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 ~  Abstract
 ~ Introduction
 ~ Patients and Methods
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~ Acknowledgement
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 1  |  Page : 60-66
 

Identification of opportunistic enteric parasites among immunocompetent patients with diarrhoea from Northern India and genetic characterisation of Cryptosporidium and Microsporidia


1 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission18-Nov-2014
Date of Acceptance20-May-2015
Date of Web Publication15-Jan-2016

Correspondence Address:
U Ghoshal
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
India
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Source of Support: DBT, New Delhi and ICMR, New Delhi, Conflict of Interest: None


DOI: 10.4103/0255-0857.174114

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 ~ Abstract 

Purpose: Enteric parasitic infestation is a major public health problem in developing countries. Parasites such as Cryptosporidium spp., Cyclospora spp., Cystoisospora spp. and Microsporidia may cause severe diarrhoea among immunocompromised patients. There is scanty data on their frequency among immunocompetent patients. Accordingly, we studied the frequency of enteric opportunistic parasites among immunocompetent patients with diarrhoea from northern India; we also performed genetic characterisation of Cryptosporidia and Microsporidia among them. Patients and Methods: Stool samples from 80 immunocompetent patients with diarrhoea, and 110 healthy controls were examined. Parasites were detected by direct microscopy, modified acid-fast (Kinyoun's) and modified trichrome stain. Polymerase chain reaction – restriction fragment length polymorphism was used for genetic characterisation of selected species such as Cryptosporidia and Microsporidia. Results: Enteric parasites were detected in 16/80 (20%) patients (mean age 28.8 ± 20 years, 45, 56% males) and in 2/110 (1.8%) healthy controls (P = 0.00007). Parasites detected were Cryptosporidium spp. (8/16, 50.0%), Cystoisospora spp. (4/16, 25%), Microsporidia (1/16, 6.25%), Cyclospora spp. (1/16, 6.25%) and Giardia spp. (1/16, 6.25%). One patient had mixed infection with Cystoisospora spp. and Giardia spp. The species of Cryptosporidia and Microsporidia detected were Cryptosporidium hominis and Enterocytozoon bieneusi, respectively. Parasites were more often detected in younger patients (≤20 years of age) than in older. Most of the parasite infected patients presented with chronic diarrhoea. Conclusion: Opportunistic enteric parasitic infestation was more common among immunocompetent patients with diarrhoea than healthy subjects. Special staining as well as molecular methods are essential for appropriate diagnosis of these parasites.


Keywords: Coccidian parasites, diarrhoea, immunocompetent, Microsporidia


How to cite this article:
Ghoshal U, Dey A, Ranjan P, Khanduja S, Agarwal V, Ghoshal U C. Identification of opportunistic enteric parasites among immunocompetent patients with diarrhoea from Northern India and genetic characterisation of Cryptosporidium and Microsporidia. Indian J Med Microbiol 2016;34:60-6

How to cite this URL:
Ghoshal U, Dey A, Ranjan P, Khanduja S, Agarwal V, Ghoshal U C. Identification of opportunistic enteric parasites among immunocompetent patients with diarrhoea from Northern India and genetic characterisation of Cryptosporidium and Microsporidia. Indian J Med Microbiol [serial online] 2016 [cited 2018 Jan 21];34:60-6. Available from: http://www.ijmm.org/text.asp?2016/34/1/60/174114



 ~ Introduction Top


Opportunistic enteric protozoan parasites such as Cryptosporidium spp., Cystoisospora spp., and Microsporidia expected to be common in tropical countries including India,[1],[2],[3] may cause severe diarrhoea, morbidity and even mortality among immunocompromised patients.[2],[4],[5],[6],[7],[8],[9],[10],[11] However, infection with these parasites is usually asymptomatic among immunocompetent subjects.

Most of the human infections are caused by two species of Cryptosporidia, namely Cryptosporidium hominis and Cryptosporidium parvum.[4],[12] However, six other species of this protozoa (Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium suis, Cryptosporidium muris and Cryptosporidium andersoni), which commonly infect specific species of animals, may occasionally spread to human beings.[5],[6],[7],[13],[14],[15] Identification of Cryptosporidium spp. by genetic characterisation among humans, particularly immunocompetent hosts, may help in understanding the mode of spread of this parasite. Since in presence of profound immune-suppression, even the most non-pathogenic species can cause infection, spectrum in immunocompromised subjects may not reflect pattern of zoonotic spread in the community. Knowledge about zoonotic spread may be of public health importance in preventing transmission of such parasitic infections from animals. Moreover, differential response of different species of Microsporidia, to its treatment with albendazole necessitates knowledge about the species infecting humans in different clinical settings.[16] Genetic characterisation of Cryptosporidium spp. and Microsporidia have been reported mostly in patients with human immunodeficiency virus infection in India.[17],[18],[19],[20],[21] However, data on genetic characterisation of Cryptosporidium spp. and Microsporidia among immunocompetent patients with diarrhoea are scanty, particularly from India.

Since existing data on genetic characterisation on Cystoisospora spp. and Cyclospora spp. suggest that these are quite homogeneous genetically even in different parts of the world,[22],[23] we thought it is not worthwhile studying their genetic variation in this study.

Accordingly, we undertook a study with the following aims: (a) To investigate the frequency of opportunistic parasites in immunocompetent patients with diarrhoea and (b) to genetically characterise the Cryptosporidium spp. and Microsporidia among them.


 ~ Patients and Methods Top


Eighty immunocompetent patients with diarrhoea, referred to parasitology laboratory from the Department of Gastroenterology and the General Hospital of a teaching Institution were included in this study from December 2008 to December 2012. Exclusion criteria included prior renal transplantation, ongoing immune-suppressive treatment, haematological malignancy and positive serological test for Human immunodeficiency virus (HIV) infection. 110 healthy subjects without diarrhoea were similarly included as controls. Data on demographic, clinical and laboratory parameters were recorded in a standard questionnaire for each patient and control. Diarrhoea was defined as more than three loose stools per day and was further categorised according to duration as follows, acute: <2 weeks; persistent: between 2 and 4 weeks; chronic: more than 4 weeks.[24],[25],[26] The study protocol was approved by Institutional Ethics Committee (Ref. nos.: EP/EC/37/20/3/2007, PGI/DIR/RC/1085/2007).

Sample collection

Three consecutive stool samples from each patient and control were subjected to microscopy and bacterial culture, as soon as possible. A part of it was stored at −40°C in normal saline for extraction of total deoxyribonucleic acid (DNA) and subsequent polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to genetically characterise the Cryptosporidium spp. and Microsporidia.

Sample processing

Stool samples were subjected to saline and iodine direct wet mount technique for detection of oocysts and trophozoites of the parasites. Thereafter, samples were concentrated by formol ether sedimentation technique and subjected to special staining techniques such as modified acid fast (Kinyoun's) stain for detection of oocysts of Cryptosporidium spp., Cystoisospora spp., Cyclospora spp. and modified trichrome stain for detecting spores of Microsporidia. Modified Kinyoun's staining was undertaken as per standard laboratory protocol,[27] with some modifications. Briefly, the smear was incubated in modified Kinyoun's stain for 20 min, decolourised with 10% acid-alcohol for 1.5–2 min and counter-stained with 3% malachite green. Weber's modified trichrome staining technique, in which smear was incubated in trichrome stain at 50°C for 10–12 min, was used with fast green as counter stain. Each stained-smear was observed under light microscopy. Stool samples were also subjected to culture on MacConkey, deoxycholate citrate agar and dextrose sorbitol rhamnose agar media as per standard microbiological technique to exclude bacterial agents causing diarrhoea such as  Salmonella More Details spp. and Shigella spp.[28]

Molecular analysis

Three stool samples from each patient were pooled into one aliquot and subjected to DNA extraction using QIAamp DNA mini kit (Qiagen Inc., Valencia, CA, USA), according to the manufacturer's protocol with some modifications. PCR was performed with gene-specific primers for detection of Cryptosporidium spp. and Microsporidia. For Cryptosporidium spp., nested PCR was carried out using primers, BCOWPF and BCOWPR for Cryptosporidium oocyst wall protein (COWP) gene, as described by Pedraza-Díaz et al.[29] to amplify a 769 bp fragment. This was followed by a secondary PCR reaction using Cry 15 and Cry 9 primers, described by Spano et al. for amplifying a 553 bp region of COWP gene.[30] An aliquot of 5 µl of each nested PCR product was subjected to digestion using RsaI restriction enzyme. The digested products were visualised on 3% agarose gel after staining with ethidium bromide. The band patterns of the digested products were matched with those described by Xiao et al.[31] The PCR-RFLP was performed to detect C. hominis, C. parvum, C. meleagridis or C. andersoni species. For Microsporidia, the extracted faecal DNA was subjected to PCR for amplification of a conserved region of small subunit rRNA gene of Enterocytozoon bieneusi and Encephalitozoon species using set of primers, previously described by Raynaud et al.[32] The amplified fragments were subjected to digestion using restriction endonucleases HinfI and HindIII to differentiate between four human Microsporidia species, Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi and Encephalitozoon hellem.[32] Ten micro litres of amplified DNA was digested with 5 units of HinfI and Hind III in a final volume of 15 µl. The digested fragments were analysed on 2.5% agarose gel stained with ethidium bromide.

Statistical analysis

Non-parametric and parametric continuous variables were analysed using Mann–Whitney U-test and unpaired t-test, respectively. Categorical variables were analysed by Chi-square test. P < 0.05 was considered significant. All the statistical analyses were done using Statistical Package for the Social Sciences - (SPSS 15, Inc., Chicago, IL, USA) and R, Epicalc and R-studio software (R development core team, Vienna, Austria).


 ~ Results Top


Detection of enteric parasites by microscopy and culture

Enteric parasites were detected in 16/80 (20%) of immunocompetent patients with diarrhoea and 2/110 (1.8%) healthy controls (P = 0.00007). Among patients with diarrhoea, the parasites detected were Cryptosporidium spp. (8/16, 50%), Cystoisospora spp. (4/16, 25%), Microsporidia (1/16, 6.25%), Cyclospora spp. (1/16, 6.25%) and Giardia spp. (1/16, 6.25%) [Figure 1]. One patient had mixed infection with Cystoisospora spp. and Giardia spp. Among healthy controls, one had Entamoeba spp. and one had Giardia spp. On stool culture, pathogenic organisms such as Shigella spp. and Salmonella spp. were not detected in any patient.
Figure 1: Opportunistic enteric parasites detected (under ×100 in light microscope): (a) Oocysts of Cryptosporidium spp., (b) oocyst of Cystoisospora belli, (c) spores of Microsporidia, (d) oocysts of Cyclospora cayetanensis

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Detection and genetic characterisation of Cryptosporidium spp. and Microsporidia by molecular analysis

The results of molecular analysis were in accordance with those of microscopy. Cryptosporidium spp. was detected in the same 8/80, 10% of immunocompetent patients and Microsporidia in 1/80, 1.25% of them, who were found positive for the above two parasites by microscopy. C. hominis was the only Cryptosporidium species detected and E. bieneusi, was the only Microsporidia species identified [Figure 2]. Other zoonotic species of Cryptosporidium spp., C. meleagridis, C. felis, C. canis, C. suis, C. muris and C. andersoni were not detected. Similarly, other species of Microsporidia, E. intestinalis, E. hellem and E. cuniculi were not detected.
Figure 2: Lane (M) 100 bp molecular marker, lane (1–4) Cryptosporidium hominis positive samples, lane (5) negative control, lane (6) C. hominis positive control

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Demographic and clinical features of patients and controls

Daily stool frequency, fever, cough, nausea, vomiting and abdominal pain were more common in immunocompetent patients with diarrhoea than among healthy controls [Table 1]. We also studied demographic, clinical and laboratory parameters of patients with and without cryptosporidiosis, such as age, gender, daily stool frequency, presence of chronic diarrhoea, total leucocyte count (TLC), neutrophil count, fever, cough, nausea and vomiting and abdominal pain [Table 2]. Parasite infested patients (16/80, 20%) had longer duration of diarrhoea than non-infected patients (64/80, 80%). Parasites were more commonly detected among patients younger than 20 years of age than among older ones. Patients with cryptosporidiosis were younger than those without (12.38 ± 13 years vs. 33.5 ± 19 years, P = 0.004). Furthermore, parasites were detected more often among patients with chronic diarrhoea than those with persistent and acute diarrhoea (13/42, 30% vs. 2/16, 12.5% vs. 1/22, 4.5%, P = 0.03).
Table 1: Demographic and clinical parameters of immunocompetent patients with diarrhoea and healthy controls

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Table 2: Demographic, clinical and laboratory parameters of immunocompetent patients having diarrhoea, with and without parasitic infection

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 ~ Discussion Top


In the current study, opportunistic enteric parasites were detected more frequently among immunocompetent patients with diarrhoea than healthy controls. The parasites detected were Cryptosporidium spp., Cystoisospora spp., Cyclospora spp. Giardia spp. and Microsporidia One patient had mixed infection with Cystoisospora spp. and Giardia spp. Parasites were more common in patients with diarrhoea than those without, and also in patients younger than 20 years than the older age groups. C. hominis and E. bieneusi were the parasite species detected.

Though there is scanty data of opportunistic enteric parasites in immunocompetent patients with diarrhoea, a few previous studies report similar results. Frequency of intestinal Coccidian parasites in Chennai has been reported to be 27.5%, and the parasites detected were Cryptosporidium spp. (21%), Cystoisospora spp. (6%) and Cyclospora spp. (1%).[33] Furthermore, in Loni, Maharashtra, prevalence of Coccidian parasites among patients hospitalised with gastrointestinal symptoms has been reported to be 31%, which included Cryptosporidium spp. (66%), mixed infection of Cryptosporidium spp. and Cyclospora spp. (31%) and only Cyclospora spp. infection (3%).[34] A study from Chennai reported Cyclospora spp. to be the only Coccidian parasite infecting (22.2%) slum dwellers.[35] Low socio-economic and educational status associated with poor hygiene practices have been indicated as the reasons for higher prevalence of enteric parasites in this community. There is scanty data on Cystoisospora belli and Cyclospora cayetanensis infections in immunocompetent people worldwide. A study from Korea reported 0.02% prevalence of C. belli in immunocompetent individuals.[36] Another study from Massachusetts, USA, on 1042 stool specimens from patients with diarrhoea, reported only five samples to show Cyclospora.[37] In the present study only a single infection due to Microsporidia was diagnosed. A study from Chile, also found no Microsporidia infection among HIV-negative patients.[38] However, its frequency in the current study was much lower than that reported in another study from India (10.9%),[39] as well as from Pakistan (5%),[40] Turkey (9.8%)[41] and Canary Islands (9.1%).[42] Thus, opportunistic enteric parasites not only causes infection in immunocompromised patients but also infects immunocompetent people, though studies are lacking in them.

All cryptosporidiosis patients in this study were found to be infected with C. hominis. This may be attributed to the geographical location and the environment. There is no study reporting genetic characterisation of Cryptosporidium spp. among immunocompetent adults in India. A study on children with diarrhoea showed C. hominis to be the most frequent Cryptosporidium sp.[20] A similar study on children from southern India showed C. hominis to be the most prevalent Cryptosporidium sp. (81%).[43] Studies from other parts of developing world such as Peru,[44] Malawi,[45] Kenya,[46] Haiti [47] and Brazil [48] reported similar results. However, our finding is in contrast to the results from temperate countries like England where C. parvum infection was most prevalent (56.1%) among patients with diarrhoea.[49] Another study from England also demonstrated higher C. parvum infection rates to be as high as 62% among patients with diarrhoea.[50] In a study from Chile, C. hominis was the most common species infecting immunocompetent individuals, while C. parvum in immunocompromised.[51] Other studies on immunocompromised patients reflect varied distribution of Cryptosporidium species worldwide. In studies from India,[5],[17] South Africa,[52] North America,[53] South America [54] and Europe,[55]C. hominis has been found to be the most prevalent species. However, in a few other studies from Kenya, Malawi, Brazil, the United Kingdom, Vietnam, Malaysia and France, C. parvum has been found to be more prevalent than C. hominis.[56],[57],[58] Thus, geographic and climatic factors might be the reason for higher frequency of a particular species. In the present study, detection of only C. hominis, and absence of the other zoonotic Cryptosporidium species indicate that anthroponotic mode of transmission is dominant among immunocompetent patients with diarrhoea, in northern India. Thus, previous studies as well as the present one suggest that immunocompetent individuals are predominantly infected with C. hominis than other Cryptosporidium species.

In our study, Microsporidia was detected in one patient only. The species detected was E. bieneusi. This is in accordance with a similar study from Canary Island, Spain, which reported E. bieneusi infection in stool samples from immunocompetent individuals.[42] Conversely, in other studies from India as well as from Pakistan, E. intestinalis was the only Microsporidia species infecting HIV negative individuals with diarrhoea.[39],[40] Among HIV infected patients E. bieneusi was reported as the most common species.[21] However, in another study E. intestinalis was reported as common causative species.[39] Among renal transplant recipients, E. bieneusi has been reported to be the most common species of Microsporidia.[10],[16],[59],[60] Thus, infecting species of an organism infecting differs according to geographical areas. As the drug of choice against Microsporidia, albendazole, is effective against Encephalitozoon spp. but not E. bieneusi, there is a need for identification of the species of Microsporidia, to ensure proper treatment to the infected patients.

Parasites were commonly detected among patients younger than 20 years of age than in the older age groups. Similar finding was reported from Chennai, where children and teenagers more commonly harboured intestinal parasites.[35]Cryptosporidium spp. was more often found in younger patients than older. It is well-known that children suffer from cryptosporidiosis more commonly than adults. This has been proved in earlier studies as well.[61],[62] Furthermore, in the current study parasites were detected more often among patients with chronic diarrhoea than those with persistent and acute diarrhoea. In immunocompromised patients, the intestinal opportunistic parasites play a major role in causing chronic diarrhoea.[63] However, this has been sparsely documented among immunocompetent individuals.[46] Thus, our study also shows the association between opportunistic enteric parasites in immunocompetent people and type of diarrhoea.


 ~ Conclusion Top


Opportunistic intestinal parasites were common among immune-competent patients with diarrhoea in our region. C. hominis and E. bieneusi were the common parasite species detected. Patients ≤20 years were frequently infected with parasites than the older ones. Most of the parasite infected patients presented with chronic diarrhoea. Thus, opportunistic enteric parasites may also be causative agents of diarrhoea, not only in immunocompromised patients but also in immunocompetent people of younger age. Proper diagnostic measures, such as special staining techniques and molecular methods should be implemented to facilitate effective management of patients.


 ~ Acknowledgement Top


The authors duly acknowledge the financial assistance from The Department of Biotechnology, New Delhi (reference no. 102/IFD/SAN 1839/2008-09) and The Indian Council of Medical Research, New Delhi (reference no. 5/3/3/3/2008-ECD-I) to Ujjala Ghoshal; Asmita Dey and Sonali Khanduja acknowledge The University Grants Commission, New Delhi and Indian Council of Medical Research, New Delhi respectively, for providing their fellowships.

 
 ~ References Top

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    Figures

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    Tables

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

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