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ORIGINAL ARTICLE
Year : 2019  |  Volume : 9  |  Issue : 1  |  Page : 18-22  

Prevalence of cryptosporidiosis in symptomatic immunocompetent children and comparative evaluation of its diagnosis by Ziehl–Neelsen staining and antigen detection techniques


1 Departments of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi University, Delhi, India
2 MBBS Student, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi University, Delhi, India

Date of Acceptance03-Aug-2019
Date of Web Publication24-May-2019

Correspondence Address:
Rumpa Saha
3rd Floor, Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi University, Delhi - 110 095
India
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DOI: 10.4103/tp.TP_59_18

PMID: 31161088

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   Abstract 


Background: Cryptosporidia is a major pathogen causing diarrhoea and with increasing morbidity and mortality. As persistent diarrhoea from intestinal cryptosporidiosis leads to increased susceptibility to recurrent diarrheal episodes further leading to chronic nutritional and cognitive sequelae or even death, diagnosis is important. Most of the studies done on Cryptosporidium worldwide have focused on immunocompromised patients which have led to a paucity of data on its prevalence among immunocompetent people.
Aims and Objectives: Keeping these facts in mind the present study was aimed to estimate prevalence of cryptosporidiosis in immunocompetent children, and do a comparative evaluation of its detection by microscopy with antigen detection methods.
Material and Methods: 80 immunocompetent children (40 OPD children presenting with diarrhea and 40 children hospitalized for diarrhea) upto age of 5 years were studies and their stool samples were compared by microscopy by mZN with copro-antigen detection methods (using rapid ICT and ELISA) for the diagnosis of Cryptosporidiosis.
Results: A Cryptosporidium prevalence rate of 22.5% was detected in the immunocompetent children upto 5 years of age. Microscopy remained the preferred method of diagnosis for Cryptosporidium being a more sensitive test and considering it's low cost in resource poor settings. Moderate agreement between mZN and ELISA in Cohen's kappa test shows that either of the tests can be used for diagnosis of Cryptosporidium from fecal sample. ELISA is time-saving method but ELISA and rapid antigen tests should not be used as the sole method of diagnosis. Keeping in view the ICT kit used in this study is species specific, and the species identification was not carried out in the present study, hence genus specific kits may be useful for diagnosis in such settings.
Conclusion: Microscopy remains the preferred method of diagnosis for Cryptosporidium having good sensitivity and specificity and considering it's low cost in resource poor settings. ELISA is time-saving method but ELISA and rapid antigen tests should not be used as the sole method of diagnosis.

Keywords: Cryptosporidium, diagnosis, immunocompetent children


How to cite this article:
Saha R, Saxena B, Jamir ST, Shekhar S. Prevalence of cryptosporidiosis in symptomatic immunocompetent children and comparative evaluation of its diagnosis by Ziehl–Neelsen staining and antigen detection techniques. Trop Parasitol 2019;9:18-22

How to cite this URL:
Saha R, Saxena B, Jamir ST, Shekhar S. Prevalence of cryptosporidiosis in symptomatic immunocompetent children and comparative evaluation of its diagnosis by Ziehl–Neelsen staining and antigen detection techniques. Trop Parasitol [serial online] 2019 [cited 2019 Aug 19];9:18-22. Available from: http://www.tropicalparasitology.org/text.asp?2019/9/1/18/258792




   Introduction Top


In India, diarrhea is a leading cause of death among children under 5 years, and Cryptosporidium is one such major pathogen causing diarrhea with increasing morbidity and mortality.[1],[2],[3] Most of the human infections are caused mainly by two species of cryptosporidia, i.e., Cryptosporidium hominis and Cryptosporidium parvum.[4],[5] It has been documented that greater shedding of oocysts for longer duration leading to more severe diarrhea is more common with C. hominis.[6],[7],[8] Although initially reported in immunocompromised individuals, it has also been found in immunocompetent persons.

A high prevalence (27.4%) of Cryptosporidium infection has been reported in immunocompetent children under the age of 5 years in India.[9] Several other studies have shown that the prevalence of Cryptosporidium is high in developing countries.[10],[11]

In immunocompetent hosts, C. parvum has been known to cause acute, self-limiting, watery diarrhea lasting 5–10 days. However, in the immunocompromised and malnourished individuals, diarrhea is usually prolonged.[2],[12] In malnourished children, persistent diarrhea leads to increased susceptibility to recurrent diarrheal episodes, which can lead to chronic nutritional and cognitive sequelae or even death.[13] It has been demonstrated in young Peruvian children that even asymptomatic C. parvum infection may lead to stunting, malnutrition, and lack of catch-up growth.[14] Hence, prompt identification of this agent is important to diagnose and study the etiology of childhood diarrhea.

Light microscopy using Kinyoun's-modified acid-fast staining is the most commonly used method for its diagnosis. Nowadays, the application of Cryptosporidium antigen detection techniques on stool samples is becoming increasingly popular. Copro-antigen techniques, include rapid antigen immunochromatographic tests (ICTs) and enzyme-linked immunosorbent assay (ELISA), act as easy diagnostic tools.

Most of the studies published on Cryptosporidium worldwide have focused on immunocompromised patients which have led to a paucity of data on its prevalence among immunocompetent people. Keeping these facts in mind, the present study was focused to estimate this parasite burden in immunocompetent children under the age of 5 years presenting with diarrhea and to evaluate modified Ziehl–Neelsen (mZN) staining with copro-antigen detection methods for its diagnosis.


   Methodology Top


This cross-sectional study conducted during May–July 2018 was done in the department of microbiology of our tertiary care hospital. The study population included children <5 years of age presenting with acute or persistent diarrhea at the pediatric outpatient department (OPD) and those requiring admission to the hospital for diarrhea. Diarrhea was defined as ≥3 loose/liquid stools in a 24-h period or when bowel movements occurred more frequently than the child's normal amount. Symptoms lasting <2 weeks were considered acute diarrhea, while 14–29 days of symptoms was considered persistent diarrhea. Children with known immunodeficiency, those with known allergy to lactose, gluten, or any other food and those receiving antiparasitic medication for the current episode of diarrhea within the past 7 days, were excluded from this study. All diarrhea patients who (i) had severe dehydration, (ii) were not accepting orally, (iii) had repeated vomiting, (iv) were not passing urine, (v) were drowsy, (vi) had any other coinfection like pneumonia, and (vii) had persistent diarrhea were admitted to the hospital.

Eighty stool samples were collected from 40 OPD children presenting with diarrhea and 40 children hospitalized for diarrhea.

Each sample was divided into three parts as follows:

  1. The first part was concentrated by formol-ether concentration technique and smear prepared from deposit. Smears were stained by mZN staining after fixation in absolute alcohol for 10 min.[15] Cryptosporidium was identified by its characteristic size (4.5–6 μm), round to slight ovoid shape, and staining variably pink with unstained sporulated crescentic forms seen within the oocyst
  2. The second part was subjected to rapid antigen detection by EpiTuub ® Fecal C. parvum antigen rapid ICT kit as per the manufacturer's instruction (Epitope Diagnostics, Inc., San Diego, USA)
  3. The third part was preserved in 10% formalin and was subjected to ELISA following manufacturer's instruction (DRG Diagnostics, Germany).


All the samples were discarded as per the BMW guidelines 2016[16] following completion of the work.

All the data collected were entered in MS-Excel and SPSS v20.0 were used for data analysis (IBM, Armonk, NY, USA). All qualitative assessment is expressed as percentage. Comparison of abilities of the various methods used for diagnosis and detection of cryptosporidiosis was done by Chi-square test. P < 0.05 was considered as statistically significant. As there was no positivity with ICT test, the diagnostic accuracy (inter-rater reliability) of mZN and ELISA was assessed by Cohen's kappa test.


   Results Top


In the present study, 80 stool samples were collected. Forty samples were collected from children hospitalized for diarrhea. Forty samples were from children attending an outpatient clinic with a history of diarrhea.

Of the total 80 samples, 18 were found positive for Cryptosporidium by any one technique (s), indicating 22.5% prevalence in immunocompetent children <5 years.

By microscopy (mZN), total positive samples were 16 (20%) (mZNs positivity in OPD: hospitalized = 6 [15%]:10 [25%]), ICT total positive samples were nil, and ELISA total positive samples were 8 (10%) (ELISA positivity in OPD: hospitalized = 3 [7.5%]: 5 [12.5%]). Eight samples were positive by microscopy alone, whereas two samples were positive by only ELISA. Six samples (7.5%) were positive by both microscopy and ELISA.

Sample positivity by various techniques in both hospitalized and OPD children are shown in [Figure 1].
Figure 1: Cryptosporidium positivity by different techniques

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Of the total 18 positive samples, 7/40 (17.5%) were from the OPD patients and 11/40 (27.5%) were from the hospitalized patients who were positive for Cryptosporidium.

Keeping ELISA as the gold standard, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value were 75%, 86.11%, 37.5%, and 96.87%, respectively, for mZN and 0%, 100%, 0%, and 90%, respectively, for ICT [Table 1].
Table 1: Comparison of modified Ziehl-Neelsen and immunochromatographic test with enzyme-linked immunosorbent assay

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As there was no positivity with ICT test, the diagnostic accuracy (inter-rater reliability) of mZN was assessed by Cohen's Kappa test, and the results were interpreted as per [Table 2].[17] The interrater reliability (kappa value) by comparing ELISA with mZN was calculated to be 0.423, thus suggesting a moderate agreement between two tests.
Table 2: Interpretation of Cohen's kappa test

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[Table 3] shows the age group distribution of children affected by Cryptosporidium. Children <1 year of age were most affected in the present study. Females were overall twice more affected with the disease (M:F = 1:2).
Table 3: Cryptosporidium positivity in different age groups

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   Discussion Top


In the present study, 18 samples were positive for Cryptosporidium by either one of the diagnostic techniques giving a prevalence rate of 22.5% in the immunocompetent children <5 years, which is comparable with studies from India (3%–30%).[18],[19] A study from the same institution 6 years back also documented a similar prevalence (27.4%) in under 5 years immunocompetent children.[9] The present study showed that children hospitalized for diarrhea had higher positivity of Cryptosporidium as compared to the OPD patients with diarrhea indicating greater severity in those who required hospitalization. This finding is comparable to a Brazilian study which reports more severe diarrhea with C. hominis.[6] Although children <1 year were most affected in the present study, most studies from India and outside India have shown the highest prevalence in children under 5 years.[20]

About 20% positivity was seen by microscopy, whereas 10% were positive by ELISA indicating higher positivity by microscopy in comparison to ELISA. Analogous results have been documented from the USA and Egypt.[21],[22] Low positivity in ELISA may be because the monoclonal antibodies used by different commercially available copro-antigen detection ELISA kits may recognize different sets of surface epitopes and thus may not react with antigens of different Cryptosporidium species resulting in false negativity in ELISA. Two samples which were positive only by ELISA, but negative microscopically, can be false positive due to clearance of oocyst from the gut, but persistence of antigen in patients who have been recently treated.[19] Low sensitivity of Cryptosporidium ELISA test has been reported from Atlanta, and hence the results should be confirmed with mZN staining, especially in low-prevalence areas.[23] Advantages of ELISA over mZN staining are that more number of samples can be tested at the same time. Moreover, microscopy skills and concentration of the fecal samples are also not required for antigen detection.

In our study, ICT (Epitope Diagnostics, Inc. San Diego, USA) gave no positive results. As the kit used in the present study detects only C. parvum antigen, so this could be one of the possible reasons that the diarrheal infections in the current study may have been caused by other species of Cryptosporidium.[18] Ideally, a genus specific would be more appropriate for routine diagnosis, especially in areas where species prevalence is unknown. A recent community-based study from Vellore found C. hominis in children to be the most common Cryptosporidium species.[24] Another reason for false negativity can be a low parasite load in the stool. A parasite load of >175 oocysts/10 μl fecal sample is necessary for ICT positivity as reported by Johnston et al.[23] The same authors suggest that the sensitivity of ICT varies between 67% and 91%, with a higher specificity of up to 99% comparable to the present study. Researchers from various studies who have evaluated the following ICT kits have detected C. parvum-specific antigen in ImmunoCard STAT (Meridian Bioscience Inc., USA); RIDA ® QUICK Cryptosporidium (R Biopharm, Germany); and CoproStrip Cryptosporidium (Savyon Diagnostics Ltd., Israel) with a 67%–84% sensitivity and 98%–100% specificity. However, Dia-Pro CA-RT cryptosporidium (Dia-pro diagnostics, Italy) and Crypto-Strip (Coris BioConcept Inc., Belgium) have used genus-specific antigen detection kits having similar sensitivity (61%–86%) and specificity (99%–100%).[22],[23],[25] The specificity of the ICT kit used in the present study although comparable with the above, a larger sample size would be needed for appropriate evaluation of this kit. To the best of our knowledge from the available literature, Epitope Diagnostics, Inc., San Diego, USA, EpiTuub ® Fecal C. parvum antigen detection kit has not been used so far by any documented studies. Hence, the use of this kit should be further evaluated by documented studies.

As none of our samples were positive for C. parvum antigen by ICT, the possibility that C. hominis can be the pathogenic Cryptosporidium in our patients cannot be ruled out. This, however, needs confirmation using molecular methods. A multicentric study from India (Delhi, Trichy, and Vellore) found C. hominis in children to be the most common Cryptosporidium species.[24] As coinfections with other diarrheal agents were not assessed in the present study, hence Cryptosporidium cannot be labeled as the sole etiological agent of diarrhea in the present study.

Moderate agreement between mZN and ELISA in Cohen's kappa test may be due to the small sample size of the size. The present study had good sensitivity and specificity of mZN and suggested that microscopy by mZN can be used for the diagnosis of Cryptosporidium from the fecal sample. Further, it detects all species although cannot differentiate between the species and hence will detect all Cryptosporidium oocysts. Studies from abroad have reported higher sensitivity of microscopy as compared to ELISA [22],[23] and studies from Chandigarh and the UK report higher specificity for mZN.[26],[27],[28] As predictive values of a disease vary with the prevalence of the disease, the low PPV of mZN in the present study can be due to the low prevalence of the disease in our study population.


   Conclusion Top


A Cryptosporidium prevalence rate of 22.5% was detected in the immunocompetent children up to 5 years of age. Microscopy remains the preferred method of diagnosis for Cryptosporidium having good sensitivity and specificity and considering its low cost in resource-poor settings. ELISA is a time-saving method, but ELISA and rapid antigen tests should not be used as the sole method of diagnosis.

Acknowledgment

The authors acknowledge the help of Mr. Room Ram Prami (technical assistant, Microbiology Laboratory, University College of Medical Sciences and Guru Teg Bahadur Hospital) for his technical assistance in the laboratory.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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