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. 2018 Jan-Mar;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  1 , Mohd Fahmi Mastuki  1 , Hesham M Al-Mekhlafi  2   3 , Norhayati Moktar  4 , Tengku Shahrul Anuar  1   5
Affiliations

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 et al. Iran J Parasitol. 2018 Jan-Mar.

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: Blastocystis; In-vitro culture; PCR; Trichrome stain.

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Conflict of interest statement

Conflict of interest The authors declare that there is no conflict of interest.

Figures

Fig. 1:
Fig. 1:
Vacuolar form with four nucleuses found at the rim of cytoplasm along with large and round vacuole at the center. a) Nucleus. b) Vacuole. c) Cytoplasm
Fig. 2:
Fig. 2:
Granular form revealed large central vacuole filled with dense granules and nucleus located along the thin rim of cytoplasm. a) Nucleus. b) Vacuole. c) Cytoplasm. d) Granules
Fig. 3:
Fig. 3:
Analysis of PCR products on a 1.5% (w/v) agarose gel. Lane 1) 100-bp DNA ladder, Lane 2) Negative control, Lane 3) Positive control, Lane 4–8) Positive Blastocystis isolates
Fig. 4:
Fig. 4:
Maximum likelihood tree displaying relationship amongst SSU rRNA gene sequences of Blastocystis from this study (bold line) with reference sequences (ST1-ST14) from GenBank. P. lacertae served as outgroup
See this image and copyright information in PMC

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