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Intestinal microsporidiosis in India: A two year study
Parasitology International 62 (2013) 53–56
Contents lists available at SciVerse ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Intestinal microsporidiosis in India: A two year study Karnika Saigal a, Aman Sharma b, Rakesh Sehgal c, Poonam Sharma c, Nancy Malla c, Sumeeta Khurana c,⁎ a b c
Dept. of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Dept. of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India Dept. of Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 17 September 2012 Accepted 18 September 2012 Available online 25 September 2012 Keywords: Microsporidiosis Gastrointestinal infection HIV seropositive Encephalitozoon intestinalis Enterocytozoon bieneusi CD4 count
a b s t r a c t Intestinal parasitic pathogens in HIV/AIDS patients include Cryptosporidium sp, Cystoisospora sp, microsporidia and less commonly other parasites. The two most common microsporidia causing intestinal infection are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Most of the Indian studies for intestinal parasitic infections in HIV/AIDS patients have not included microsporidia, due to difficult staining and identification of the parasite. The aim of the present study was to find the prevalence of intestinal microsporidiosis and their species identification along with correlation of CD4 count with parasite positivity and diarrhoea in HIV positive individuals. Stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various out-patient departments and in-patients admitted to a tertiary care hospital from August 2008 to October 2009. The stool samples were subjected to examination by wet mount, modified acid fast stain for coccidian parasites and multiplex nested PCR for microsporidia. The overall prevalence of all intestinal parasites among HIV patients in our study was 26.5%. The prevalence of intestinal parasitic pathogens in HIV positive patients with diarrhoea was 43.2%. Microsporidia were the most common parasites detected (14%) in all patients, while in HIV infected patients 15.9% patients had microsporidia infection. The most common species causing intestinal microsporidiosis in our study was E. intestinalis (10.5%). In HIV seropositive individuals with diarrhoea, E. intestinalis was 20.8% and E. bieneusi 8.0% while in HIV-seropositive individuals without diarrhoea, E. intestinalis was 3.8% and E. bieneusi 1.9%. E. intestinalis was present in 10.9% of HIV negative individuals with diarrhoea in whom E. bieneusi was not found. There was a significant association between CD4 count ≤200/μl and intestinal parasite positivity. Thus, it can be concluded that intestinal microsporidiosis is under reported but an important disease in India. The predominant species in our study is E. intestinalis , in contrast to other parts of the world where E. bieneusi is more common. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Opportunistic parasitic infections have gained prominence and they are a leading cause of high morbidity and mortality in HIV/AIDS and in immunosuppressed patients. Chronic diarrhoea is present in about 60% of HIV patients in developed countries and in 95% of those living in developing countries [1,2]. The most commonly reported intestinal parasitic pathogens include Cryptosporidium sp, Cystoisospora sp, microsporidia, and less commonly other parasites. However, very few studies conducted in India have focused on detecting microsporidia by special staining methods or molecular techniques. The two most common microsporidia causing gastrointestinal infection worldwide are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Staining techniques cannot differentiate between different genera of microsporidia and there is a vast difference in antimicrobial ⁎ Corresponding author at: Department of Parasitology, P.G.I.M.E.R., Chandigarh, Pin: 160012, India. Tel.: +91 172 2755165. E-mail address: [email protected] (S. Khurana). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2012.09.005
susceptibility among species of microsporidia causing gastrointestinal infections [1]. The aim of the present study was to assess the prevalence of intestinal parasites including microsporidia in both immunocompetent and immunocompromised individuals and to identify the species of microsporidia by nested PCR.
2. Materials and methods 2.1. Sample collection The stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various outpatient departments or patients admitted to Nehru Hospital, Post Graduate Institute of Medical Education and Research, Chandigarh from August 2008 to October 2009. A written informed consent was obtained from each patient and relevant history and CD4+ cell counts were recorded.
54
K. Saigal et al. / Parasitology International 62 (2013) 53–56
2.2. Methods Unconcentrated stool samples and those after formol ether concentration were subjected to wet mount examination and trichrome staining for demonstration of all intestinal parasites. The modified acid fast staining was done for coccidian parasites [3] and multiplex nested PCR was performed for microsporidia.
mean CD4+ count of HIV seropositive patients with diarrhoea was 228.7/μl and that of HIV seropositive patients without diarrhoea was 343.6/μl. CD4 counts of b 500/μl were found in 92.6% of HIV patients with diarrhoea and 74.8% of HIV patients without diarrhoea. Severely low CD4 counts (of less than 200/μl) were found in 53.6% of HIV patients with diarrhoea and 36.6% of patients without diarrhoea. 3.2. Intestinal parasite positivity
2.2.1. Molecular identification of microsporidia Genomic DNA was extracted from the stool samples using the QIAamp DNA Stool Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The extracted DNA was stored at − 20 °C. Microsporidia were detected by Multiplex Nested PCR using outer primers MSP-1 (TGA ATG GGT CCC TGT), MSP-2A (TCA CTC GCC GCT ACT) and MSP-2B (GTT CAT TCG CAC TAC T) and inner primers MSP-3 (GGA ATT CAC ACC GCC CGT CAT TAT), MSP-4A (CCA AGC TTA TGC TTA AGT CAA AAG GGT) and MSP-4B (CCA AGC TTA TGC TTA AGT CCA GGG AG) to amplify E. bieneusi and E. intestinalis DNA as reported in the literature [4,5]. 2.3. Ethical clearance The study was approved by the institutional ethics committee of Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Overall, parasites were detected in 100 of 363 (27.5%) faecal samples. The parasite distribution in various groups is presented in Table 1. There was a statistically significant difference in the presence of microsporidiosis (both E. intestinalis and E. bieneusi) in patients with diarrhoea 42/180 (23.3%) vis a vis those without diarrhoea 6/183 (3.3%) (pb 0.05) (Table 1, Fig. 1). 3.3. Age and sex In age groups b 14 years, 15–45 years, and >45 years; overall parasite positivity was 38%, 27% and 34% respectively but there was no significant relationship between intestinal parasite positivity and age or sex of the subject. 3.4. Presence of multiple infections
3. Results
A total of 7 subjects were having infection with ≥2 parasites. Co-infection with Giardia intestinalis, Hymenolepis nana and Cystoisospora belli was present in two patients—one was HIV seropositive with diarrhoea and the other was HIV seronegative with diarrhoea. There were another two cases in which co-infection with E. intestinalis and E. bieneusi was present, both of whom were HIV seropositive individuals although one suffered from diarrhoea and the other did not have diarrhoea. Coinfection of G. intestinalis with E. bieneusi was present in one HIV seropositive patient with diarrhoea.
3.1. Demographic details of study population
3.5. CD4 + count of subjects with intestinal parasites
A total of 363 subjects were included in the study. The mean age of the study population was 32.5 years. The male:female ratio was 1.6:1. A total number of 180 patients were affected with diarrhoea. The
CD4+ counts were available for only 262 HIV seropositive individuals, not all, thus the following observations pertain to these patients only. Among the 123 HIV seropositive individuals with diarrhoea, 51
2.4. Statistical analysis Chi square test and Student-t test were used. SPSS 15 software was used for analysis. Patients with diarrhoea were compared with those without diarrhoea, and comparisons were made between HIV infected and nonHIV infected patients.
Table 1 Intestinal parasite positivity in each group. Parasite
Microsporidia 1. Encephalitozoon intestinalis 2. Enterocytozoon bieneusi Cryptosporidium sp Cystoisospora belli Cyclospora cayetanensis Giardia intestinalis Hymenolepis nana Hookworm Strongyloides stercoralis Total
HIV seropositive with diarrhoea (n = 125)
HIV seropositive without diarrhoea (n = 158)
HIV seronegative with diarrhoea (n = 55)
Healthy controls (n = 25)
Total (% = total cases /subjects under study) (n = 363)
26 (20.8%) 10 (8%) 6 (4.8%) 3 (2.4%) 0
6 (3.8%) 3 (1.9%) 2 (1.3%) 4 (2.5%) 0
6 (10.9%) 0
0
0
8 (6.4%) 0
6 (3.8%) 0
1 (0.8%) 0
0
54 (43.2%)
21 (13.3%)
3 (5.5%) 2 (3.6%) 1 (1.8%) 7 (12.7%) 3 (5.5%) 1 (1.8%) 2 (3.6%) 25 (45.5%)
38 (10.5%) 13 (3.6%) 11 (3%) 9 (2.5%) 1 (0.3%) 21 (5.7%) 3 (0.8%) 2 (0.8%) 2 (0.6%) 100 (27.5%)
0
0
0 0 0 0 0 0 0
Note—All parasites were detected by wet mount examination except coccidian parasites, which were detected by modified Ziehl–Neelsen stain, and microsporidia, which were detected by multiplex nested polymerase chain reaction.
K. Saigal et al. / Parasitology International 62 (2013) 53–56
Fig. 1. Correlation of intestinal parasite positivity with diarrhoea in both HIV and nonHIV patients (*p b 0.05).
(41.5%) had infection with at least one of the intestinal parasites. Among these, 30 (58.8%) had CD4 + count ≤ 200/μl, 20 (39.2%) between 201 and 500/μl and only 1 (2%) had CD4 + count > 500/μl. The difference in the parasite positivity of patients with CD4 count ≤ 200/μl and > 501/μl was statistically significant (p b 0.05). Among the 139 HIV seropositive individuals without diarrhoea for whom CD4 counts were available, 21 (15%) were positive for at least one of the intestinal parasites. Among these, CD4+ counts were ≤200/μl in 11 (52.4%), between 201 and 500/μl in 7 (33.3%) and >500/μl in 3 (14.3%) subjects. There was a significant difference in intestinal parasite positivity in patients with CD4 count ≤200/μl or 201– 500/μl and those with CD4 count >501/μl (pb 0.05). However, there was no significant difference in parasite positivity in patients with CD4 counts b 200/μl and 201–500/μl. 4. Discussion In our study, the overall prevalence of intestinal parasites in HIV positive patients was 26.5% (75/283) and in HIV positive patients with diarrhoea, it was 43.2%. Our results are in concordance with reports from other parts of the world. Parasitic infections have been reported in 37.2%–69.2% of all the infectious causes of diarrhoea in HIV patients worldwide [3,6–11]. Microsporidia were the most common cause (14%) of intestinal parasitic infection in both HIV positive and negative patients in our study. Studies from other parts of the world have reported a prevalence of microsporidia infection of 5% to 50% [7–11]. A handful of studies from India have reported prevalence of 1.5 to 30% [12–16]. All these studies from India detected microsporidia by microscopy, which is much less sensitive than PCR and speciation is also not possible. The most common reported microsporidian associated with gastrointestinal infection is E. bieneusi. However, in our study E. intestinalis was much more common and E. bieneusi was conspicuously absent in HIV-negative patients with diarrhoea. We analysed the association of diarrhoea with intestinal parasite positivity and it was found that in patients with diarrhoea, the overall parasite positivity was 43.9% (79/180) while in patients without diarrhoea it was 13.3% excluding healthy controls. There is a lack of
55
demonstrated clear association between gastrointestinal tract microsporidia infection and diarrhoea, worldwide [17]. Microsporidia infections were significantly higher in patients with diarrhoea (23.3%) compared to those without diarrhoea (3.3%). The same difference was there in HIV positive patients with and without diarrhoea (28.8% vs 5.7%) thus supporting the role of microsporidia in HIV-associated diarrhoea. In HIV negative individuals with diarrhoea, intestinal microsporidia were present in 10.9% patients, second only to Giardia, thus highlighting the fact that microsporidia can cause diarrhoea even in HIV negative individuals. It is generally seen that in HIV/AIDS patients, when CD4 count drops to b 200/μl, clinical symptoms of watery, non-bloody diarrhoea, nausea, diffuse abdominal pain and fever appear or become more pronounced while, diarrhoea is self-limited in immunocompetent individuals or in those with CD4 counts >200/μl [18]. In our study, there was a significantly higher intestinal parasite positivity in patients with CD4 count b200/μl compared to those with CD4 count >500/μl (pb 0.05%). However, there was no significant difference in parasite positivity in patients with CD4 counts b 200/μl and those between 201 and 500/μl. The results of CD4 counts in our study are in concordance with other studies where decreasing parasite positivity has been reported as CD4 count of HIV/ AIDS patient increases [13]. In HIV/AIDS patients, treatment of cryptosporidiosis should accompany HAART therapy to decrease the incidence of parasitic infection and chances of diarrhoea. Intestinal microsporidiosis is a significant cause of diarrhoea in both HIV positive and HIV negative individuals. With increasing prevalence of HIV positive individuals, the clinical laboratories need to be equipped to diagnose microsporidiosis in patient samples. There is an urgent need to upgrade the knowledge base of microsporidia species prevalent in different geographical areas of India so that the diagnosis is not missed. References [1] Didier ES, Weiss LM. Microsporidiosis: current status. Current Opinion in Infectious Diseases 2006;19:485-92. [2] Knox TA, Spiegelman D, Skinner SC, Gorbach S. Diarrhoea and abnormalities of gastrointestinal function in a cohort of men and women with HIV infection. The American Journal of Gastroenterology 2000;95:3482-9. [3] Garcia C, Rodriguez E, Do N, Lopez de Castilla D, Terashima A, Gotuzzo E. Intestinal parasitosis in patients with HIV–AIDS. Revista de Gastroenterología del Perú 2006;26:21-4. [4] Katzwinkle-Wladarsch S, Lieb M, Helse W, Löscher T, Rinder H. Direct amplification and species determination of microsporidian DNA from stool specimens. Tropical Medicine & International Health 1996;1:373-8. [5] Verweij JJ, Ten Hove R, Brienen EA, van Lieshout L. Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR. Diagnostic Microbiology and Infectious Disease 2007;57:163-7. [6] Kaushik K, Khurana S, Wanchu A, Malla N. Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients. Acta Tropica 2008;107:1-7. [7] Mariam ZT, Abebe G, Mulu A. Opportunistic and other intestinal parasitic infections in AIDS patients, HIV seropositive healthy carriers and HIV seronegative individuals in southwest Ethiopia. East African Journal of Public Health 2008;5: 169-73. [8] Stark D, Fotedar R, van Hal S, Beebe N, Marriott D, Ellis JT, et al. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney, Australia. The American Journal of Tropical Medicine and Hygiene 2007;76:549-52. [9] Vignesh R, Balakrishnan P, Shankar EM, Murugavel KG, Hanas S, Cecelia AJ, et al. High proportion of isosporiasis among HIV-infected patients with diarrhoea in southern India. The American Journal of Tropical Medicine and Hygiene 2007;77: 823-4. [10] Wiwanitkit V. Intestinal parasitic infections in Thai HIV-infected patients with different immunity status. BMC Gastroenterology 2001;1:3. [11] Zali MR, Mehr AJ, Rezaian M, Meamar AR, Vaziri S, Mohraz M. Prevalence of intestinal parasitic pathogens among HIV-positive individuals in Iran. Japanese Journal of Infectious Diseases 2004;57:268-70. [12] Dwivedi KK, Prasad G, Saini S, Mahajan S, Lal S, Baveja UK. Enteric opportunistic parasites among HIV infected individuals: associated risk factors and immune status. Japanese Journal of Infectious Diseases 2007;60:76-81. [13] Kulkarni SV, Kairon R, Sane SS, Padmawar PS, Kale VA, Thakar MR, et al. Opportunistic parasitic infections in HIV/AIDS patients presenting with diarrhoea by the level of immunesuppression. The Indian Journal of Medical Research 2009;130:63-6. [14] Mohandas, Sehgal R, Sud A, Malla N. Prevalence of intestinal parasitic pathogens in HIV-seropositive individuals in Northern India. Japanese Journal of Infectious Diseases 2002;55:83-4.
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[15] Kumar SS, Ananthan S, Joyee AG. Detection of Enterocytozoon bieneusi (Microsporidia) by polymerase chain reaction (PCR) using species-specific primer in stool samples of HIV patients. The Indian Journal of Medical Research 2005;121:215-9. [16] Sadraei J, Rizvi MA, Baveja UK. Diarrhoea, CD4+ cell counts and opportunistic protozoa in Indian HIV-infected patients. Parasitology Research 2005;97:270-3.
[17] Rabeneck L, Gyorkey F, Genta RM, Gyorkey P, Foote LW, RN, et al. The role of microsporidia in the pathogenesis of HIV-related chronic diarrhoea. Annals of Internal Medicine 1993;119:895-9. [18] Smith PD, Janoff EN. Infectious diarrhoea in human immunodeficiency virus infection. Gastroenterology Clinics of North America 1988;17:587-98.
Contents lists available at SciVerse ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Intestinal microsporidiosis in India: A two year study Karnika Saigal a, Aman Sharma b, Rakesh Sehgal c, Poonam Sharma c, Nancy Malla c, Sumeeta Khurana c,⁎ a b c
Dept. of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Dept. of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India Dept. of Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 17 September 2012 Accepted 18 September 2012 Available online 25 September 2012 Keywords: Microsporidiosis Gastrointestinal infection HIV seropositive Encephalitozoon intestinalis Enterocytozoon bieneusi CD4 count
a b s t r a c t Intestinal parasitic pathogens in HIV/AIDS patients include Cryptosporidium sp, Cystoisospora sp, microsporidia and less commonly other parasites. The two most common microsporidia causing intestinal infection are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Most of the Indian studies for intestinal parasitic infections in HIV/AIDS patients have not included microsporidia, due to difficult staining and identification of the parasite. The aim of the present study was to find the prevalence of intestinal microsporidiosis and their species identification along with correlation of CD4 count with parasite positivity and diarrhoea in HIV positive individuals. Stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various out-patient departments and in-patients admitted to a tertiary care hospital from August 2008 to October 2009. The stool samples were subjected to examination by wet mount, modified acid fast stain for coccidian parasites and multiplex nested PCR for microsporidia. The overall prevalence of all intestinal parasites among HIV patients in our study was 26.5%. The prevalence of intestinal parasitic pathogens in HIV positive patients with diarrhoea was 43.2%. Microsporidia were the most common parasites detected (14%) in all patients, while in HIV infected patients 15.9% patients had microsporidia infection. The most common species causing intestinal microsporidiosis in our study was E. intestinalis (10.5%). In HIV seropositive individuals with diarrhoea, E. intestinalis was 20.8% and E. bieneusi 8.0% while in HIV-seropositive individuals without diarrhoea, E. intestinalis was 3.8% and E. bieneusi 1.9%. E. intestinalis was present in 10.9% of HIV negative individuals with diarrhoea in whom E. bieneusi was not found. There was a significant association between CD4 count ≤200/μl and intestinal parasite positivity. Thus, it can be concluded that intestinal microsporidiosis is under reported but an important disease in India. The predominant species in our study is E. intestinalis , in contrast to other parts of the world where E. bieneusi is more common. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Opportunistic parasitic infections have gained prominence and they are a leading cause of high morbidity and mortality in HIV/AIDS and in immunosuppressed patients. Chronic diarrhoea is present in about 60% of HIV patients in developed countries and in 95% of those living in developing countries [1,2]. The most commonly reported intestinal parasitic pathogens include Cryptosporidium sp, Cystoisospora sp, microsporidia, and less commonly other parasites. However, very few studies conducted in India have focused on detecting microsporidia by special staining methods or molecular techniques. The two most common microsporidia causing gastrointestinal infection worldwide are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Staining techniques cannot differentiate between different genera of microsporidia and there is a vast difference in antimicrobial ⁎ Corresponding author at: Department of Parasitology, P.G.I.M.E.R., Chandigarh, Pin: 160012, India. Tel.: +91 172 2755165. E-mail address: [email protected] (S. Khurana). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2012.09.005
susceptibility among species of microsporidia causing gastrointestinal infections [1]. The aim of the present study was to assess the prevalence of intestinal parasites including microsporidia in both immunocompetent and immunocompromised individuals and to identify the species of microsporidia by nested PCR.
2. Materials and methods 2.1. Sample collection The stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various outpatient departments or patients admitted to Nehru Hospital, Post Graduate Institute of Medical Education and Research, Chandigarh from August 2008 to October 2009. A written informed consent was obtained from each patient and relevant history and CD4+ cell counts were recorded.
54
K. Saigal et al. / Parasitology International 62 (2013) 53–56
2.2. Methods Unconcentrated stool samples and those after formol ether concentration were subjected to wet mount examination and trichrome staining for demonstration of all intestinal parasites. The modified acid fast staining was done for coccidian parasites [3] and multiplex nested PCR was performed for microsporidia.
mean CD4+ count of HIV seropositive patients with diarrhoea was 228.7/μl and that of HIV seropositive patients without diarrhoea was 343.6/μl. CD4 counts of b 500/μl were found in 92.6% of HIV patients with diarrhoea and 74.8% of HIV patients without diarrhoea. Severely low CD4 counts (of less than 200/μl) were found in 53.6% of HIV patients with diarrhoea and 36.6% of patients without diarrhoea. 3.2. Intestinal parasite positivity
2.2.1. Molecular identification of microsporidia Genomic DNA was extracted from the stool samples using the QIAamp DNA Stool Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The extracted DNA was stored at − 20 °C. Microsporidia were detected by Multiplex Nested PCR using outer primers MSP-1 (TGA ATG GGT CCC TGT), MSP-2A (TCA CTC GCC GCT ACT) and MSP-2B (GTT CAT TCG CAC TAC T) and inner primers MSP-3 (GGA ATT CAC ACC GCC CGT CAT TAT), MSP-4A (CCA AGC TTA TGC TTA AGT CAA AAG GGT) and MSP-4B (CCA AGC TTA TGC TTA AGT CCA GGG AG) to amplify E. bieneusi and E. intestinalis DNA as reported in the literature [4,5]. 2.3. Ethical clearance The study was approved by the institutional ethics committee of Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Overall, parasites were detected in 100 of 363 (27.5%) faecal samples. The parasite distribution in various groups is presented in Table 1. There was a statistically significant difference in the presence of microsporidiosis (both E. intestinalis and E. bieneusi) in patients with diarrhoea 42/180 (23.3%) vis a vis those without diarrhoea 6/183 (3.3%) (pb 0.05) (Table 1, Fig. 1). 3.3. Age and sex In age groups b 14 years, 15–45 years, and >45 years; overall parasite positivity was 38%, 27% and 34% respectively but there was no significant relationship between intestinal parasite positivity and age or sex of the subject. 3.4. Presence of multiple infections
3. Results
A total of 7 subjects were having infection with ≥2 parasites. Co-infection with Giardia intestinalis, Hymenolepis nana and Cystoisospora belli was present in two patients—one was HIV seropositive with diarrhoea and the other was HIV seronegative with diarrhoea. There were another two cases in which co-infection with E. intestinalis and E. bieneusi was present, both of whom were HIV seropositive individuals although one suffered from diarrhoea and the other did not have diarrhoea. Coinfection of G. intestinalis with E. bieneusi was present in one HIV seropositive patient with diarrhoea.
3.1. Demographic details of study population
3.5. CD4 + count of subjects with intestinal parasites
A total of 363 subjects were included in the study. The mean age of the study population was 32.5 years. The male:female ratio was 1.6:1. A total number of 180 patients were affected with diarrhoea. The
CD4+ counts were available for only 262 HIV seropositive individuals, not all, thus the following observations pertain to these patients only. Among the 123 HIV seropositive individuals with diarrhoea, 51
2.4. Statistical analysis Chi square test and Student-t test were used. SPSS 15 software was used for analysis. Patients with diarrhoea were compared with those without diarrhoea, and comparisons were made between HIV infected and nonHIV infected patients.
Table 1 Intestinal parasite positivity in each group. Parasite
Microsporidia 1. Encephalitozoon intestinalis 2. Enterocytozoon bieneusi Cryptosporidium sp Cystoisospora belli Cyclospora cayetanensis Giardia intestinalis Hymenolepis nana Hookworm Strongyloides stercoralis Total
HIV seropositive with diarrhoea (n = 125)
HIV seropositive without diarrhoea (n = 158)
HIV seronegative with diarrhoea (n = 55)
Healthy controls (n = 25)
Total (% = total cases /subjects under study) (n = 363)
26 (20.8%) 10 (8%) 6 (4.8%) 3 (2.4%) 0
6 (3.8%) 3 (1.9%) 2 (1.3%) 4 (2.5%) 0
6 (10.9%) 0
0
0
8 (6.4%) 0
6 (3.8%) 0
1 (0.8%) 0
0
54 (43.2%)
21 (13.3%)
3 (5.5%) 2 (3.6%) 1 (1.8%) 7 (12.7%) 3 (5.5%) 1 (1.8%) 2 (3.6%) 25 (45.5%)
38 (10.5%) 13 (3.6%) 11 (3%) 9 (2.5%) 1 (0.3%) 21 (5.7%) 3 (0.8%) 2 (0.8%) 2 (0.6%) 100 (27.5%)
0
0
0 0 0 0 0 0 0
Note—All parasites were detected by wet mount examination except coccidian parasites, which were detected by modified Ziehl–Neelsen stain, and microsporidia, which were detected by multiplex nested polymerase chain reaction.
K. Saigal et al. / Parasitology International 62 (2013) 53–56
Fig. 1. Correlation of intestinal parasite positivity with diarrhoea in both HIV and nonHIV patients (*p b 0.05).
(41.5%) had infection with at least one of the intestinal parasites. Among these, 30 (58.8%) had CD4 + count ≤ 200/μl, 20 (39.2%) between 201 and 500/μl and only 1 (2%) had CD4 + count > 500/μl. The difference in the parasite positivity of patients with CD4 count ≤ 200/μl and > 501/μl was statistically significant (p b 0.05). Among the 139 HIV seropositive individuals without diarrhoea for whom CD4 counts were available, 21 (15%) were positive for at least one of the intestinal parasites. Among these, CD4+ counts were ≤200/μl in 11 (52.4%), between 201 and 500/μl in 7 (33.3%) and >500/μl in 3 (14.3%) subjects. There was a significant difference in intestinal parasite positivity in patients with CD4 count ≤200/μl or 201– 500/μl and those with CD4 count >501/μl (pb 0.05). However, there was no significant difference in parasite positivity in patients with CD4 counts b 200/μl and 201–500/μl. 4. Discussion In our study, the overall prevalence of intestinal parasites in HIV positive patients was 26.5% (75/283) and in HIV positive patients with diarrhoea, it was 43.2%. Our results are in concordance with reports from other parts of the world. Parasitic infections have been reported in 37.2%–69.2% of all the infectious causes of diarrhoea in HIV patients worldwide [3,6–11]. Microsporidia were the most common cause (14%) of intestinal parasitic infection in both HIV positive and negative patients in our study. Studies from other parts of the world have reported a prevalence of microsporidia infection of 5% to 50% [7–11]. A handful of studies from India have reported prevalence of 1.5 to 30% [12–16]. All these studies from India detected microsporidia by microscopy, which is much less sensitive than PCR and speciation is also not possible. The most common reported microsporidian associated with gastrointestinal infection is E. bieneusi. However, in our study E. intestinalis was much more common and E. bieneusi was conspicuously absent in HIV-negative patients with diarrhoea. We analysed the association of diarrhoea with intestinal parasite positivity and it was found that in patients with diarrhoea, the overall parasite positivity was 43.9% (79/180) while in patients without diarrhoea it was 13.3% excluding healthy controls. There is a lack of
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demonstrated clear association between gastrointestinal tract microsporidia infection and diarrhoea, worldwide [17]. Microsporidia infections were significantly higher in patients with diarrhoea (23.3%) compared to those without diarrhoea (3.3%). The same difference was there in HIV positive patients with and without diarrhoea (28.8% vs 5.7%) thus supporting the role of microsporidia in HIV-associated diarrhoea. In HIV negative individuals with diarrhoea, intestinal microsporidia were present in 10.9% patients, second only to Giardia, thus highlighting the fact that microsporidia can cause diarrhoea even in HIV negative individuals. It is generally seen that in HIV/AIDS patients, when CD4 count drops to b 200/μl, clinical symptoms of watery, non-bloody diarrhoea, nausea, diffuse abdominal pain and fever appear or become more pronounced while, diarrhoea is self-limited in immunocompetent individuals or in those with CD4 counts >200/μl [18]. In our study, there was a significantly higher intestinal parasite positivity in patients with CD4 count b200/μl compared to those with CD4 count >500/μl (pb 0.05%). However, there was no significant difference in parasite positivity in patients with CD4 counts b 200/μl and those between 201 and 500/μl. The results of CD4 counts in our study are in concordance with other studies where decreasing parasite positivity has been reported as CD4 count of HIV/ AIDS patient increases [13]. In HIV/AIDS patients, treatment of cryptosporidiosis should accompany HAART therapy to decrease the incidence of parasitic infection and chances of diarrhoea. Intestinal microsporidiosis is a significant cause of diarrhoea in both HIV positive and HIV negative individuals. With increasing prevalence of HIV positive individuals, the clinical laboratories need to be equipped to diagnose microsporidiosis in patient samples. There is an urgent need to upgrade the knowledge base of microsporidia species prevalent in different geographical areas of India so that the diagnosis is not missed. References [1] Didier ES, Weiss LM. Microsporidiosis: current status. Current Opinion in Infectious Diseases 2006;19:485-92. [2] Knox TA, Spiegelman D, Skinner SC, Gorbach S. Diarrhoea and abnormalities of gastrointestinal function in a cohort of men and women with HIV infection. The American Journal of Gastroenterology 2000;95:3482-9. [3] Garcia C, Rodriguez E, Do N, Lopez de Castilla D, Terashima A, Gotuzzo E. Intestinal parasitosis in patients with HIV–AIDS. Revista de Gastroenterología del Perú 2006;26:21-4. [4] Katzwinkle-Wladarsch S, Lieb M, Helse W, Löscher T, Rinder H. Direct amplification and species determination of microsporidian DNA from stool specimens. Tropical Medicine & International Health 1996;1:373-8. [5] Verweij JJ, Ten Hove R, Brienen EA, van Lieshout L. Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR. Diagnostic Microbiology and Infectious Disease 2007;57:163-7. [6] Kaushik K, Khurana S, Wanchu A, Malla N. Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients. Acta Tropica 2008;107:1-7. [7] Mariam ZT, Abebe G, Mulu A. Opportunistic and other intestinal parasitic infections in AIDS patients, HIV seropositive healthy carriers and HIV seronegative individuals in southwest Ethiopia. East African Journal of Public Health 2008;5: 169-73. [8] Stark D, Fotedar R, van Hal S, Beebe N, Marriott D, Ellis JT, et al. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney, Australia. The American Journal of Tropical Medicine and Hygiene 2007;76:549-52. [9] Vignesh R, Balakrishnan P, Shankar EM, Murugavel KG, Hanas S, Cecelia AJ, et al. High proportion of isosporiasis among HIV-infected patients with diarrhoea in southern India. The American Journal of Tropical Medicine and Hygiene 2007;77: 823-4. [10] Wiwanitkit V. Intestinal parasitic infections in Thai HIV-infected patients with different immunity status. BMC Gastroenterology 2001;1:3. [11] Zali MR, Mehr AJ, Rezaian M, Meamar AR, Vaziri S, Mohraz M. Prevalence of intestinal parasitic pathogens among HIV-positive individuals in Iran. Japanese Journal of Infectious Diseases 2004;57:268-70. [12] Dwivedi KK, Prasad G, Saini S, Mahajan S, Lal S, Baveja UK. Enteric opportunistic parasites among HIV infected individuals: associated risk factors and immune status. Japanese Journal of Infectious Diseases 2007;60:76-81. [13] Kulkarni SV, Kairon R, Sane SS, Padmawar PS, Kale VA, Thakar MR, et al. Opportunistic parasitic infections in HIV/AIDS patients presenting with diarrhoea by the level of immunesuppression. The Indian Journal of Medical Research 2009;130:63-6. [14] Mohandas, Sehgal R, Sud A, Malla N. Prevalence of intestinal parasitic pathogens in HIV-seropositive individuals in Northern India. Japanese Journal of Infectious Diseases 2002;55:83-4.
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