Coccidian intestinal parasites among immunocompetent children presenting with diarrhea: Are we missing them?

Abstract

Background:

Diarrhoea is an important cause of both morbidity and mortality among children in India. Coccidian parasitic infections are an important cause of diarrhea in immunocompromised patients, but their investigations are rarely sought by the treating physicians in seemingly immunocompetent children. This study was aimed to find the incidence rate of coccidian parasites in all children presented with diarrhoea, irrespective of their immune status.

Materials and Methods:

Between December 2015 and May 2016, all fecal samples from children aged between 0 and 15 years presenting with diarrhoea, irrespective of their immune status, were examined using conventional wet mount and modified acid-fast staining. At the end of the study, records of their clinical history and immune status including HIV positivity were evaluated. Findings of wet-mount and modified acid-fast stained smear microscopy were analyzed in relation with clinical details.

Results:

During the study, samples from 200 children (single sample) with diarrhea were processed. Their mean age was 5.7 ± 3.3 years (range 4–168 months). Seventeen out of 200 (8.5%) samples were positive for acid-fast coccidian parasites. Eight (4%) samples were found to be positive for Cryptosporidium hominis, while 5 (2.5%) were positive for Cyclospora cayetonensis and 4 (2%) samples for Isospora belli oocysts. Half (50%) of the children who were tested positive for Cryptosporidium and Cyclospora were found to be otherwise immunocompetent. However, all four cases of Isosporiasis were immunocompromised patients.

Conclusion:

We highlight the high incidence of coccidian parasites among immunocompetent children with diarrhea. The clinicians need to be aware that coccidian parasites are a potential cause of childhood diarrhea even in immunocompetent children.

INTRODUCTION

Intestinal parasitic infections are a major public health problem in developing countries like India. Infections with opportunistic parasitic organisms such as Cryptosporidium, Cyclospora, and Isospora are common in immunocompromised patients but have occasionally been seen in immunocompetent individuals also.[1,2] Cryptosporidium hominis has been found to be most commonly detected intestinal protozoa in India.[3] The frequency of Cryptosporidium infection has been reported to be variable (4%–29.2%) in patients presenting with diarrhea in urban Northern India.[4,5] Similarly, studies have reported the variable prevalence of Isospora belli ranging from 2.4% to 58.3% and Cyclospora ranging from 2.1% to 7.8% in North India.[2,5,6,7] There have been reports describing Isospora as a cause of diarrhea in immunocompromised as well as in immunocompetent individuals.[6] Children with HIV infection and those with severe malnutrition have shown higher frequencies of protozoan infections. Fontanet et al.[8] found that the prevalence of intestinal parasites in immunosuppressed patients was same as that in immunocompetent individuals. Therefore, it may not be out of context to suggest that identification of these so-called opportunistic pathogens should be an essential part of investigations for childhood diarrhea, irrespective of the immune status of the child. With this aim, we conducted an analysis of all diarrhea cases from pediatric population presenting in our laboratory to find out the prevalence of such microorganisms in immunocompetent pediatric cases.

MATERIALS AND METHODS

With the primary aim of assessing the utility of modified acid-fast staining of stool samples, in cases of childhood diarrhea, this prospective, observational study was conducted from December 2015 to May 2016 at the All India Institute of Medical Sciences (AIIMS), New Delhi. Stool samples, from all children aged between 0 and 15 years seen in children OPD or hospitalized in pediatric wards of AIIMS, New Delhi, who presented with diarrhea, irrespective of their clinical history of immune suppression, were processed. As a standard protocol, after receiving the samples in the laboratory, stool samples were processed and wet-mount preparations with both saline and iodine were prepared and screened within 2 h of sample collection to look for motile trophozoites, larvae, ova, and cyst. Stool samples were also processed by the formalin-ether concentration technique and reexamined with saline and iodine wet-mount preparations as well as stained with Kinyoun's acid-fast stain[5] to look for Cryptosporidium, Cyclospora, and Isospora oocysts. Each wet-mount preparation and stained fecal smears were examined by a clinical microbiologist and a trained technologist independently and findings were cross-checked. After completion of the study, their clinical data were retrospectively retrieved from the register and analyzed.

RESULTS

During the study, a total of 200 stool samples from 200 children with diarrhea (single sample) were examined. Repeat samples, if any, were not counted as different. The mean age of children comprising the study population was 5.7 (SD +3.3) years (range: 4 months to 168 months). Seventeen samples out of 200 were positive (8.5%) for any one or more acid-fast coccidian parasite. The data of other parasites are not shown here. Eight of the 200 samples (4%) were found to be positive for Cryptosporidium, while 5 (2.5%) and 4 (2%) samples were positive for Cyclospora and Isospora, respectively. On further tracking their clinical records, 3 of the 8 children who were Cryptosporidium positive were found to be HIV seropositive while one child was a patient of Wiskott–Aldrich syndrome. Remaining 5 (40%) children did not have any immunosuppressive condition. Similarly, 2 out of 4 (50%) patients who tested positive for Cyclospora were immunocompetent. However, all the four children whose stool samples tested positive for Isospora were found to be HIV positive.

DISCUSSION

Our study reports that in 4% of children, the main causative agent of diarrhea was Cryptosporidium, while Cyclospora was next commonest occidian pathogen. Of these, almost half were immunocompetent children. This is an important observation. Another recent study on diarrheal cases including adults reported its prevalence to be 10% (8/80) in immunocompetent patients.[3] Similarly, we found that the Isospora was main etiological agent of diarrhea in 2% (4/200), which was similar to other recent studies but much lower than earlier studies wherein these parasites were found to be highly prevalent in AIDS patients.[3,4,5] In recent years, the incidence of Isospora has decreased significantly mainly due to administration of prophylactic co-trimoxazole. Cyclospora spp. was found in 2.5% cases, which is also comparable to other studies in this region.[3,6,9,10] Although all the cases of Isospora in our study were associated with immunocompromised state and were symptomatic, cases of asymptomatic infection in nonimmunocompromised children have been reported earlier.[11,12] It should be noted that cases of prolonged diarrhea, especially from immunocompetent children, are usually not referred by clinicians for laboratory investigations for acid-fast coccidian parasites. This might be due to the fact that most of the clinicians do not suspect these parasitic infections as a prominent cause of diarrhea in immunocompetent children.

Intestinal coccidian parasitic infections can be acquired from contaminated water sources as they are resistant to most methods of chemical disinfection and usually cause a self-limiting diarrheal illness in healthy persons.[13] These infections can however be life-threatening in immunocompromised individuals.[14] Patients with low CD4+ counts, as in HIV infection, those receiving immunosuppressive agents or suffering from other causes of immunodeficiency, such as solid organ transplantation or primary immunodeficiency disorders such as Wiskott–Aldrich syndrome, hyper-IgM syndrome, severe combined immunodeficiency, selective IgA deficiency, and possibly gamma interferon (IFN-γ) deficiency, are at particular risk for cryptosporidiosis.[15] Various experimental studies with mice have shown that immunity against these parasites depends on the number of intestinal intraepithelial CD4+ lymphocytes which produce IFN-γ, important for clearance of these organisms which is affected in immunocompromised states. T-cell immunity plays a critical role in the resolution of Cryptosporidium infection mainly by CD4+ cells and IFN-γ-mediated pathways.[16] In the intestines, it causes villous atrophy, crypt hyperplasia, and infiltration of lymphocytes, neutrophils, plasma cells, and macrophages into the lamina propria, which causes the severity of symptoms. Infection with these parasites in immunocompetent individuals is confined to the intestine, while in immunocompromised patients, it spreads to the entire gastrointestinal tract, resulting in increased intestinal permeability and chloride secretion within the epithelial cells of biliary tree, pancreatic duct, and even in airways.[15,16]

The results of this study highlight the fact that coccidian parasites should not be overlooked by the clinical microbiologists, while investigating cases of diarrhea, even if these are not requested by the physician. The fact that 50% of Cryptosporidium cases were seen among immunocompetent cases shows the existence of this pathogen even in nonimmunocompromised pediatric population. Cryptosporidium is an important etiological agent and its diagnosis is of utmost importance as this is a useful guide for the prompt treatment of such cases. More importantly, it can be diagnosed by relatively simple and inexpensive techniques such as modified acid-fast staining, which can also help in avoiding invasive procedures such as colonoscopy and intestinal biopsies in cases of persistent diarrhea.[17,18] Since the acid-fast staining was not requested from the clinician's side, especially for the immunocompetent cases, our study also points to the importance of writing complete and proper clinical history on the requisition forms, which can guide a laboratory so as to perform these tests when needed, especially in resource-limited settings. Ours was a tertiary care and referral laboratory where modified acid-fast staining in stool samples is a standard protocol in diarrheal cases, but not all laboratories might be doing this in routine, until they are asked for. Hence, it might be possible that many such cases might be missed by them.

We would also like to point out the fact that the data presented by us here might be an underestimation of the actual burden of these parasitic infections because parasitic oocysts are shed intermittently, which might not necessarily correspond to periods of clinical symptoms. Similarly, oocysts may not be shed during the initial phase of infection when the asexual phase of life cycle predominates and the clinical symptoms become apparent.[4] This is most relevant to a laboratory, as we rarely receive three stool samples from a patient, as recommended for diagnosing parasites in stool, and mostly, the stool sample is sent for examination only once. Singh et al. reported that in such circumstances, three coverslips or three smears from a single sample can be prepared on single day or multiple days to increase the diagnostic yield.[19] Nevertheless, it is always proper to send at least three stool samples for three consecutive days, to increase the diagnostic yield.

CONCLUSION

Intestinal infections caused by organisms such as Cryptosporidium, Cyclospora, and Isospora can be diagnosed by a simple and inexpensive method of modified acid-fast staining. Awareness must be spread among the clinicians about the occurrence of these coccidian parasites not only in immunocompromised but even in immunocompetent patients, causing gastrointestinal symptoms. It is also important that requisition forms with proper history must be sent to the laboratories because all laboratories might not be able to do this exercise for all the stool samples, until they are asked for, and hence these infections may be missed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.