|Year : 2013 | Volume
| Issue : 2 | Page : 154-160
Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa
NH Ahmed1, A Chowdhary2
1 Consultant Microbiology, Hospital lab, Apollo BSR Hospitals, Bhilai, Chhatisgarh, India
2 Department of Microbiology, Grant Medical College and Sir JJ Group of Hospitals, Mumbai, India
|Date of Submission||13-Aug-2012|
|Date of Acceptance||08-Jan-2013|
|Date of Web Publication||19-Jul-2013|
Department of Microbiology, Grant Medical College and Sir JJ Group of Hospitals, Mumbai
Source of Support: None, Conflict of Interest: None
Purpose: The study was conducted to compare different methods of detection of pathogenic protozoan parasites in stool specimens of People Living with HIV/AIDS (PLHA). Materials and Methods: Stool specimens of 242 HIV sero-positive patients were examined using the wet mount technique, modified Ziehl-Neelsen's (ZN) staining, auto-fluorescence and auramine fluorescence staining. Patient specimens, 94 and 40 out of 242, were also subjected to Giardia antigen detection using an enzyme immunoassay and Cryptosporidium antigen detection by immuno-chromatography, respectively. For calculation of sensitivity, specificity, positive and negative predictive values, light microscopy of wet mounts and modified ZN stained smears for Giardia and Coccidia, respectively, were considered as gold standards. Results: Sensitivity of auto-fluorescence, auramine-O staining and antigen detection techniques was found to be 100% as compared to the routine standards. The specificity of auto-fluorescence was 90.6% and 100% for Cyclospora and Isospora, respectively; that of auramine-O staining was 98.9% for Cryptosporidium, 99.30% for Cyclospora and 100% for Isospora; and that of antigen detection was 90.6% and 97.7% for Cryptosporidium and Giardia, respectively. Conclusion: In laboratories requiring screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection can be useful techniques. Confirmation of positive results, however, needs to be done with the standard techniques.
Keywords: Auto-fluorescence, auramine staining, antigen detection, modified ZN staining, people living with HIV/AIDS (PLHA)
|How to cite this article:|
Ahmed N H, Chowdhary A. Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa. Indian J Med Microbiol 2013;31:154-60
|How to cite this URL:|
Ahmed N H, Chowdhary A. Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa. Indian J Med Microbiol [serial online] 2013 [cited 2020 Nov 30];31:154-60. Available from: https://www.ijmm.org/text.asp?2013/31/2/154/115242
| ~ Introduction|| |
Intestinal protozoa are important cause of diarrhoea in developing countries, notable pathogens being Entamoeba histolytica and Giardia intestinalis. In immuno-compromised patients, especially in People Living with HIV/AIDS (PLHA); coccidian parasites and microsporidia also cause significant cases of diarrhoea. In such patients, the parasitic diarrhoeas are usually chronic and/or persistent and add to the morbidity and mortality of PLHA. , However, early detection of the causative parasites plays a significant role in implementing timely and correct treatment, which relieves the patients' symptoms and also prevents recurrences. While light microscopy is the standard diagnostic technique for diagnosis of enteric protozoa, other techniques are also picking up recognition. In the present study, we have compared different methods of detection of enteric protozoa in stool specimens of PLHA.
| ~ Materials and Methods|| |
Specimen collection and pathogen detection techniques
The study was conducted in a 1350-bedded tertiary care medical institute over a period of two years. Approval of Institutional Ethics Committee was taken prior to beginning the study. Stool specimens from 242 HIV sero-positive/AIDS patients attending out-patient department or admitted in wards, who presented with diarrhoea, were received in the microbiology laboratory. The specimens which met the standard criteria of acceptance were examined using light microscopy, fluorescence microscopy and antigen detection. For all microscopy, processing was done directly from fresh specimens of watery stools. In case of semi-solid and formed stools, processing was done from fresh specimen, as well as concentrated stool suspensions.
The following techniques were employed in order to detect the parasitic infections. ,,, Briefly;
- Wet mounts: Saline and iodine wet preparations were examined under low power (10X) and high power (40X) objectives of light microscope for detection of protozoal trophozoites and cysts.
- Modified Ziehl-Neelsen's (ZN) staining: Modified ZN staining (Kinyoun's modification of acid fast staining) was done on smears made from fresh specimen/concentrated suspension after methanol fixation. The slides were screened under low power (10X), high power (40X) and oil immersion (100X) objectives of light microscope for identification of the coccidian parasites - Cryptosporidium spp., Isospora belli and Cyclospora cayetanensis.
- Auto-fluorescence: This technique was used for identification of Isospora belli and Cyclospora cayetanensis oocysts. Saline wet mounts of the stool specimens were scanned under low power and high power objectives of fluorescence microscope with blue excitation filter (Wavelength = 450-490 nm) to detect the oocysts of Isospora and Cyclospora. The walls of the oocysts appeared fluorescent yellow-green due to property of auto-fluorescence [Figure 1] and [Figure 2].
|Figure 1: Auto-fluorescence image showing oocyst of Isospora belli; the arrow indicates the oocyt in faecal mass, wavelength 450-490 nm, ×10|
Click here to view
|Figure 2: Auto-fluorescence image showing oocyst of Cyclospora cayetanensis. The arrow indicates a typical auto-fluorescent oocyst wall, wavelength 450-490 nm, ×40|
Click here to view
- Auramine-O staining: This technique was used for identification of Cryptosporidium spp., I. belli and C. cayetanensis. In this, heat-fixed smears were primarily stained with auramine-O stain and were then decolorised with acid alcohol. Counter staining was done using potassium permanganate. The oocysts of Cryptosporidium spp., I. belli and C. cayetanensis showed apple green fluorescence with blue excitation filter (Wavelength = 450-490 nm), under high power and oil immersion objectives of fluorescence microscope [Figure 3], [Figure 4] and [Figure 5].
|Figure 3: Auramine-O stain showing oocysts of Cryptosporidium sp, wavelength 450-490 nm, ×100|
Click here to view
|Figure 4: Auramine-O stain showing oocysts of Cyclospora cayetanensis, wavelength 450-490 nm, ×100|
Click here to view
|Figure 5: Auramine-O stain showing oocyst of Isospora belli, wavelength 450-490 nm, ×100|
Click here to view
- Antigen detection techniques: Antigen detection techniques were employed for diagnosing Cryptosporidium and Giardia infections using the commercially available kits. For detection of Cryptosporidial antigen, rapid test based on quick immune-chromatography (RIDA Quick Cryptosporidium [N 1203], Ridascreen biopharmaceuticals, Darmstadt, Germany) was used. This was done in 40 out of 242 specimens using manufacturer's protocol. Giardia antigen detection was done using an enzyme immunoassay (RIDASCREEN Giardia [C 1101], Ridascreen biopharmaceuticals, Darmstadt, Germany), in 94 out of 242 stool specimens following manufacturers protocol.
For comparative analysis of different techniques for identification of parasitic protozoa, standard light microscopy techniques used routinely for respective parasites were taken as gold standard. For coccidian parasites, the routine standard is Kinyoun's modification of ZN staining; and for Giardia, it is the examination of saline and iodine wet mounts. The sensitivities, specificities, negative and positive predictive values of the tests were calculated by Fisher's exact test using Graphpad Prism software.
| ~ Results|| |
A total of 242 patient specimens were studied in the present study for comparing techniques for detection of four pathogenic protozoa, including Cryptosporidium spp., I. belli, C. cayetanensis and G. intestinalis in PLHA. In the study population, identification of Cryptosporidium spp. was done by auramine-O fluorescence staining for all 242 stool specimens and Cryptosporidium antigen detection in 40 out of 242 specimens. Considering modified ZN as standard, the sensitivity, specificity, positive and negative predictive values of auramine-O fluorescence staining were 100%, 98.9%, 72.7% and 100%, respectively (P < 0.0001); while identification of Cryptosporidium antigen in 40 samples showed 100% sensitivity, 90.6% specificity, 72.7% positive predictive value and 100% negative predictive value (P < 0.0001) [Table 1] and [Table 5].
|Table 1: Evaluation of auramine-O and rapid antigen detection techniques for identification of Cryptosporidium spp. compared to modified ZN staining as standard|
Click here to view
For identification of I. belli, all 242 stool specimens were subjected to modified ZN staining, wet mount examination, auto-fluorescence detection and auramine-O staining. Sensitivity and specificity of wet mount light microscopy for detection of Isospora was 54.5% and 100%, respectively, while the positive and negative predictive values were 100% and 96.4%, respectively, when compared with modified ZN staining (P < 0.001). Whereas sensitivity, specificity, and positive and negative predictive values of using auto-fluorescence and Auramine-O staining for Isospora detection were all 100% (P < 0.0001) [Table 2] and [Table 5].
|Table 2: Evaluation of different techniques for identification of Isospora belli compared to modified ZN staining as standard|
Click here to view
The sensitivity of light microscopy of wet mounts for detection of Cyclospora cayetanensis was very low when compared with modified ZN staining for Coccidia, i.e., 33.3%, however; specificity, positive and negative predictive values were high, 100%, 100% and 92.7%, respectively (P < 0.0124). Auto-fluorescence for detection of Cyclospora cayetanensis showed sensitivity and specificity of 100% and 98.9%, respectively. Positive and negative predictive values were 50% and 100%, respectively (P < 0.0001). The sensitivity, specificity, positive and negative predictive values of auramine-O fluorescence staining for detection of Cyclospora cayetanensis were 100%, 99.3%, 60% and 100%, respectively (P < 0.0001) [Table 3] and [Table 5].
|Table 3: Evaluation of different techniques for identification of Cyclospora cayetanensis compared to modified ZN staining as standard|
Click here to view
Identification of Giardia antigen detection by enzume linked immunosorbent assay was done for 94 specimens, and the findings of ELISA were compared with the light microscopic findings of the wet mounts. Considering light microscopy results as standard, the sensitivity, specificity, positive and negative predictive values were 100%, 97.7%, 77.7% and 100%, respectively (P < 0.0001) [Table 4] and [Table 5].
|Table 4: Evaluation of antigen detection by ELISA for identification of Giardia intestinalis compared to wet mount technique as standard|
Click here to view
|Table 5: Comparison of the diagnostic techniques for identification of the parasitic protozoa using Fisher's exact test|
Click here to view
| ~ Discussion|| |
Examination of temporary wet mounts and smears stained with modified ZN technique under light microscope is the standard technique used for detecting intestinal parasites in most of the laboratories. This technique is user friendly and good for detection of ova, larvae, trophozoites, cysts and oocysts of pathogenic intestinal parasites. However, light microscopic detection is time consuming and entails a trained and experienced person to screen the slides to identify any pathogenic protozoa. The sensitivity of the technique varies depending on the expertise and skill of the person screening the slides. Moreover, in high throughput laboratories, screening all specimens using light microscopy becomes a tedious job. Using highly sensitive fluorescence microscopy and antigen detection techniques for initial screening of stool specimens is a constructive alternative, with the provision of confirmation of positive findings using standard light microscopy.
Fluorescence microscopic examination has an innate obligation of a fluorescence microscope; also, it requires a well trained person to view the preparation objectively and requires dexterous interpretation which includes some amount of subjectivity. However, in existence of such facility auto-fluorescence and auramine-O fluorescence microscopy are highly valuable for initial screening of stool specimens for diagnosing coccidial infections. The time taken for screening large number of specimens is greatly reduced using fluorescence microscopy. Nevertheless, positive results need to be confirmed with conventional standards; which can be done using the same mounts used for auto-fluorescence, and after re-staining the auramine-O stained smears with modified ZN technique.
In the present study, we have looked into the worth of fluorescence microscopy and antigen detection tests for rapid initial screening of stool samples for enteric protozoan parasites. [Table 6] summarises the findings of present study and those of previous similar studies. In our study, auramine-O fluorescence microscopy, used for detection of coccidian parasites showed 100% sensitivity, the specificity was low for Cryptosporidium and Cyclospora (98.9% and 99.3%, respectively) while it was 100% for Isospora. Auto-fluorescence, for detection of I. belli and C. cayetanensis showed 100% sensitivity. The specificity for detection of Isospora was 100%, whereas that for Cyclospora was 98.9%.
|Table 6: Comparison of key findings of present study with previous similar studies|
Click here to view
In the literature, there are studies highlighting the effectiveness of fluorescent antibody assays for diagnosing coccidian parasites. , MacPherson et al. and Tuli et al. have previously reported high sensitivity and specificity of fluorescence microscopy for detection of Cryptosporidium and Cyclospora. , These assays are no doubt highly sensitive as well specific, and are less time consuming; however, in the setting of developing countries, where the prevalence of these parasites is also high, the cost of investigation also needs to be taken care of. In our experience, auto-fluorescence and auramine-O fluorescence microscopy have the advantages of high sensitivity and rapid screening; the results can be made more specific by doing light microscopy of the specimens showing positive results with fluorescence microscopy.
In our study, antigen detection techniques for detection of Cryptosporidium and Giardia were found to be 100% sensitive, but less specific (specificity 90.6% and 97.7% for Cryptosporidium and Giardia, respectively). Various studies in the literature have reported sensitivity of antigen detection techniques for enteric protozoan parasites to be ranging from 44% to 98.3% and the specificity from 90% to 100%. ,,,, The disparity can in part be attributed to the selection of "gold standard" for the calculation of results.
The available antigen detection assays along with high sensitivity and specificity, also have advantages of being high throughput, rapid and easy to interpret compared to the conventional standards. Using antigen detection kits for diagnosis of intestinal amoebiasis can support in differentiating pathogenic Entamoeba histolytica from the non pathogenic and more common Entamoeba dispar, which is not possible using morphological examination; but is none-the-less vital in predicting clinical course and prescribing treatment.  Antigen detection kits are available which can diagnose multiple protozoan parasites, thus making the antigen tests more cost-effective; which is once again a valuable benefit for developing countries. , Moreover, the limitation of person to person variability in interpretation of results, as observed in microscopy based techniques, is also overcome using antigen detection kits.
| ~ Conclusion|| |
For rapid, cost effective and proficient initial screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection techniques can be useful; positive results, however, need to be confirmed by standard techniques.
| ~ Acknowledgment|| |
We gratefully acknowledge the help from Ms. Tabish Hussain (ACTREC) for her valuable inputs in corrections.
| ~ References|| |
|1.||Kolter DP, Gaet HP, Lange M. Enteropathy associated with the Acquired Immuno Deficiency Syndrome. Ann Intern Med 1984;101:421-8. |
|2.||Mannheimer SB, Soave R. Protozoal infections in patients with AIDS Cryptosporidium, Isospora, Cyclospora and Microspoidium. Infect Dis Clin North Am 1991;8:483-8. |
|3.||DPDx- Centers for disease control and prevention. Identification and diagnosis of parasites of public health concern. Available from: http://www.dpd.cdc.gov/dpdx. Last accessed on 2012 July 23]. |
|4.||Parija SC. Textbook of medical parasitology: 3 rd ed. New Delhi: All India Publishers and Distributors; 2008. p. 156-83, 294-9. |
|5.||Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, et al. Parasitology (Chapter 22). In: Color Atlas and Textbook of Diagnostic Microbiology. 6 th ed. New York: Lippincott Williams and Wilkins; 2006. p. 1245. |
|6.||Baveja UK. Parasitic infections of gastrointestinal tract associated with HIV/AIDS. In: Manual on laboratory diagnosis of common opportunistic infections associated with HIV/AIDS. 1999;48-56. |
|7.||Weitzel T, Dittrich S, Mo¨hl I, Adusu E, Jelinek T. Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples. Clin Microbiol Infect 2006;12:656-9. |
|8.||Fedorko DP, Williams EC, Nelson NA, Calhoun LB, Yan SS. Performance of Three Enzyme Immunoassays and Two Direct Fluorescence Assays for Detection of Giardia lamblia in Stool Specimens Preserved in ECOFIX. J Clin Microbiol 2000;38:2781-3. |
|9.||MacPherson DW, McQueen R. Cryptosporidiosis: Multiattribute Evaluation of Six Diagnostic Methods. J Clin Microbiol 1993;31:198-202. |
|10.||Tuli L, Singh DK, Gulati AK, Sundar S, Mohapatra TM. A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhoea in AIDS patients. BMC Microbiol 2010;10:11. |
|11.||Al-Saeed AT, Issa SH. Detection of Giardia lamblia antigen in stool specimens using enzyme-linked immunosorbent assay. East Mediterr Health J 2010;16:362-4. |
|12.||Gonin P, Trudel L. Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay. J Clin Microbiol 2003;41:237-41. |
|13.||Garcia LS, Shimizu RY, Bernard CN. Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the Triage Parasite Panel Enzyme Immunoassay. J Clin Microbiol 2000;38:1267-8. |
|14.||Johnston SP, Ballard MM, Beach MJ, Causer L, Wilkins PP. Evaluation of three commercial assays for detection of Giardia and Cryptosporidium in stool specimens. J Clin Microbiol 2003;41:623-6. |
|15.||Jayalakshmi J, Appalaraju B, Mahadevan K. Evaluation of an enzyme linked immunoassay for the detection of Cryptosporidium antigen in fecal specimens of HIV/AIDS patients. Indian J Pathol Microbiol 2008;51:137-8. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]