Scandinavian Journal of Infectious Diseases, 2007; 39: 862 866
ORIGINAL ARTICLE
Prevalence of Campylobacter jejuni and enteric bacterial pathogens among hospitalized HIV infected versus non-HIV infected patients with diarrhoea in southern India
HAYATH KOWNHAR1, ESAKI MUTHU SHANKAR1, RAMACHANDRAN RAJAN1, APPASAMY VENGATESAN2 & USHA ANAND RAO1 From the 1Bacteriology Laboratory, Department of Microbiology, Faculty of Medicine, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, and 2Clinical Epidemiology Unit, Government Stanley Medical College, and Hospital, Chennai, India
Abstract A prevalence study on Campylobacter jejuni and other enteric bacterial pathogens was carried out in 200 HIV infected and 200 non-HIV infected subjects with diarrhoeal symptoms at an AIDS Hospital in southern India. Diarrhoeal specimens were inoculated onto standard culture media as well as onto Columbia and Campylobacter blood agar media for C. jejuni isolation. All the C. jejuni isolates were tested for antimicrobial susceptibility using Kirby-Bauer’s method. A significant difference in recovery rates was observed between the 2 groups in relation to C. jejuni (p 50.02; 95% CI 5.5 (1 10) and Shigella spp. (p 50.02; 95% CI 6.5 (1 12). 21 isolates of Shigella spp., 16 C. jejuni, 5 Salmonella typhi, 3 Arcobacter spp., 3 Yersinia enterocolitica, and 2 Aeromonas hydrophila were recovered from the HIV infected cases. All the C. jejuni isolates were sensitive to ciprofloxacin whereas 1 strain was resistant to nalidixic acid. Interestingly, all the 29 Shigella spp. (21 from HIV and 8 from non-HIV cases) were resistant to erythromycin and most were resistant to many other antibiotics used. Our observations underline the need for epidemiological investigations to screen C. jejuni and Shigella spp. in HIV infected subjects with diarrhoea and analyse their antibiograms periodically to minimize disease burden in HIV/AIDS.
Introduction Progressive decline in immunological response makes HIV infected patients vulnerable to myriads of opportunistic infections (OI). Diarrhoea is 1 of the major complications, occurring in almost 90% of persons living with HIV/AIDS (PLWHA) in third world countries [1]. Intestinal pathogens are widespread in Indian public water supplies and probably infect a significant proportion of the human population. Campylobacter is an invasive microorganism that has been associated with both diarrhoeal and systemic disease worldwide. It is estimated that about 2.4 million cases of human campylobacteriosis occur each y in the United States [3]. Gastrointestinal infections due to campylobacters are an extremely common complication of HIV/AIDS in India [1], which often leads to wasting, malnutrition and cachexia in the afflicted population [2]. This infec-
tion occurs primarily in infants, elderly people, and patients with underlying disease. Immunocompromized individuals are at higher risk of acquiring severe campylobacteriosis [4]. The disease is usually self-limiting and antimicrobial therapy may not be required generally. However, treatment can decrease the duration and the severity of illness, if initiated early in the course of infection [510]. Campylobacters are important opportunist pathogens in HIV disease and may sometimes cause a septicaemic illness in the absence of enteric disease [11]. Campylobacter spp. are non-spore-forming, motile, Gram-negative rod organisms that have a comma or S-shape morphology. Campylobacters are microaerophilic and grow best at 378C; however, C. jejuni grows best at 428C. Enrichment medium is not required for recovery, since infected humans usually excrete 106 to 109 cfu of C. jejuni per g of stool [12].
Correspondence: U. A. Rao, Department of Microbiology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, India 600 113. E-mail:
[email protected]
(Received 4 January 2007; accepted 10 April 2007) ISSN 0036-5548 print/ISSN 1651-1980 online # 2007 Taylor & Francis DOI: 10.1080/00365540701393096
Campylobacter diarrhoea in HIV/AIDS India, being a developing nation with many risk factors, is likely to account for much of the increased incidence of campylobacteriosis [1316]. However, the role of the C. jejuni and related morbidity in HIV/AIDS is still unclear [17]. In Chennai, southern India, the prevalence rate of enteric pathogens particularly among the HIV infected population is yet to be documented, a lack of adequate public health surveillance strategies being the reason. Moreover, the prevalence of C. jejuni among HIV infected and non-HIV infected subjects has never, to the best of our knowledge, been compared previously. Therefore, we investigated the prevalence of C. jejuni and other enteric bacterial pathogens from the stool specimens of each of 200 HIV infected and 200 non-HIV infected subjects with diarrhoea.
Patients and methods Patients The case control study was carried out in 200 HIV-1 infected subjects and 200 non-HIV study subjects with diarrhoea at an AIDS speciality hospital in Chennai between April 2001 and March 2004. The study was carried out upon approval of the study protocols by the human subjects Ethical Review Committee of the University of Madras. Written, informed consent was obtained from all the study participants, or their legal representatives, before study enrolment. All the patients were sequential and were randomly selected according to the inclusion criteria. Individuals ]18 y of age, male or female subjects randomly falling within the inclusion criteria, were considered. Subjects negative for HIV1 antibodies with diarrhoea were considered under the non-HIV infected group, while those positive for HIV-1 and diarrhoea were considered under the HIV infected group. Selection criteria for diarrhoea were based on the presence of persistent febrile diarrhoea for more than 2 d with nausea, headache and malaise. The study was carried out in compliance with good clinical laboratory practice (GCLP), including the International Conference on Harmonization Guidelines and the Declaration of Helsinki. HIV-1 infection in the patients was determined using HIV-1/2 ELISA and confirmed using Western blot assay (positive for HIV-1 antibody) (Immunetics Inc., MA, USA). CD4 lymphocyte counts and plasma viral load were not available as the setting was resource-constrained. All enteric bacterial pathogens were isolated from faecal specimens of HIV infected and HIV uninfected adults with diarrhoea, and the strains were from sporadic cases and no outbreak of diarrhoea was observed during the study period.
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Bacteriological procedures Briefly, the stool specimens collected were directly inoculated on 5% Columbia sheep blood agar and MacConkey agar plates and incubated at 378C aerobically overnight. The plates with samples from the patients were evaluated daily for 3 d. The specimens inoculated on Campylobacter blood agar (CAB) were incubated at 428C for 48 72 h in an anaerobic jar with Campy Pak (BBL Inc., USA) without a catalyst. Suspicious catalase-positive and/ or oxidase-positive colonies were Gram stained to look for the ‘comma’ or ‘gull wing’ morphology, motility by dark-field illumination; susceptibility to nalidixic acid and rapid hippurate hydrolysis was used as a confirmatory test for the identification of C. jejuni [19]. All the other enteric bacterial pathogens were identified using standard protocols [18]. Briefly, Arcobacter spp. were identified based on oxidase, catalase and growth on MacConkey agar. Polyvalent grouping antisera were used to support the tentative identification of isolates as Shigella spp. after isolation in culture [18]. Blood culture investigations were not performed.
Antibiotic susceptibility Antibiotic susceptibility testing was performed by the Kirby-Bauer’s disk diffusion method, adhering to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (now known as Clinical and Laboratory Standards Institute, CLSI) [20,21], using commercially available antibiotic discs with C. jejuni NCTC11168 as the control. The isolates were tested on Mueller-Hinton blood agar supplemented with 5% sheep blood. The other bacterial isolates were tested according to the NCCLS guidelines with the conventional battery of antibiotics against enteric organisms.
Statistical analysis Isolation rates of enteric bacterial pathogens among HIV infected and uninfected study subjects and antibiogram pattern among the 2 groups were given in frequencies with their percentages. Prevalence of enteric bacterial pathogens and HIV infected and uninfected study subjects were given in percentage of occurrence with their 95% confidence interval (CI). Statistically significant difference for enteric bacterial pathogens among the 2 groups and antibiogram pattern were analysed using 2-samples binomial test and occurrence percentage difference with 95% CI.
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Results HIV infected (n 200) patients with diarrhoea comprised 131 male and 69 female (mean age 37 y) subjects. The non-HIV infected group included 102 males and 98 females (mean age 39.3 y). The HIV infected group presented a total number of 50 (25%) bacterial isolates (95% CI 19 32%) whereas the non-HIV infected group yielded only 26 (13%) isolates (95% CI 9 19%). When the isolation rate of C. jejuni (16%) was compared with all the other bacterial isolates, it came second only to Shigella spp. (21%) among the HIV infected cases. Culture yielded a total of 21 C. jejuni isolates (16 from HIV infected and 5 from the non-HIV group) from the diarrhoeal specimens screened. 21 isolates of Shigella spp., 5 Salmonella typhi, 3 Arcobacter spp., 3 Yersinia enterocolitica, 2 Aeromonas hydrophila isolates were also recovered from the HIV infected cases. The occurrence of C. jejuni in HIV diarrhoeal specimens was higher than the non-HIV diarrhoeal cases. An occurrence percentage difference (8% 2.5% 5.5%) with 95% CI was observed. A significant difference in isolation rates was seen between the 2 groups with regard to C. jejuni (p 50.02; 95% CI 5.5 (110)) and Shigella spp. (p 50.02; 95% CI 6.5 (112)) using the 2-samples binomial test. The profile of enteric bacterial isolates encountered in the investigation is shown in Table I. Multiple infections were seen in 11 HIV infected cases. Concurrent Shigella and C. jejuni infection was noticed in the cases. In addition, 2 non-HIV infected cases presented C. jejuni and Shigella coinfection. Three cases revealed S. typhi and Shigella coinfection, whereas no S. typhi and C. jejuni coinfection was seen. Of the 5 (2.5%) C. jejuni isolates recovered from HIV uninfected subjects, 1 was resistant to erythromycin, ciprofloxacin and nalidixic acid. All the 29 Shigella spp. (21 from the HIV infected group and 8 from the non-HIV infected group) were resistant to erythromycin. Multiple resistance was found to exist among Shigella isolates. The resis-
tance pattern of C. jejuni and Shigella spp. is shown in Tables II and III, respectively. Discussion Diarrhoea represents a well-recognized component of HIV related morbidity and has been cited as a clinical case definition of AIDS by the National AIDS Control Organization (NACO) & World Health Organization (WHO) [22,23]. The prevalence of HIV/AIDS associated infections is on the increase in third world nations compared with industrialized countries and contributes to substantial mortality [22]. Campylobacter-associated diarrhoea can increase the number of cases of campylobacteriosis in these countries [24]. These observations further support the need for improved understanding of the epidemiology of enteric pathogens and particularly campylobacteriosis in developing countries. The occurrence of C. jejuni in HIV diarrhoeal specimens was higher than in the nonHIV diarrhoeal cases. A significant difference in isolation rates was documented with regard to C. jejuni (p 50.02; 95% CI 5.5 (110)) and Shigella spp. (p 50.02; 95% CI 6.5 (112)). Reports in the southern Indian context are still unclear and therefore the present study assumes importance. When the isolation rate of C. jejuni (16%) was compared with all other isolates, it came next to Shigella spp. (21%) in the HIV infected cases. A study from a north Indian normal population has shown the prevalence of Campylobacter spp. to be 13.5% among subjects with diarrhoea and 0.6% from asymptomatic controls [18]. However, our study did not focus on asymptomatic individuals. Studies by Quinn [24] have shown that campylobacters other than C. jejuni could be associated with diarrhoea in HIV/AIDS and we did not look for other Campylobacter spp. as our isolation method used was not favourable for their isolation. Antimicrobial resistance patterns in our study showed that all the Campylobacter isolates were sensitive to erythromycin and aminoglycosides used, thereby maintaining the susceptibility pattern [18].
Table I. Prevalence of enteric bacterial pathogens in HIV infected and uninfected study subjects.
Bacterial isolates Campylobacter spp. Shigella spp. Salmonella typhi Arcobacter spp. Yersinia spp. A. hydrophila.
HIV with diarrhoea (n 200) 16 21 5 3 3 2
(8%) (10.5%) (2.5%) (1.5%) (1.5%) (1%)
Non-HIV with diarrhoea (n 200) 5 8 9 2
(2.5%) (4%) (4.55) (1%) NI 2 (1%)
NI: No. isolates obtained; p value 50.05 reveals statistical significance.
2- samples binomial test p value
Proportion with 95%CI
0.02 0.02 0.40 1.00 0.10 1.00
5.5 (1 10) 6.5 (1 12) 2.05 ( 2 6) 0.5 ( 2 6) 1.5 ( 0.2 3) 0
Campylobacter diarrhoea in HIV/AIDS Table II. Antibiogram pattern of C. jejuni among HIV positive and negative cases. Resistance Antibiotics
HIV (n 16)
Ciprofloxacin Erythromycin Cephalothin Nalidixic acid Tetracycline Chloramphenicol Trimethoprim Imipenem Gentamicin Clindamycin Ampicillin
0 0 15 (93.75%) 1 (6.25%) 0 0 16 (100%) 0 0 0 0
Non-HIV (n 5) 1 1 5 1 0 0 5 0 0 0 3
(20%) (20%) (100%) (20%)
(100%)
(60%)
Furthermore, all were sensitive to ciprofloxacin and gentamicin except 1 strain in the non-HIV infected group that was resistant to ciprofloxacin. The study, however, is in contrast to a previous north Indian study carried out in the general population, which has reported a 71% ciprofloxacin resistance [18]. Our study among the non-HIV infected cases revealed 1 case with ciprofloxacin resistance, which seems negligible. Some studies have focused on quinolone resistance in Campylobacter spp. In Spain, a rapid increase in quinolone resistance was observed after 1988, with up to 50% of C. jejuni isolates resistant by 1991 [26,27]. Tee and Mijch [25] suggested that the prolonged and persistent nature of Campylobacter diarrhoea in patients with AIDS has led to emergence of multidrug resistant C. jejuni, and suggested that HIV infected patients require more aggressive treatment with multiple antibiotics. In contrast to the findings reported, where all isolates were sensitive to ciprofloxacin, Tee and Mijch [25] reported that 2 HIV Table III. Antibiogram pattern of Shigella spp. among HIV positive and negative cases. Resistance Antibiotics
HIV (n 21)
Ciprofloxacin Erythromycin Cephalothin Nalidixic acid Ceftriaxone Chloramphenicol Cefotaxime Augmentin Gentamicin Cotrimoxazole Cefuroxime Doxycycline Amikacin
21 21 18 12 19 15 4 21 14 15 11 10 7
(100%) (100%) (85.71%) (57.14%) (90.40%) (71.42%) (19.04%) (100%) (66.66%) (71.4%) (52.38%) (47.62%) (33.34%)
Non-HIV (n 8) 4 8 8 8 6 6 6 8 5 7 6 6 2
(50%) (100%) (100%) (100%) (75%) (75%) (75%) (100%) (62.5%) (87.5%) (75%) (75%) (25%)
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infected patients with ongoing Campylobacter enteritis prior to bacteraemia had ciprofloxacin-resistant C. jejuni. Others [4] suggested that the failure of C. jejuni to respond to antibiotic therapy was due to the lack of an adequate humoral response to infection. With regard to Shigella spp., the antimicrobial resistance profiles of the isolated strains showed multidrug resistance to certain antimicrobial agents, notably ciprofloxacin (HIV isolates 100% and nonHIV isolates 50%), erythromycin (100%), augmentin (100%), ceftriaxone (HIV isolates 90.4% and non-HIV isolates 75%) and nalidixic acid (HIV isolates 57% and non-HIV isolates 100%). Emergence of drug-resistant species is of concern to clinicians treating HIV infected cases with shigellosis. Concomitant search for alternative new drugs should be continued because, although newer antimicrobial drugs can offer hope for treatment of shigellosis, emergence of resistance to the new drugs may also be close. The more effective way of reducing the impact of diarrhoeal diseases in HIV/ AIDS and the risk of contracting infections lies in improving poor living conditions and supplying safe drinking water. However, accomplishing those objectives is not an easy task in developing countries. This study has several limitations. First, the study design failed to assess the role of laboratory parameters such as CD4 lymphocyte counts and plasma viral loads that might have helped classify the stage of HIV among the HIV-positive study participants. Secondly, the study did not give the outcome variables, viz. mortality, recurrence etc., on the HIV group compared to the controls. Furthermore, the study also lacks details on the importance of blood culture, which might have revealed more isolation rates in regard to bacterial pathogens. Campylobacters are associated with a high mortality rate in bacteraemic cases among HIV patients, and are reported to be more common and more severe in the HIV-seropositive population as reported previously [25]. Thirdly, the study has not looked at the rates of parasites and other aetiologies that might have thrown more light on our existing knowledge of diarrhoeal pathogens in the south Indian HIV context. Also, the study has not identified Shigella to the species level. In conclusion, our study shows that the prevalence of C. jejuni in HIV/AIDS patients was 8% and 2.5% in non-HIV infected patients admitted with diarrhoea in Chennai, southern India. Ciprofloxacin proved to be the drug of choice for C. jejuni in HIV infected patients, as all the isolates were sensitive to the drug, which is in contrast to a north Indian report on high ciprofloxacin resistance. The prevalence of campylobacteriosis among HIV infected patients is higher than in the general population and, as studies
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in the Indian context are unclear, the present study assumes significance. More epidemiological investigations on diarrhoeal pathogens should be attempted to evaluate risk factors for enteric pathogens in HIV/ AIDS in India. In addition, laboratory detection capabilities also need to be strengthened at all levels to increase the baseline surveillance data for improved isolation of the pathogen.
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