Behera MK¹, Nath P² and Meher LK^3*
*Correspondence: Meher LK, Bhim Bhoi Medical College, India, Tel: +919861184752, Email:

Keywords

Diarrhea; HIV/AIDS; Mycobacterium tuberculosis

Introduction

HIV continues to be a major global public health issue, affecting more than 35.3 million people worldwide. India has a distinction to be the third largest contributor to global HIV burden after South Africa and Nigeria [1]. It remains one of the most significant problems haunting India over the past decade with an estimated adult prevalence of 0.26% [2].

Gastrointestinal involvement in HIV/AIDS is almost a universal and significant disease occurring in 50-70% of patients [3]. HIV/AIDS is characterized by opportunistic infection by various pathogens, especially in the gastrointestinal tract [4]. Diarrhea is a major gastrointestinal symptom in HIV infection affecting more than 90% of patients and it becomes more frequent as immunodeficiency progresses during the course of disease [5].

Diarrhea in AIDS could be due to infectious or noninfectious etiology. Noninfectious diarrhea could be due to ART-related adverse effects and HIV enteropathy [6,7]. The etiologic spectrum of enteric pathogens is numerous including bacteria, parasites, fungi and viruses. Several species of protozoa are associated chronic diarrhea which includes Isospora spp, Cryptosporidium parvum, Microsporidia spp, Giardia lamblia, Entamoeba histolytica, Blastocystis hominis and many more [4]. While reviewing the available information, a need was felt to study the prevalence of enteric pathogens in HIV patients with chronic diarrhea of this part of the world. Hence, the study was conducted to determine the prevalence and microbiological profile of pathogens associated with diarrhea in human immunodeficiency virus (HIV) positive patients and their relation to CD4 counts.

Materials and Methods

Study population

One hundred twenty-six consecutive HIV seropositive adult subjects with diarrhea attending the ART clinic of MKCG Medical College and Hospital, Berhampur, Odisha, India were recruited for this study, irrespective of their ART status. Hundred age- and sex-matched HIV seronegative subjects with symptoms of diarrhea were also enrolled as control group who came for routine examinations of their stool samples. Patients who were on medications (such as magnesium containing antacids, proton pump inhibitors, laxatives, antibiotics etc.) which can cause diarrhea were excluded from the study.

Study design

This study was conducted from August 2012 to June 2015. This was a cross-sectional analysis to determine the microbiological profile of diarrhea in AIDS cases and HIV seronegative control subjects. Informed consent was taken, and each study participant was asked to complete a questionnaire which consisted of socio-demographic and personal details, history of diarrheal episodes. The socio-economic classification was done according to the Kuppuswamy’s socioeconomic status scale [8]. Fecal specimens were collected from all the participants in a clean wide mouth screw capped disposable plastic container and transported to the microbiology laboratory by the patients themselves on the same day avoiding any unnecessary delay.

Definition of diarrhea

Diarrhea was defined as the passage of three or more loose or watery bowel movements in a 24-hour period. Acute diarrhea was defined as diarrhea which lasted 7 days or less at the time of presentation. Persistent diarrhea was defined as diarrhea which lasted for more than 7 days but less than 4 weeks at presentation. Diarrhea was called chronic if it lasted for more than 4 weeks.

Laboratory examination

Stool sample was emulsified in a drop of saline and Lugol’s iodine on a slide and examined under the microscope for the presence of trophozoites of Entamoeba histolytica, Giardia lamblia, RBCs, pus cells, helminthic ova, and cyst. Stool smears were prepared, heat fixed, and stained by the Gram stain, ZN stain, Modified ZN stain and Mansons Trichrome stain. Modified ZN staining was done to detect oocysts of microsporidia spp, Isospora belli and Cryptosporidia spp. CD4+ T cell counting was done in all HIV patients using flow cytometry on a BD FACS Caliber cytometer.

All patients were subjected to ultrasound of abdomen and pelvis to look for any intraabdominal pathologies. Patients who had microbiologic or radiologic evidence of tuberculosis either were evaluated with colonoscopy and ileal intubation for biopsy caecum and ileum. Specimens obtained from colonoscopic biopsy were sent to the laboratory for histopathologic analysis and TB culture using the BACTEC System method and LJ media. Biopsied specimens were examined with H&E, Giemsa, Gram, PAS and AFB stains.

Statistical analysis

Analysis was performed using SPSS version 17.0 statistical software. A bivariate analysis was performed to look an association between organism’s isolated and chronic Diarrhea. Two groups of patients were compared with independent t test and three groups of patients were compared with ANOVA t-test. The significance level was set at P<0.05.

Results

In this study, 100 HIV patients presented which chronic Diarrhea were taken as cases and 60 age sex matched HIV negative patients with diarrhea were selected as controls. The mean age group among cases was 30.19 ± 7.127, whereas controls had a mean age of 28.32 ± 9.63. Among cases, male to female ratio was 2.5:1 while controls had a ratio of 1.8:1. This was not statistically significant (Table 1). The sociodemographic characteristics (including socio-economic classes) in both groups were depicted in Table 1. Intestinal pathogens were detected in 81 cases (64.29%) with chronic diarrhea in HIV positive patients and 8 cases (8%) with diarrhea in HIV negative controls (Table 2). Besides diarrhea, fever, pain abdomen and weight loss (>10% of body weight) were observed in higher frequency in the cases as compared to the controls (p<0.001) (Table 2). In the HIV positive group, 60 cases (47.6%) had single or multiple parasites, whereas in the HIV negative patients only 11 (11%) had single or multiple parasites (p<0.05) (Table 3).

The most common enteric pathogen detected in HIV positive diarrhea subjects was Mycobacterium tuberculosis [41 cases (32.53%)] as evidenced in stool samples, imaging studies, histopathology study of colonoscopic biopsy samples. Isospora belli and Cryptosporidum parvum were detected in 27 cases (21.49%) and in 15 cases (11.9%) respectively. As compared to controls, the observed incidence of these organisms in HIV patients in diarrhea was significantly high (P<0.05). Less frequently found pathogens were Cyclospora in 4.76% cases and Microsporidia, Giardia and Strongyloides in 2.38% cases each (Table 4).

Among diarrheal patients with an identifiable cause, 60 cases had CD4<200, 12 patients had CD4 within 200-350 and 4 patients had CD4>350. Among diarrheal patients without an identifiable cause 20 cases had CD4<200, 18 cases had CD4 within 200-300 and 12 cases had CD4>350. This was statistically significant (P<0.001) (Table 5).

All cases of Isosporiasis had CD4<200 and 80% cases of Cryptosporidiosis had CD4<200 and 20% had CD4 within 200- 350. Correlation between Isospora & Cryptosporidium with CD4 count was significant (P<0.05). Among diarrheal stool samples with Mycobacterium tuberculosis, 73.3% cases had CD4 <200, 23.3% cases had CD4 200-350 and 3.33% case had CD4 >350 and it was not statistically significant. Thus, Mycobacterium tuberculosis was identified as a cause of chronic diarrhea at any CD4 count (Table 6).

Discussion

Available literatures on HIV epidemic in India, the number of people living with HIV in India in 2015 were 2.11 million with an estimated adult HIV prevalence of 0.26% [2]. Diarrhea is among the most common symptoms of HIV infection and is experienced by over 90% of patients with AIDS. It becomes more frequent as immune deficiency progresses. HIV infected patients are becoming more susceptible to variety of opportunistic infections with increasing severity and frequency. Several intestinal parasites previously thought to be non-pathogenic in immunocompetent persons are now opportunistically becoming aggressive in immunosuppressed HIV/ AIDS patients.

Most of the HIV patients were in the age range of 25-35 years which was almost similar to the current study. The present study showed a male preponderance with 91 males and 35 females (Male: Female ratio of 2.6:1) among case group and 39 males and 21 females (Male to Female ratio of 1.8:1) among control group which is compatible. Predominance of male cases may be due to migration to other cities in search of work, staying away from families for longer periods and males being promiscuous by habit resulted in HIV infection. Generally, females particularly of low-socio-economic classes have a tendency to avoid health check-ups leading to a low detection rate.

The high prevalence of intestinal parasites like Cryptosporidium, Isospora and Microspora were consistently found in HIV/AIDS patients with diarrhea. These pathogens were reported as most common cause of diarrhea in HIV/AIDS in most of the previous studies [4,9]. Mycobacterium tuberculosis was identified in 23.8% cases. Infection with common intestinal pathogens Ascaris lumbricoides, Entamoeba histolytica and Strongyloides stercolis were lower (3% each) in HIV/AIDS patients with diarrhea when compared to diarrhea in HIV negative persons as reported in previous studies [4,9,10]. This discrepancy in HIV patients are due to structural and functional alteration in the gut that may not be suitable for growth of common intestinal parasites. Our study did not reveal any other bacterial or fungal pathology.

In our study, mycobacterium TB was the most commonly isolated pathogen i.e., 32.53% followed by Isospora 21.49% and Cryptosporidium 11.9%. As compared to the controls, the observed incidence of these organisms in HIV patients with chronic diarrhea was significantly higher (P<0.05). Our study differed strongly from other similar studies with respect to Mycobacterium tuberculosis as a predominant cause of chronic diarrhea in HIV. A study done at Nairobi Kenya by Mwchari et al. showed Cryptosporidium as the leading cause followed by Mycobacterium tuberculosis [10]. There are many reports regarding frequency of various pathogens causing diarrhea in different parts of India. Vignesh et al from south India detected Isospora belli as the most frequently encountered parasite [11]. Kotgire et al. from India found Cryptosporidium parvum as the most common intestinal parasite in HIV positive diarrhea patients [12]. But no studies have depicted Mycobacterium tuberculosis as the predominant cause of diarrhea in HIV/AIDS patients. These differences can be explained by geographic variation, underestimation of TB as a contributor to development of this condition as most of the studies focuses on intestinal protozoa as a cause of chronic diarrhea.

The cause of diarrhea in intestinal tuberculosis is postulated to be due to diminished absorptive surface due to multiple ulcerations in the Intestine. Bile salt deconjugation, and involvement of lymphatics and lymph nodes may also contribute in causing diarrhea in GI tuberculosis [13]. An autopsy study from India revealed that Mycobacterium tuberculosis was cause of gastrointestinal infection in 14% of total patients [14]. Antinori et al. reported two cases of intestinal tuberculosis presenting as chronic diarrhoea in patients infected with HIV [15]. Another study from India reported that Mycobacterium tuberculosis was isolated from 3.7% of stool samples of HIV positive patients with diarrhea.

Our study also demonstrated a higher pathogen detection rate at a low CD4 count. There was an inverse correlation between CD4 counts and parasite isolation rates from diarrhea patients. It was also observed that the CD4 cell count influenced the cause of diarrhea as well as the diagnostic yield. The maximum parasitic isolation was in the group of patients who had CD4 cell counts below 200 cells/μL and Isospora was found to be the most commonly acquired protozoa causing chronic diarrhea. The isolation rates decreased with the increase in the CD4 cell counts. This finding is in accordance with the study conducted by Lekha Tuli et al. from Varanasi, India [16]. The low diagnostic yield of stool analysis in patients with higher CD4 cell counts was probably due to effective HAART eradicating opportunistic protozoal infection and causing the influx of CD4 positive cells into the lamina propria. Mycobacterium tuberculosis was second to Isospora as a pathogenic organism in HIV positive diarrhea with CD4 cell count <200. One interesting observation in our study was that Mycobacterium tuberculosis was identified as a cause of chronic diarrhea irrespective of any level of CD4 cell count.

Conclusion

In the present study, Mycobacterium tuberculosis has been found to be the most common pathogen causing chronic diarrhea in HIV patients in this part of world. Among parasitic infections, Isospora belli followed by Cryptosporidium are most commonly found in our study. Chronic diarrhea as a manifestation in HIV patients is more common with low CD4 count. Gastroenteric pathogen detection is more common in patients with low CD4 count.

References

1. World Health Organization (2009) United Nations Children's Fund, UNAIDS: Towards universal access: scaling up priority HIV/AIDS interventions in the
2. National AIDS Control Organization (NACO) Annual Report 2016-2017.
3. Cello JP, Day LW (2009) Idiopathic AIDS enteropathy and treatment of gastrointestinal opportunistic pathogens. Gastroenterology 136: 1952-1965.
4. Smith PD, Quinn TC, Strober W, Janoff EN, Masur H (1992) Gastrointestinal infections in AIDS. Ann Intern Med 116: 63-77.
5. Siddiqui U, Bini EJ, Chandarana K, Leong J, Ramsetty S, et al. (2007) Prevalence and impact of diarrhea on health-related quality of life in HIV-infected patients in the era of highly active antiretroviral therapy. J Clin Gastroenterol 41: 484-490.
6. Call SA, Heudebert G, Saag M, Wilcox CM (2000) The changing etiology of chronic diarrhea in HIV-infected patients with CD4 cell counts less than 200 cells/mm³Am J Gastroenterol 95: 3142-3146.
7. MacArthur RD, DuPont HL (2012) Etiology and pharmacologic management of noninfectious diarrhea in HIV-infected individuals in the highly active antiretroviral therapy era. Clin Infect Dis 55: 860-867.
8. Mishra D, Singh HP (2003) Kuppuswamy’s socio-economic status scale-A revision. Indian J Pediatr 70: 273-274.
9. Fontanet AL, Sahlu T, Rinke de Wit T, Messele T, Masho W, et al. (2000) Epidemiology of infections with intestinal parasites and huma immunodeficiency virus (HIV) among sugar-estate residents in Ethiopia. Ann Trop Med Parasitol 94: 269-278.
10. Mwachari C, Batchelor BI, Paul J, Waiyaki PG, Gilks CF (1998) Chronic diarrhoea among HIV-infected adult patients in Nairobi, Kenya. J Infec 37: 48-53.
11. Vignesh R, Balakrishnan P, Shankar EM, Murugavel KG, Hanas S, et al. (2007) High proportion of isosporiasis among HIV-infected patients with diarrhea in Southern India. Am J Trop Med Hyg 77: 823-824.
12. Kotgire SA, Tankhiwale N, Tankhiwale S, De A, Das D (2011) Evaluation of parasites causing gastro-intestinal tract infestations in HIV/AIDS patients. Indian J Public Health 55: 337-338.
13. Lanjewar DN, Anand BS, Genta R, Maheshwari MB, Ansari MA, et al. (1996) Major differences in the spectrum of gastrointestinal infections associated with AIDS in India versus the west: An autopsy study. Clin infect dis 23: 482-485.
14. Shah S, Kongre V, Kumar V, Bharadwaj R (2016) A study of parasitic and bacterial pathogens associated with diarrhea in HIV-positive patients. Cureus 8: e807.
15. Antinori S, Galimberti L, Parente F (2001) Intestinal tuberculosis as a cause of chronic diarrhoea among patients with human immunodeficiency virus infection: A report of two cases. Dig Liver Dis 33: 63-67.
16. Tuli L, Gulati AK, Sundar S, Mohapatra TM (2008) Correlation between CD4 counts of HIV patients and enteric protozoan in different seasonsâ??An experience of a tertiary care hospital in Varanasi (India). BMC Gastroenterol 8: 36.