD.19In our study we used ELISA for toxin A, B and culture for diagnosis of C. difficileinfection.
As observed from a previous study conducted in the same laboratory on the patient population of the same hospital, the prevalence of C. difficile in HIV negative diarrheal patients was 7.19 % (63/876). In our study, prevalence of C. difficile in HIV positive diarrheal patients was found to be 5.06%. This lower rate of prevalence could be explained by the fact that most of our patients already had started empirical metronidazole and that a lower percentage of our study population was hospitalized. Also, the prevalence of C. difficile (CdAD) in HIV negative patients is also variable in different studies as reported by Choudhary et.al8as 5% in 2005, and 4% by Uppal et.al6. Variability in prevalence of C.difficile could also be due to variation in population studied and geographical area selected.
The lower rate of enteric bacterial pathogens detected is consistent with the result of studies by Uppal et al6 and Becker et al20.The prevalence of enteric bacterial pathogens is very low at our tertiary care hospital and may be due to use of antibiotics prior to or during the hospital visit.
Prevalence of C. difficile is variable. Traditional risk factors for C.difficile arecommon, and are more prominent in patients with advanced HIV disease. Any hospitalized HIV infected patient with diarrhea should submit stool samples for C.difficile toxin testing and culture and should be treated early empirically, if clinical symptoms are consistent with CdAD to prevent complications associated with it.