Comparative Study of Wheatley’s Trichrome Stain and In-vitro Culture against PCR Assay for the Diagnosis of Blastocystis sp. in Stool Samples

  • Nabilah Amelia MOHAMMAD Center of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, Se-langor, Malaysia
  • Mohd Fahmi MASTUKI Center of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, Se-langor, Malaysia
  • Hesham M. AL-MEKHLAFI Endemic and Tropical Diseases Unit, Medical Research Center, Jazan University, Jazan, Kingdom of Saudi Arabia Dept. of Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, Sana’a, Yemen
  • Norhayati MOKTAR Dept. of Pre-Clinical Sciences, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Sungai Long Campus, Selangor, Malaysia
  • Tengku Shahrul ANUAR Center of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, Se-langor, Malaysia Integrative Pharmacogenomics Institute, Universiti Teknologi MARA, Puncak Alam Campus, Selangor, Malaysia
Keywords: Trichrome stain, In-vitro culture, PCR, Blastocystis

Abstract

Background: This study evaluated the performance of routine permanent stain and cultivation method in comparison with polymerase chain reaction assay as the reference technique to detect Blastocystis sp.Methods: A cross-sectional study was conducted among aboriginal populations that reside in Pahang, Peninsular Malaysia in Feb to Mar 2015. A total of 359 stool samples were examined using Wheatley’s trichrome stain, in-vitro cultivation in Jones’ medium and PCR assay. Positive amplicons were subjected to sequencing and phylogenetic analysis.Results: Fifty-six (15.6%) samples were detected positive with Blastocystis sp. by Wheatley’s trichrome stain and 73 (20.3%) by in-vitro culture, while PCR assay detected 71 (19.8%) positive samples. Detection rate of Blastocystis sp. was highest in combination of microscopic techniques (27.9%). The sensitivity and specificity of Wheatley’s trichrome staining and in-vitro culture techniques compared to PCR assay were 49.3% (95% CI: 37.2-61.4) and 92.7% (95% CI: 89.1-95.4) and 39.4% (95% CI: 28.0-51.8) and 84.4% (95% CI: 79.7-88.4), respectively. However, the sensitivity [60.6% (95% CI: 48.3-71.9)] of the method increased when both microscopic techniques were performed together. False negative results produced by microscopic techniques were associated with subtype 3. The agreement between Wheatley’s trichrome stain, in-vitro culture and combination of microscopic techniques with PCR assay were statistically significant by Kappa statistics (Wheatley’s trichrome stain: K = 0.456, P<0.001; in-vitro culture: K = 0.236, P<0.001 and combination techniques: K = 0.353, P<0.001).Conclusion: The combination of microscopic technique is highly recommended to be used as a screening method for the diagnosis of Blastocystis infection either for clinical or epidemiological study to ensure better and accurate diagnosis.

References

Li LH, Zhou XN, Du ZW et al. Molecular epidemiology of human Blastocystis in a vil-lage in Yunnan province, China. Parasitol Int. 2007;56(4):281-6.

Wong KH, Ng GC, Lin RT et al. Predom-inance of subtype 3 among Blastocystis iso-lates from a major hospital in Singapore. Parasitol Res. 2008;102(4):663-70.

Anuar TS, Ghani MK, Azreen SN, Salleh FM, Moktar N. Blastocystis infection in Ma-laysia: Evidence of waterborne and hu-man-to-human transmissions among the Proto-Malay, Negrito and Senoi tribes of Orang Asli. Parasit Vectors. 2013;6:40.

Stensvold CR, Arendrup MC, Nielsen HV, Bada A, Thorsen S. Symptomatic infection with Blastocystis sp. subtype 8 successfully treated with trimethoprim–sulfamethoxazole. Ann Trop Med Parasi-tol. 2008;102(3):271-4.

Tan KS. New insights on classification, identification, and clinical relevance of Blas-tocystis spp. Clin Microbiol Rev. 2008;21(4):639-65.

Lee LI, Chye TT, Karmacharya BM, Go-vind S. Blastocystis sp.: waterborne zoonotic organism, a possibility. Parasit Vectors. 2012;5:130.

Souppart L, Moussa H, Cian A et al. Sub-type analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitol Res. 2010;106(2):505-11.

Alfellani MA, Taner-Mulla D, Jacob AS et al. Genetic diversity of Blastocystis in live-stock and zoo animals. Protist. 2013;164(4):497-509.

Boorom KF, Smith H, Nimri L E et al. Oh my aching gut: irritable bowel syn-drome, Blastocystis, and asymptomatic infec-tion. Parasit Vectors. 2008;1(1):40.

Tan TC, Suresh KG, Smith HV. Pheno-typic and genotypic characterization of Blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential. Parasitol Res. 2008;104(1):85-93.

Eida AM, Eida MM. Identification of Blas-tocystis hominis in patients with irritable bow-el syndrome using microscopy and culture compared to PCR. Parasitol United J. 2008; 1(2):87-92.

Santos HJ, Rivera WL. Comparison of direct fecal smear microscopy, culture, and polymerase chain reaction for the detec-tion of Blastocystis sp. in human stool sam-ples. Asian Pac J Trop Med. 2013;6(10):780-4.

Elghareeb AS, Younis MS, El Fakahany AF et al. Laboratory diagnosis of Blastocystis spp. in diarrheic patients. Trop Parasitol. 2015;5(1):36-41.

Rajamanikam A, Govind SK. Amoebic forms of Blastocystis spp.-evidence for a pathogenic role. Parasit Vectors. 2013; 6(1):295.

Stensvold R, Brillowska-Dabrowska A, Nielsen HV, Arendrup MC. Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reac-tion. J Parasitol. 2006;92(5):1081-7.

Roberts T, Barratt J, Harkness J, Ellis J, Stark D. Comparison of microscopy, cul-ture, and conventional polymerase chain reaction for detection of Blastocystis sp. in clinical stool samples. Am J Trop Med Hyg. 2011;84(2):308-12.

Anuar TS, Al-Mekhlafi HM, Abdul Ghani MK et al. Evaluation of formalin-ether sedimentation and trichrome staining techniques: its effectiveness in detecting Entamoeba histolytica/dispar/moshkovskii in stool samples. J Microbiol Methods. 2013;92(3):344-8.

Salleh FM, Anuar TS, Yasin AM, Moktar N. Wintergreen oil: a novel method in Wheatley's trichrome staining technique. J Microbiol Methods. 2012;91(1):174-8.

JONES WR. The experimental infection of rats with Entamoeba histolytica; with a method for evaluating the anti-amoebic properties of new compounds. Ann Trop Med Parasitol. 1946;40:130-40.

Böhm‐Gloning B, Knobloch J, Walderich B. Five subgroups of Blastocystis hominis iso-lates from symptomatic and asymptomatic patients revealed by restriction site analysis of PCR‐amplified 16S‐like rDNA. Trop Med Int Health 1997; 2(8):771-778.

Stensvold CR, Arendrup MC, Jes-persgaard C, Mølbak K, Nielsen HV. De-tecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagn Microbiol Infect Dis. 2007; 59(3):303-7.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolu-tionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725-9.

Fegan DF. Evaluation of diagnostic tests: the epidemiological approach. DNA-based molecular diagnostic techniques. Research needs for standardization and validation of the detection of aquatic animal patho-gens and diseases. FAO Fish Tech Pap. 2000; 395: 30-37.

Markell EK, Udkow MP. Association of Blastocystis hominis with human disease? J Clin Microbiol. 1990; 28(5):1085.

Tan ZN, Wong WK, Nik Zairi Z et al. Identification of Entamoeba histolytica troph-ozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites. Trop Biomed. 2010; 27(1):79-88.

Bart A, Wentink-Bonnema EM, Gilis H et al. Diagnosis and subtype analysis of Blas-tocystis sp. in 442 patients in a hospital set-ting in the Netherlands. BMC Infect Dis. 2013;13:389.

Adıyaman Korkmaz G, Doğruman Al F, Mumcuoğlu İ. Investigation of the pres-ence of Blastocystis spp. in stool samples with microscopic, culture and molecular methods. Mikrobiyol Bul. 2015;49(1):85-97.

Dogruman-Al F, Simsek Z, Boorom K et al. Comparison of methods for detection of Blastocystis infection in routinely submit-ted stool samples, and also in IBS/IBD Patients in Ankara, Turkey. PLoS One. 2010;5(11):e15484.

Termmathurapoj S, Leelayoova S, Aimpun P et al. The usefulness of short-term in vitro cultivation for the detection and mo-lecular study of Blastocystis hominis in stool specimens. Parasitol Res. 2004;93(6):445-7.

Parkar U, Traub RJ, Kumar S M et al. Di-rect characterization of Blastocystis from fae-ces by PCR and evidence of zoonotic po-tential. Parasitology. 2007;134(Pt 3):359-67.

Leelayoova S, Taamasri P, Rangsin R et al. In-vitro cultivation: a sensitive method for detecting Blastocystis hominis. Ann Trop Med Parasitol. 2002;96(8):803-7.

Zierdt CH, Swan JC. Generation time and growth rate of the human intestinal para-site Blastocystis hominis. J Protozool. 1981; 28(4):483-485.

Suresh K, Smith H. Comparison of meth-ods for detecting Blastocystis hominis. Eur J Clin Microbiol Infect Dis. 2004;23(6):509-11.

Abu-Madi M, Aly M, Behnke JM, Clark CG, Balkhy H. The distribution of Blasto-cystis subtypes in isolates from Qatar. Para-sit Vectors. 2015;8:465.

Popruk S, Udonsom R, Koompapong K et al. Subtype Distribution of Blastocystis in Thai-Myanmar Border, Thailand. Korean J Parasitol. 2015;53(1):13-9.

How to Cite
1.
MOHAMMAD NA, MASTUKI MF, M. AL-MEKHLAFI H, MOKTAR N, ANUAR TS. Comparative Study of Wheatley’s Trichrome Stain and In-vitro Culture against PCR Assay for the Diagnosis of Blastocystis sp. in Stool Samples. IJPA. 13(1):127-36.
Section
Short Communication(s)