Iranian Journal of Parasitology 2018. 13(1):127-136.

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, Mohd Fahmi MASTUKI, Hesham M. AL-MEKHLAFI, Norhayati MOKTAR, Tengku Shahrul ANUAR

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.

Keywords


Trichrome stain; In-vitro culture; PCR; Blastocystis

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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.


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