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Year : 2015  |  Volume : 58  |  Issue : 1  |  Page : 69-71
Comparative analysis of enzyme-linked immunosorbent assay and direct microscopy for the diagnosis of Giardia intestinalis in fecal samples


1 Department of Microbiology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

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Date of Web Publication11-Feb-2015
 

   Abstract 

Context: Giardiasis is one of the most common nonviral infections causing diarrheal illness worldwide. In this prospective cross-sectional study, we evaluated the RIDASCREEN ® Giardia kit for detection of Giardia intestinalis in stool samples and compared the results with direct microscopy. Materials and methods: A total of 360 fecal samples were collected. They were then processed by wet film, iodine preparation and an enzyme-linked immunosorbent assay (ELISA) kit to determine the presence of Giardia trophozoites and cysts. Statistical analysis was performed by sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy. Results and Conclusion: Of the 360 cases, 17.2% samples were positive for Giardia by direct microscopy and 23.6% were found to be positive by ELISA (sensitivity ~97%), but specificity was ~92% only. Because of less specificity, we need to perform ELISA in congruence with direct microscopy, etc. Further studies need to be performed on a larger sample size using other molecular tests in order to get more accurate estimations.

Keywords: Direct microscopy, enzyme-linked immunosorbent assay, Giardia intestinalis

How to cite this article:
Singhal S, Mittal V, Khare V, Singh YI. Comparative analysis of enzyme-linked immunosorbent assay and direct microscopy for the diagnosis of Giardia intestinalis in fecal samples. Indian J Pathol Microbiol 2015;58:69-71

How to cite this URL:
Singhal S, Mittal V, Khare V, Singh YI. Comparative analysis of enzyme-linked immunosorbent assay and direct microscopy for the diagnosis of Giardia intestinalis in fecal samples. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Sep 26];58:69-71. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/69/151192



   Introduction Top


Giardiasis is one of the most common nonviral infections causing diarrheal illness in humans worldwide. The infection rate is 2-7% in developed countries and 20-30% in developing countries. [1] In India, the prevalence of Giardia is 3.8-23.5%, and it is also a common protozoan infection. [2]

With the increasing potential of waterborne outbreaks caused by Giardia intestinalis, which can cause severe symptoms in humans, laboratories are reviewing their option with regard to diagnostic modalities that can be incorporated into routine testing protocols. G.intestinalis is usually diagnosed from stool samples by visualizing the organism, either the trophozoites or the cysts, in unstained wet smears by microscopy. Special techniques like concentration by formalin ethyl acetate centrifugation or by zinc sulfate floatation and special staining like trichrome staining can also be used in conjugation with microscopy. But, even after application of these techniques, the sensitivity of ova and parasites microscopy is dependent on the skill of the microscopist and the time spent scanning each preparation. In recent years, efforts have been made to improve the sensitivity of the diagnosis of Giardia. Some of the methods have been investigated for automating the detection of Giardia species, including immunofluorescent assay, enzyme immunoassay, counter immunoelectrophoresis and radioimmune precipitation assay. [3]

In patients who show clinical symptoms of Giardiasis, enzyme-linked immunosorbent assay (ELISA) is a rapid, sensitive and economic method to confirm infection and copro-antigens of Giardia, which could be traced and diagnosed even if the live parasite is absent in the fecal samples. [1] ELISA for G. intestinalis is approved by the World Health Organization and is also a highly sensitive and specific method. [4] Presently, very few studies have been performed on ELISA for Giardia, and all of them have been conducted abroad. This study was undertaken to evaluate the performance of the RIDASCREEN ® (R-Biopharm AG, Landwehrstr Inc. 2006, Darmstadt, Germany) Giardia kit in India for the prevalence of Giardiasis.


   Materials and Methods Top


This prospective, cross-sectional study was conducted on fecal samples received from patients in a tertiary care center at the suburban areas of Lucknow. The study was conducted in accordance with the ethical rules of the institute. Informed written consent from patients was not required as the stool samples were taken directly from the microbiology laboratory and we did not involve patients directly in our study.

A total of 360 stool samples were taken from different departments of the Era Lucknow Medical College and Hospital from November 2012 to October 2013. Patients presenting with gastrointestinal symptoms with more than 5 years of age were included and children <5 years were excluded from the study as it is seen that in this age group Rotavirus and bacteria like  Escherichia More Details coli are mainly responsible for diarrheal infections. [5]

A stool specimen was taken in a 25 mL screw-capped sterile plastic container, observing that the rim or outside of the container remains clean. In those patients who were not ambulatory, feces were collected from a clean bedpan, unmixed with urine or disinfectant. One to two milliliters of feces was then transferred into a 25 mL screw-capped wide-mouthed sterile plastic container. After tightening the cap, the specimens were transported to the microbiology laboratory as quickly as possible.

Gross examination of the stool sample was performed for color, consistency, mucous, blood and adult parasite. The sample was then divided into two parts. From the first part, direct wet mount and iodine mount examinations were carried out. Five milligrams of feces was picked on tip of a wooden applicator stick and mixed in a drop of 0.05 mL of saline for wet mount. It was then covered with a cover slip to view under a low-power objective and then with a high-power objective. The iodine preparation was also processed in a similar way using Lugol's iodine. The second part of the stool was refrigerated at 4°C for ELISA of G. intestinalis. The kit used in the study was a RIDASCREEN ® Giardia kit for detection of G. intestinalis.

The procedure was performed as per the manufacturer's instructions.

Statistics

Statistical analysis was performed using direct microscopy as the gold standard test for diagnosis of Giardiasis. We evaluated the RIDASCREEN ® Giardia ELISA kit for sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy using the statistical software SPSS version 18.


   Results Top


A total of 360 samples were included in study. Of these, 62 samples (17.3%) were positive by direct microscopy although 85 samples (23.6%) were positive by the RIDASCREEN ® Giardia ELISA kit [Table 1]. Among the later, 60 were positive by direct microscopy also and 25 were positive only by ELISA. [Table 2] shows the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of the RIDASCREEN ® Giardia ELISA kit.
Table 1: Comparison of the RIDASCREEN® Giardia ELISA kit results with microscopy


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Table 2: Analysis of the RIDASCREEN® Giardia ELISA kit


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   Discussion Top


G. intestinalis is a common protozoan parasite in humans, causing intestinal infections with watery diarrhea, abdominal pain, weight loss, malabsorption and bloating, which may last for week to months. [3]

In our study, the prevalence of Giardia was 17.3% by direct microscopy and as high as 23.6% by ELISA.Our direct microscopy result is higher than other studies of India, where it was 14.8% and 14.4%, respectively. [6],[7] But, this was lower than that in the study by Nitin et al. from Lucknow, who showed 22% Giardia spp. in their study. [2] By the ELISA method, 23.6% of the subjects were positive for Giardia, which was <43.8% in the study by Terri-Lynn Duffy. [8] This may be because of the only child population in the later study aged 1.5-7 years, contrary to ours where both adults and children were included. The results may be different due to the different study area, sample size, age group, etc. in our study.

Comparison of fecal diagnostic methods is problematic due to the lack of a true gold standard method. However, in our study, we have taken microscopy as the gold standard as cited by other studies. [4],[8],[9] But, the conventional microscopical examination, which is the most commonly used method to diagnose Giardiasis by detection of cysts in the stool requires the services of experienced personnel, is time consuming and sensitivity could be affected by intermittent excretion of Giardia as compared with ELISA. [9] Copro-antigenic identification of G. intestinalis was introduced in the early 90s. [9] Since then, several attempts have been made to establish sensitive and cost-effective methods to diagnose intestinal infections with Giardia. [10] Amongst copro-antigenic detection tests, ELISA has the highest sensitivity and specificity for the detection of G. intestinalis.[9]

The sensitivity and specificity of the RIDASCREEN Giardia ELISA kit was found to be as high as 96.8% and 91.6%, respectively. The diagnostic accuracy was 92.5%. In our study, the sensitivity was higher than the specificity. It is comparable to studies performed by Duque-Beltrαn et al., Ozekinci et al. and Guimarães and Sogayar, where the sensitivity of ELISA for Giardia was 100%, 96.4% and 82%, respectively, and the specificity was 95%, 80.8% and 39%, respectively. [11],[12],[13] In another study performed in Turkey, the sensitivity was almost equal to the specificity. [14] A study performed by Weitzel et al showed the sensitivity of RIDASCREEN ® Giardia to be higher compared with other copro-antigen tests like the Rida Quick Giardia, Rida Quick Combi and Giardia strip. However, in the same study, the specificity of all the three other copro-antigen tests was ≥98%. [15]

In our study, ELISA has a high sensitivity (96.8%) but a comparatively low specificity (91.6%). This means that it is a very good diagnostic test at finding the disease because it is sensitive. But, because of its lower specificity, it can give positive results when the disease is not actually present. Accordingly, false positive cases can be present because it is not very specific. This may be due to some cross-reactions with other intestinal parasites and some past infection with Giardiasis. However, if the ELISA result is negative, we can be fairly certain that the patient does not have Giardiasis. This type of test is therefore most informative to us diagnostically when it gives a negative result and rules out false-negative results caused by lack of training and experience of the laboratory personnel responsible for microscopic examination, untimely processing of samples and intermittent excretion of Giardia cysts and trophozoites in feces.


   Conclusion Top


Copro-antigen test ELISA for detection of Giardia can be used in screening and in carrying bulk tests where microscopy can be time consuming and more emphasis is laid on true negatives. However, due to false-positive cases, we need to further perform ELISA in congruence with direct microscopy or some other tests. Because of its lower specificity and high cost, this test cannot be implemented in routine laboratory practices in an Indian scenario. To the best of our knowledge, this is the first study that uses the RIDASCREEN ® Giardia copro-antigenic test to detect Giardia infection in humans and to find its sensitivity and specificity in India, a geographic region with a high prevalence of Giardiasis. However, the study has its limitations as this study was conducted using a single ELISA kit and small sample size. Further studies need to be performed on a larger sample size using other molecular tests in order to get more accurate estimation of serological tests and any cross-reactions with other intestinal parasites that could be beneficial for implementing effective treatment and control measures for G. intestinalis in the future.

 
   References Top

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Bilenko N, Levy A, Dagan R, Deckelbaum RJ, El-On Y, Fraser D. Does co-infection with Giardia lamblia modulate the clinical characteristics of enteric infections in young children? Eur J Epidemiol 2004;19:877-83.  Back to cited text no. 1
    
2.
Nitin S, Venkatesh V, Husain N, Masood J, Agarwal GG. Overview of intestinal parasitic prevalence in rural and urban population in Lucknow, north India. J Commun Dis 2007;39:217-23.  Back to cited text no. 2
    
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Garcia LS, Shimizu RY. Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens. J Clin Microbiol 1997;35:1526-9.   Back to cited text no. 3
    
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Duffy TL, Montenegro-Bethancourt G, Solomons NW, Belosevic M, Clandinin MT. Prevalence of giardiasis in children attending semi-urban daycare centres in Guatemala and comparison of 3 giardia detection tests. J Health Popul Nutr 2013;31:290-3.  Back to cited text no. 8
    
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Chakarova B. Comparative evaluation of the diagnostic methods for detection of Giardia intestinalis in human fecal samples. Trakia JSci 2010;8:174-9.  Back to cited text no. 9
    
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Ozekinci T, Uzun A, Suay A, Elçi S, Akpolat N, Atmaca S. Comparison of microscopy and EIA in the diagnosis of Giardia intestinalis in stool specimens. Turkiye Parazitol Derg 2005;29:89-92.  Back to cited text no. 12
    
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Guimarães S, Sogayar MI. Detection of anti-Giardia lamblia serum antibody among children of day care centers. Rev Saude Publica 2002; 36:63-8.  Back to cited text no. 13
    
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Correspondence Address:
Dr. Shipra Singhal
Junior Resident III, Department of Microbiology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.151192

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    Tables

  [Table 1], [Table 2]

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