Evaluation for the Clinical Diagnosis of Pythium insidiosum Using a Single-Tube Nested PCR

Yordhathai Thongsri,Petr Hamal,Lucie Svobodova,Chularut Prariyachatigul,Angkana Chaiprasert,Lumyai Wonglakorn,Maitree Pakarasang

doi:10.1007/s11046-013-9695-3

Abstract

Pythiosis is a rare infectious disease caused by Pythium insidiosum, which typically occurs in tropical and subtropical regions. The high mortality rate may be in consequence of the lack of diagnosis. The objective of this study was to evaluate reliability of a new single-tube nested PCR for detection of P. insidiosum DNA. A total of 78 clinical isolates of various fungi and bacteria, 106 clinical specimens and 80 simulated positive blood samples were tested. The developed primer pairs CPL6–CPR8 and YTL1–YTR1 are located on 18S subunit of the rRNA gene of P. insidiosum. The specificity, negative and positive predictive values were 100, 100 and 87.5 %, respectively, as compared with direct microscopy and cultivation. The detection limit of the single-tube nested PCR was 21 zoospores corresponding to 2.7 pg of the DNA. The results demonstrate that the new single-tube nested PCR offers a highly sensitive, specific and rapid genetic method for detecting P. insidiosum.

Highlights

The results demonstrate that the new single-tube nested PCR offers a highly sensitive, specific and rapid genetic method for detecting P. insidiosum
Pythiosis is an emerging and life-threatening infectious disease caused by the oomycete Pythium insidiosum, which can develop in humans and animals [1,2,3,4]
If P. insidiosum DNA was presented in clinical material, four amplicons sized 512, 452, 340 and 240 bp were detected by our nested PCR

Summary

Introduction

Pythiosis is an emerging and life-threatening infectious disease caused by the oomycete Pythium insidiosum, which can develop in humans and animals [1,2,3,4]. Human pythiosis can be divided into four types: cutaneous/subcutaneous, vascular, ocular and disseminated. Nested PCR has been developed for the diagnosis of pythiosis using the internal transcribed spacer 1 (ITS1) of the gene for rRNA [14]. It is highly sensitive, the main problem of this PCR is a high risk of contamination as the product of the first reaction needs to be transferred into another tube for the second reaction. The purpose of this study was to solve this problem by developing a nested PCR for detecting P. insidiosum in a single tube and to evaluate its reliability using various clinical specimens, including simulated positive blood samples and clinical isolates of bacteria and fungi

Objectives

Methods

Conclusion