Intestinal protozoans in adults with diarrhea.

BACKGROUND: Diarrhea is one of the most common presenting complaints in human immunodeficiency virus-infected individuals. AIMS: The study was designed to determine the magnitude of opportunistic and nonopportunistic intestinal parasitic infections among diarrheal patients and association between CD4(+) T-cell counts and human immunodeficiency virus (HIV)-infected intestinal parasites.
MATERIALS AND METHODS: A cross-sectional study was conducted among 207 enrolled diarrheal patients attending HIV integrated counseling and testing center from January 2012 to December 2012. Stool samples were subjected to special modified Ziehl-Neelsen and chromotrope staining method for detection of opportunistic protozoans. Blood samples were also collected from all study subjects for HIV testing and CD4(+) T-cell counts were estimated by only in HIV-infected patients.
RESULTS: Intestinal parasitic pathogens were detected in 46.1% HIV-infected patients and the major pathogens were opportunistic protozoans 32.2% (37/115), most common being Isospora belli 16.5% (19/115) followed by Cryptosporidium parvum 12.2% (14/115). In HIV noninfected diarrheal patients, major pathogens detected were Entamoeba histolytica/Entamoeba dispar 8.7% (8/92) and Ascaris lumbricoides 3.3% (3/92).
CONCLUSIONS: The opportunistic intestinal protozoans especially I. belli and C. parvum were most commonly isolated in HIV-infected patients with diarrhea. Majority of the infections occurred in patients when a CD4(+) T-cell counts were less than 200 cells/μl.

Introduction

The global pandemic of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) in its third decade has shown public health concerns especially infections by opportunistic pathogens including various forms of intestinal parasitosis. Diarrhea is one of the most common presenting complaints in HIV-infected individuals, reaching a rate up to 60% in developed countries and 90% in developing countries.[1] The infectious etiological agents include both opportunistic agents that consistently cause severe, often frequent or chronic diarrhea and nonopportunistic agents that usually cause acute, treatable gastro-intestinal illness.[2] Many self-limiting and sporadic intestinal parasites have now become opportunistic parasites causing uncontrollable life threatening diarrhea among people living with HIV/AIDS.[3] Opportunistic intestinal protozoan parasites such as Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis and Microsporidia including Enterocytozoon bieneusi and Encephalitozoon intestinalis have been reported in HIV infection.[4] Recent studies have also opined that mucosa dwelling parasites may benefit from HIV-induced pathological changes and reduced immune response due to HIV infection, which creates suitable environment for opportunistic intestinal parasites in HIV/AIDS patients.[34]

Intestinal parasites are frequently transmitted by low level of environmental and personal hygiene, contamination of food and drinking water and poor sanitary conditions in developing countries.[5] Proper investigation of the parasitic etiology of diarrhea leading to prompt and effective management can help in decreasing the morbidity and mortality in such patients.[6]

Only a few studies have been reported regarding the prevalence of intestinal parasites from eastern part of India. Against the above background as well as the generated data that will improve the management of opportunistic infections in HIV/AIDS patients, this study is aimed to determine the prevalence of intestinal parasitic infections with special emphasis on opportunistic coccidian parasites among HIV-infected and HIV noninfected patients presenting with diarrhea as well as effect of CD4+ (cluster of differentiation 4) T-cell counts on prevalence of the disease among HIV-infected patients.

Materials and Methods

Study design and subjects

A cross-sectional study was carried out between January 2012 and December 2012 in a 900-bedded tertiary health institution, Odisha state with antiretroviral therapy (ART) facilities for HIV/AIDS management. This study was conducted after due approval of institutional ethical committee.

A total of 5973 subjects who attended integrated counseling and testing center (ICTC) were enrolled for HIV testing during study period and among them 372 suffered from diarrhea. Stool samples were collected from 207 consenting HIV-infected and HIV noninfected patients suffered from diarrhea were included in this study in order to determine the magnitude and prevalence of intestinal parasites among HIV-infected patients. Study patients were interviewed using a structured questionnaire and information was obtained on demographic characteristics, present and past history of diarrhea and antibiotic treatment. Diarrhea was defined as two or more liquid or three or more soft stools per day. Patients already received antiparasitics and antibiotics treatment and less than 18 years were excluded from the study. Verbal informed consent was obtained from all patients prior to collection of stool sample.

Blood sample collection and processing, HIV serology, and CD4+ T cell count

All the ICTC attendees received relevant pretest counseling and written informed consent was obtained from each of them before HIV testing was carried out. Five milliliters venous blood sample was collected in a sterile plain container from all patients suffering from diarrhea who attended ICTC. Blood was allowed to clot for 30 minutes at room temperature (25–30 C) and serum was separated after centrifugation at low speed. The serum samples were then stored at 4 C and were tested within 48 hours.

HIV antibodies were tested by the three rapid tests protocol as per the guidelines laid down by the World Health Organization (WHO Testing strategy III) and National AIDS Control Organization, Government of India.[7] All positive test results were disclosed only after post test counseling of the patients. Antibodies to HIV (1 and 2) were tested initially with a SD BIOLINE HIV-1/2 3.0 rapid test [Standard Diagnostics, Inc. Korea]. The samples tested positive in the first method were subjected to tests with two different rapid tests, that is, PAREEKSHAK HIV 1/2 Triline Card Test [Bhat Bio-Tech India (P) Ltd., Bangalore, India] and PAREEKSHAK HIV 1/2 Rapid Test Kit (TRISPOT) [Bhat Bio-Tech India (P) Ltd., Bangalore, India]. The samples were considered as positive when found reactive by all three different methods. All tests were done according to manufacturer's instructions.

CD4+ T-lymphocytes count was carried out in HIV-infected patients by using BD FACS™ Calibour system (Becton Dickinson, Singapore). In brief, a three-color direct immunofluorescence reagent, that is, CD3 fluorescein isothiocyanate (FITC)/CD4 phycoerythrin (PE)/CD45 peridinin chlorophyll protein (PerCP) was used to identify and determine the absolute counts of helper T-lymphocyte (CD4+) subsets in erythrocyte-lysed whole-blood.[8]

Stool sample collection and examination

From consenting ICTC diarrheal attendees, single stool sample was collected for this study. All stool specimens were subjected to fresh direct wet mounts, formol-ether concentration technique, modified Ziehl–Neelsen stain and chromotrope stain for detection of intestinal parasites.

Direct wet mount

A direct saline and iodine wet mount of each sample was used to detect intestinal parasites microscopically. The wet mounts were examined under light microscope at ×100 and ×400 magnifications. A small portion of the stool samples were also preserved in 10% formalin for repeating the tests whenever required.

Formol ether concentration method

A portion of each fresh stool sample was taken and processed. Briefly, 1 g of stool was placed in a clear 15 ml conical centrifuge tube containing 7 ml formalin saline by using applicator stick. The resulting suspension was filtered through a sieve into another conical tube. After adding 3 ml of diethyl ether to the formalin solution, the content was centrifuged at 3200 rpm for 3 minutes. The supernatant was poured away and the tube was replaced in its rack. Finally smear was prepared from the sediment and observed under light microscope with a magnification of ×100 and ×400. Fresh stool specimens were used as saline wet mounts to detect motile trophozoites and concentrated stool specimens were used as iodine wet mounts to detect ova, larva, and cysts.

Modified Ziehl-Neelsen staining method

Thin smear was prepared directly from sediment of formol-ether concentrated stool and allowed to air dry. The slides were then fixed with methanol for 5 minutes and stained with carbol fuchsin for 30 minutes. After washing the slides in tap water, they were decolorized with acid alcohol (99 ml of 96% ethanol and 1 ml HCL) for 3 minutes and counterstained in methylene blue for 1 minute. The slides were then washed in tap water and observed for Cryptosporidium parvum, Isospora belli, and Cyclospora cayetanensis under light microscope with a magnification of 1000×.[9]

Chromotrope staining method

A small part of 10% formalin fixed unconcentrated stool suspension was smeared on a glass slide. The thin smear was heat fixed on a slide warmer at 60 C until completely dry (5-10 minutes) followed by absolute methanol fixation for 5 minutes. The slides were stained with chromotrope for 90 minutes. After washing in tap water, they were destained with acid alcohol (995.5 ml of 90% ethanol and 4.5 ml glacial acetic acid) only for 1-3 seconds, then immediately rinsed with 95% ethanol by dipping. Subsequently the slides were changed twice in 100% ethanol for 3 minutes each and changed twice in xylene for 10 minutes each. The slides were then drained and mounted and observed for Microsporidia under light microscope with a magnification of 1000×.[9]

Quality control

A control slide of Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, and Microsporidia from a 10% preserved specimen were included along with each staining run.

Statistical analysis

GraphPad Inc. statistical software (2236 Avenida de la Playa La Jolla, CA 92037 USA) was used for calculation of mean, median, standard deviation, range, and P value using Fisher's exact test. Statistical significance was defined when P value is less than 0.05.

Results

A total of 207 diarrheal stool samples were examined for presence of intestinal parasites, 115 (55.6%) were collected from HIV-infected patients and the rest 92 (44.4%) were from HIV negative patients. The mean age of HIV-infected patients was 37.7 (9.7) (median 38, range 20-62 years) and HIV noninfected was 34.6 (12.8) (median 32, range 18–67 years). Intestinal parasites were detected in 53 (46.1%) out of 115 samples from HIV-infected patients, whereas only 16 (17.4%) parasites were found from 92 HIV negative samples. Among HIV-infected patients, majority 32.2% (37 out of 115) had infected with opportunistic protozoans (Coccidia and Microsporidia) and the rest 16 (13.9%) suffered from nonopportunistic protozoans and nematodes [Table 1]. From 92 HIV negative stool samples, 16 (17.4%) were positive for intestinal parasites and only 1 (1.1%) had revealed opportunistic protozoan infection. The association of opportunistic protozoans with HIV-infected patients had shown statistically significant P value (P < 0.01).

Table 1

Distribution of different intestinal parasites detected from HIV-infected and HIV-negative diarrheal patients

Among HIV-infected patients, I. belli 16.5% (19/115) was most commonly detected parasites followed by C. parvum 12.2%, E. histolytica/E. dispar 7.8%, Microsporidia (2.6%), S. stercoralis (2.6%), and 0.9% each of C. cayetanensis, A. duodenale, and G. lamblia, respectively. From 16 (17.4%) parasite positive stool samples of 92 HIV-negative patients, E. histolytica/E. dispar was the common parasite (8.7%), followed by A. lumbricoides (3.3%), A. duodenale (2.2%), and 1.1% each of E. vermicularis, T. trichiura, and C. parvum respectively [Table 1].

CD4+ T-cell count was performed in all 115 HIV-infected patients. The prevalence of opportunistic intestinal protozoans among HIV-infected patients with CD4 counts <200 cells/μl and ≥ 200 cells/μl were 61.9% (26/42) and 15.1% (11/73), respectively. The CD4+ T-cell count was significantly associated with an increased prevalence of opportunistic intestinal protozoans in HIV-infected diarrheal patients (P < 0.001) [Table 2]. The mean CD4+ T-cell count among HIV-infected opportunistic protozoans was 135.2 (80.7) cells/μl (median 112, range 37–389 cells/μl).

Table 2

Correlation of CD4+ T-cell counts with opportunistic intestinal protozoans among HIV-infected diarrheal patients

The HIV-seropositive subjects were grouped by 18-27, 28-37, 38-47, and ≥ 48 years. The percentage of HIV-infected study participants who were males with diarrhea was more (61.7%, 71/115) than that of females (38.3%, 44/115). Similarly, the occurrence of opportunistic protozoans was higher in ≥ 48 years of age group followed by 38-47 years [Table 3].

Table 3

Age and sex wise distribution of HIV-infected diarrheal patients

Discussion

HIV-infected individuals become susceptible to a variety of opportunistic parasite infections that occur with greater frequency and severity due to down regulation of immune system. Almost 80% of AIDS patients die from AIDS-related infections including intestinal parasites rather than HIV infection itself.[10] The Coccidian parasites (C. parvum, I. belli and C. cayetanensis) and Microsporidia (Enterocytozoon bieneusi and Encephalitozoon intestinalis) are the foremost among enteric parasites in these patients.[2] Gastrointestinal parasitic infections largely present with diarrhea leading to life threatening complications.[11]

In this present study, enteric parasites were detected in 46.1% of HIV-infected patients with diarrhea. Various studies from India and other countries have reported low, similar, or higher prevalence of intestinal parasites, ranging from 15.3% to 77.14%.[121314151617] The opportunistic protozoans (69.8%) predominated among all intestinal parasites with I. belli (51.3%) being the most commonly detected parasite followed by C. parvum (37.8%), Microsporidia (8.1%), and C. cayetanensis (2.8%). The overall high prevalence of 16.5% was shown by I. belli followed by C. parvum (12.2%), E. histolytica/dispar (7.8%), Microsporidia (2.6%), S. stercoralis (2.6%), A. lumbricoides (1.7%), and 0.9% each by G. lamblia, C. cayetanensis and A. duodenale in HIV-infected diarrheal patients. This present study differed from most of the studies within and outside India where they have found out C. parvum was the most commonly prevalent protozoan.[12131516171819202122232425262728] Few studies within India have reported I. belli was the predominant opportunistic intestinal protozoan.[1429] The reason for these large difference in prevalence of opportunistic intestinal infections that tend to vary from one locality to the other and from one country to the other depending on the level of contamination of water, foodstuff, and contact with animals, which are important factors in the dissemination of the parasites.[30] The variation in the prevalence of opportunistic intestinal protozoans in HIV diarrheal patients could also be related to the immune status of the HIV patients examined, the sensitivity of the diagnostic techniques used and the experience of the microscopist.[23]

HIV-seronegatives were predominantly infected by common intestinal parasites such as E. histolytica/E.dispar (8.7%) followed by A. lumbricoides (3.3%) and A. duodenale (2.2%). Single (1.1%) case of C. parvum was found in HIV noninfected diarrheal patient, which is in agreement with studies conducted by Ramakrishnan et al. and Mohandas et al. in India where they found out C. parvum in 3.7% and 2% cases, respectively.[518] Our study differed from the Gupta et al. study in India, the Akinbo et al. study in Nigeria, and the Alemu et al. study in Ethiopia where they did not find any opportunistic protozoans in control groups.[121423]

The findings of Coccidia and Microsporidia among HIV-infected patients indicate that opportunistic parasites readily cause infection in immune-deficient individuals. In this present study, HIV-infected patients with CD4+ T-cell count <200 cells/μl are at an increased risk of acquiring opportunistic infections, which is similar to all previous studies.[5121415] Indeed, an immune-deficient state has been reported by other authors as risk factor for opportunistic protozoan infections.

Majority, that is, 67.7% of opportunistic intestinal parasites were detected in males in comparison to females (32.3%). This may be due to males utilizing more integrated counseling and testing services. They usually go undiagnosed for long periods and present late in the course of disease with severe persistent infections and low CD4+ T-cell counts.[14]

There were few limitations in this present study. The study was done on a small sample size as majority of the patients were referred from general practitioners and from secondary care centers, therefore they have already received some amount of antiparasitics and antibiotics. Also the single stool specimen processing might underestimate the actual parasite prevalence. As the etiology of diarrhea could not be determined in 53.9% of HIV-infected patients and 82.6% of HIV noninfected patients, comprehensive etiological studies are needed to cover bacterial, fungal, viral, and noninfective causes of diarrhea among patients.

Conclusion

In conclusion, intestinal parasitic infections caused diarrhea in 46.1% of HIV-infected individuals. The opportunistic intestinal protozoans especially I. belli and C. parvum were most commonly isolated in HIV-infected patients with diarrhea. Majority of the infections occurred in patients when CD4+ T-cell counts were less than 200 cells/μl.