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Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay
  1. Samson S Y Wong1,2,
  2. Rosana W S Poon3,
  3. Kelvin K W To1,2,4,
  4. Jasper F W Chan1,2,4,5,
  5. Gang Lu5,6,7,
  6. Fanfan Xing4,
  7. Vincent C C Cheng3,
  8. Kwok-Yung Yuen1,2,3,4,5,8,9
  1. 1Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
  2. 2Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China
  3. 3Department of Microbiology, Queen Mary Hospital, Hong Kong, China
  4. 4Department of Clinical Microbiology and Infection Control, The University of Hong Kong–Shenzhen Hospital, Shenzhen, China
  5. 5Hainan Medical University–The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, Hainan Medical University, Haikou, Hainan, and The University of Hong Kong, Hong Kong, China
  6. 6Department of Pathogen Biology, Hainan Medical University, Haikou, Hainan, China
  7. 7Key Laboratory of Translational Tropical Medicine, Hainan Medical University, Haikou, Hainan, China
  8. 8State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China
  9. 9Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong, China
  1. Correspondence to Dr Samson S Y Wong, Department of Microbiology, The University of Hong Kong, Hong Kong, China; samsonsy{at}hku.hk

Abstract

Aims Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay.

Methods We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested.

Results The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples.

Conclusions The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

  • infections
  • microbiology
  • parasites
  • diagnostics

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Footnotes

  • Handling editor Dr Tony Mazzulli.

  • Contributors SSYW is responsible for the identification of parasites in the corresponding institution, acquisition of data, and prepared and approved the final version of the manuscript. RWSP is responsible for all aspects of molecular identification of the parasites. KKWT, JFWC and VCCC are responsible for acquisition of data and review of the manuscript. GL and FFX are responsible for acquisition of data and identification of parasites in the corresponding institutions. K-YY is responsible for initiation and supervision of the study and critical evaluation of the manusript.

  • Funding The study was partly funded by the High Level Hospital-Summit Program in Guangdong, China; donations from the Shaw Foundation Hong Kong, Dr Richard Yu and Carol Yu, Michael Seak-Kan Tong, Hui Ming, Hui Hoy, Chow Sin Lan Charity Fund Limited and Chan Yin Chuen Memorial Charitable Foundation.

  • Competing interests None declared.

  • Patient consent for publication Not required

  • Provenance and peer review Not commissioned; externally peer reviewed.

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