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

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Pages (from-to)127-136
Number of pages10
JournalIranian Journal of Parasitology
Volume13
Issue number1
Publication statusPublished - 1 Jan 2018
Externally publishedYes

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Blastocystis
Polymerase Chain Reaction
Blastocystis Infections
Mars
Culture Techniques
Malaysia
Epidemiologic Studies
Coloring Agents
Cross-Sectional Studies
trichrome stain
In Vitro Techniques
Staining and Labeling
Sensitivity and Specificity
Population

Keywords

  • Blastocystis
  • In-vitro culture
  • PCR
  • Trichrome stain

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

Cite this

Comparative study of wheatley’s trichrome stain and in-vitro culture against PCR assay for the diagnosis of Blastocystis sp. In stool samples. / Mohammad, Nabilah Amelia; Mastuki, Mohd Fahmi; Al-Mekhlafi, Hesham M.; Moktar, Norhayati; Anuar, Tengku Shahrul.

In: Iranian Journal of Parasitology, Vol. 13, No. 1, 01.01.2018, p. 127-136.

Research output: Contribution to journalArticle

Mohammad, Nabilah Amelia ; Mastuki, Mohd Fahmi ; Al-Mekhlafi, Hesham M. ; Moktar, Norhayati ; Anuar, Tengku Shahrul. / Comparative study of wheatley’s trichrome stain and in-vitro culture against PCR assay for the diagnosis of Blastocystis sp. In stool samples. In: Iranian Journal of Parasitology. 2018 ; Vol. 13, No. 1. pp. 127-136.
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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.",
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N2 - 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.

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