Pakistan Journal of Biological Sciences

Year: 2009 | Volume: 12 | Issue: 18 | Page No.: 1277-1281
DOI: 10.3923/pjbs.2009.1277.1281

Prevalence of Intestinal Parasites and Profile of CD₄⁺ Counts in HIV⁺/AIDS People in North of Iran, 2007-2008

A. Daryani, M. Sharif, M. Meigouni, F. Baba Mahmoudi, A. Rafiei, Sh. Gholami, A. Khalilian, Sh. Gohardehi and A.M. Mirabi

Abstract: In this study 142 stool samples (64 HIV⁺/AIDS patients and 78 non-HIV infected individuals) collected from Mazandaran province and screened for intestinal parasites, using direct wet mont, formalin-ether sedimentation concentration, modified Ziehl Neelsen and modified trichrome techniques. Each person in this study was examined for CD4⁺ counts. Intestinal parasites were found in 11/64 (17.2%) of patients in HIV⁺/AIDS group and in 14/78 (17.9%) of controls. Prevalence of parasites detected in HIV⁺/AIDS individuals was as follow: Cryptosporidium sp. 9.4%, Giardia lamblia 3.1%, Entamoeba coli 1.6%, E. histolytica 1.6% and Chilomastix mesnili 1.6%. Prevalence of parasites in controls was as follow: Trichostrongylus sp. 6.4%, G. lamblia 3.8%, Cryptosporidium sp. 2.5% , E. coli 2.5% , E. histolytica 1.2% , Hookworms 1.2%. The mean of CD4⁺ counts in HIV-positive group (430 cells μL^-1) was remarkedly less than controls (871 cells μL^-1) (p = 0.00l). As patients usually belong to poor socio-economic backgrounds and they can hardly afford treatment, therefore, it is suggested screening and free treatment of intestinal parasites in these individuals should be taken by health centers to prevent the occurrence of these diseases in HIV⁺/AIDS patients, as often the disease may take a fulminant form.

Keywords: Intestinal parasites, profile of CD4+, HIV+/AIDS and Iran

INTRODUCTION

Different infections in Human Immunodeficiency Virus (HIV) positive/AIDS people reduce their quality of life and life span (Kurniawan et al., 2009). Among them intestinal parasitic infections cause morbidity and mortality in HIV positive individuals (Mukhopadya et al., 1999). In these patients, progressive decline in their immunological responses make them extremely susceptible to a variety of common and opportunistic infections (Mukhopadya et al., 1999; Cegielski et al., 1999), protozoan parasites, namely Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, Microsporidia, E. histolytica/dispar and G. lamblia account for a significant number of cases of diarrhoea in this population (Mohandas et al., 2002). In AIDS patients, opportunistic parasitic gut infections cause sever diarrhoea, profoundly compromise the absorptive function of the small intestine and cause significant mortality (Castello-Branco et al., 1996). With the impaired immunity especially in the patients with low immune level (CD₄⁺<200 cells μL^-1) infestation with intestinal organisms resulting in diarrhoeal symptom is commonly seen (Wiwanikit, 2001). In two surveys performed in Iran, prevalences of intestinal parasites in HIV/AIDS patients have been reported 11.4 and 18.4% (Meamar et al., 2007; Zali et al., 2004). Because of important of intestinal parasites in HIV⁺/AIDS patients and also there is only few studies regarding the prevalence of intestinal parasites and their association with CD₄⁺ in HIV-infected patients are available from Iran at the present. We took up this study in North of Iran to evaluate the prevalence of such infections in HIV/AIDS patients and to emphasize the importance of stool examination for the detection of intestinal parasites.

MATERIALS AND METHODS

This cross-sectional study was conducted in Northern Iran between September 2007 and October 2008. This study was previously assessed and approved by the Ethics Committee at the Mazandaran University of Medical Sciences.

Patients and control group selection: There were two groups, one composed of 64 HIV⁺ infected patients detected by ELISA and confirmed by western blot, the other composed of 78 individuals without clinical signs of HIV infection and AIDS. In case group, 49 (76.5%) were male and 15 (23.5%) were female. In control group, 58 (74.4%) were male and 20 (25.6%) were female. There wasn’t significant difference in two groups (p>0.05; Table 1). The mean age of HIV⁺/AIDS group was 33.5 years (range: 17-58, SD: 7.44) and in the other group, the mean age of controls was 32.7 years (range: 12-62, SD: 9.48). There was no statistically significant difference by age in the HIV⁺/AIDS and control groups (p>0.05; Table 1). All of these people gave their consent to participate in this investigation.

Data collection and stool and blood examinations: All individuals in this study were interviewed by using a questionnaire to collect sociodemographic and clinical data. Stool samples were collected in 10% buffered formalin and SAF (sodium acetate acetic formaldehyde) in two sterile plastic containers and were transported to research laboratory of Mazandaran University of Medical Sciences. Stool speciments were collected and examined microscopically following direct and formalin-ether concentration methods (Garcia and Bruckner, 1993). In brief, stool samples were collected in labeled, leak-proof and clean plastic stool cups and brought to the laboratory. Direct microscopy of smears was performed for the detection of ova, larvae, trophozoites and cysts of intestinal parasites. Furthermore, a concentration procedure was employed that involved mixing the stool samples with formalin, treating with ether and centrifuging. The layers of ether, formalin and debris were discarded and the residues were used to investigate for the presence of intestinal parasites (Garcia and Bruckner, 1993). Detection of Cryptosporidium oocysts in the concentrated stool was done using the modified cold Ziehl Neelsen staining technique (Smith, 1995).

Briefly, a concentrated smear of the stool was made on a clean grease- free slide and fixed in methanol for 3 min. The slide was immersed in cold carbol fuchsin and stained for 15 min. It was then thoroughly rinsed in tap water and decolorized in 1% HCL in methanol for 10-15 min. After rising again in tap water, the slide was counter stained with 0.4% malachite green for 30 sec. The slide was then air-dried and observed under the compound light microscope using 40x objective lens for the presence of Cryptosporidium oocysts, which was confirmed under the oil- immersion objectives, as small pink to red spherules on pale green background (Yemisi et al., 2007). Modified trichrome stain was used for detection of Microsporidia spores (Garcia and Bruckner, 1993). Five milliliters of EDTA from each subjects was collected to count WBC, Eosinophil and CD₄⁺. Determination of CD4⁺ counts was performed by flow cytometry that include of whole blood samples collected in EDTA from 78 normal and 64 HIV⁺/AIDS individuals. Immunophenotyping of the lymphocyte subsets was done by using three color flow cytometric methods (partech, Germany). The blood samples were stained by using FITC conjugated mouse anti- human CD₄ (clone MT 310) and mouse anti-human CD₃ (clone UCHTI) conjugated with RPE.CYS monoclonal antibodies. All, reagents were purchased from Dakocytomation (Denmark).

Statistical analysis: Comparisons were made between the HIV⁺/AIDS and control group. Differences were compared by χ², fisher exact tests and student t-test by using SPSS. 15.00. The p<0.05 was considered significant.

RESULTS AND DISCUSSION

A total of 142 of individuals were enrolled in the study. Sixty four HIV⁺/AIDS patients (case group) and 78 without clinical signs (control group) were examined for intestinal parasitic infections. Among the 64 HIV⁺/AIDS patients, intestinal parasites were detected in 11 cases (17.2%); Cryptosporidium sp. (9.4%) and G. lamblia (3.1%) were the most common parasites. Among 78 controls analyzed, Trichostrongylus sp. (6.4%), G. lamblia (3.8%), Cryptosporidium sp. (2.5%) and E. coli (2.5%) were the most common parasites. Intestinal helminthes have been only detected in control group. Prevalence of intestinal parasites detected was shown in Table 2.

There was no significant difference in prevalence of intestinal parasite infections between HIV⁺/AIDS and control groups (p>0.05). Prevalence of intestinal parasites detected in different ages has been shown in Table 3. There was no significant difference between intestinal infection in different age groups between HIV⁺/AIDS and control groups (p>0.05). CD₄⁺ T- cell counts were done in all HIV-positives and controls. The CD₄⁺ counts were remarkedly depressed in all HIV-positive patients. The mean of CD₄⁺ counts in HIV⁺/AIDS was 430 cells μL^-1 and in control group was 871 cells μL^-1 (p = 0.001). Although, some other studies evaluate association between AIDS (with diarrhea) and CD₄ in patients, in our study most of patients suffering from constipation due to drug abusing, with the exception of the most individuals infected with Cryptosporidium had diarrhoea and the CD₄⁺ counts in these patients were significantly lower than control group (p>0.05). The mean of CD₄⁺ counts for each parasite, in HIV⁺/AIDS patients and control individuals infected with intestinal parasite have been shown in Table 4.

Opportunistic infections constitute a major health problem in patients infected with HIV. Among these, intestinal parasitic diseases are the commonest and a major cause of morbidity and mortality in HIV positive individuals worldwide (Chaisson et al., 1998). The coccidian parasites (Cryptosporidium sp., Isospora belli, Cyclospora sp. and Microsporidium sp.) are foremost among the enteric parasites in these patients (Smith et al., 1988). Almost all of the HIV-positive patients enrolled in this study were adults, a predominance of them were male (76.5%) and most of them were intravenous drug abusers.

In our study, prevalence of intestinal parasites in HIV⁺/AIDS patients was 17.2%. In Iran, Zali et al. (2004) also have been showed same prevalence rate, 18.4%. Of course Meamar et al. (2007) have been reported prevalence rate of 11.4 and 11.6% in HIV⁺/AIDS and healthy people, respectively. In other countries, its prevalence rates have been reported 30-84.3% (Mohandas et al., 2002; Cimerman et al., 1999; Wiwanikit, 2001; Ibrahim et al., 2007; Kurniawan et al., 2009). Different results show that there is no special pattern of occurance of parasites among HIV⁺/AIDS patients in different countries, even in different regions in the same country.

Such as our survey, Meamar et al. (2007) have been showed that there was no significant difference in prevalence of intestinal parasites between HIV⁺/AIDS and control groups. The similarity in frequencies of extra cellular intestinal parasites in both groups, could be explained by the fact that control Th₂ CD₄⁺ lymphocytes, which are highly necessary to protect the host against such parasites, remain less affected than Th₁ in HIV seropositive patients (ZU et al., 1992).

In a study performed in Iran, prevalence of Cryptosporidium was 0.9% in HIV⁺ individuals (Meamar et al., 2007) and in other previous study, its prevalence in HIV-positive patients was 1.5% (Zali et al., 2004). Other studies show that in Brazil 7% (Cimerman et al., 1999), Nigeria 16.83% (Ibrahim et al., 2007) and in other study in HIV⁺ individuals with diarrhoea in the same country 52.7% (Yemisi et al., 2007) infected with Cryptosporidium. Prevalence of Cryptosporidium in HIV⁺ patients in Indonesia, Cuba and India has been reported 4.9, 11.9 and 23.6%, respectively (Kurniawan et al., 2009; Escobedo and Nunez, 1999; Gupta et al., 2008). In other study in India, in 39.8% of HIV positive cases Cryptosporidium was detected (Tuli et al., 2008). In this study although the prevalence of Cryptosporidium sp. in HIV⁺/AIDS (9.4%) was more than control group (2.5%), there was no significant difference between them. Some other studies have been shown Cryptosporidium as the predominant pathogen with significant association to diarrhoea cases (Tarimo et al., 1996). Since, most of HIV⁺/AIDS individuals in our survey were without diarrhoea, therefore these carrier hosts is not diagnosed and cured and this parasite may be a cofactor for improvement of HIV to AIDS.

Giardia lamblia, in comparison with intestinal coccidian is not considered as an opportunistic agent and less frequently observed to cause sever illness in HIV⁺/AIDS patients (Gupta et al., 2008). In the present study, G. lamblia was the second most prevalent parasite detected in both groups. Meamar et al. (2007) also have been reported similar finding in HIV⁺ and HIV¯ individuals in Iran. In other study in Iran, this rate in HIV⁺ patients has been reported between 1-7.3% (Zali et al., 2004). Prevalence of Giardia in HIV-positive patients in other countries was as follow: 1.9% in Indonesia (Kurniawan et al., 2009), 1.98% in Nigeria (Ibrahim et al., 2007), 3.8% in Ethiopia (Hailmeriam et al., 2004), 6% in Cuba (Escobedo and Nunez, 1999), 11.8% in India (Gupta et al., 2008) and 16% in Brazil (Cimerman et al., 1999). Our findings in this study show giardiasis doesn’t occur in greater prevalence in HIV-positive than control group individuals.

No case of Microsporidium was found in our study. This may attribute to the difficulty these organisms in fecal specimens by conventional staining techniques due to their small size (1-3 μL) (Brandonoisio et al., 1999) and partly due to different risk factors for HIV infection among this Iranian population, which included more intravenous drug abusers than homosexuals (Zali et al., 2004).

Although, according to CDC the coccidian parasites are considered as opportunistic pathogens in AIDS patients, due to the patients usually belong to poor socio-economic backgrounds and they can hardly afford treatment, therefore, it is suggested screening and free treatment of intestinal parasites in these individuals should be taken by health centers to prevent the occurrence of these diseases in HIV⁺/AIDS patients, as often the disease may take a fulminant form.

ACKNOWLEDGMENTS

This study was supported by the grant No. 86-115 from Deputy of Research, Mazandaran University of Medical Sciences, Sari, Iran. We wish to thank Ali Jafari Balalami and Resa Bastani for their kind cooperation in this survey.

REFERENCES