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

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

doi:10.1007/s11046-013-9695-3

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

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Author(s): Yordhathai Thongsri , Lumyai Wonglakorn , Angkana Chaiprasert , Lucie Svobodova , Petr Hamal , Maitree Pakarasang , Chularut Prariyachatigul

Publication date (Electronic): 15 August 2013

Journal: Mycopathologia

Publisher: Springer Netherlands

Keywords: Pythium insidiosum, Pythiosis, Single-tube nested PCR, 18S rRNA

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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.

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Theerapong Krajaejun, Boonmee Sathapatayavongs, Roongnapa Pracharktam … (2006)

Pythiosis is an emerging and life-threatening infectious disease in humans and animals that is caused by the pathogenic oomycete Pythium insidiosum. Human pythiosis is found mostly in Thailand, although disease in animals has been increasingly reported worldwide. Clinical information on human pythiosis is limited, and health care professionals are unfamiliar with the disease, leading to underdiagnosis, delayed treatment, and poor prognosis. To retrospectively study the clinical and epidemiological features of human pythiosis, we analyzed clinical data from patients with pythiosis diagnosed during the period of January 1985 through June 2003 at 9 tertiary care hospitals throughout Thailand. A total of 102 cases of human pythiosis were documented nationwide. A substantial proportion (40%) of cases occurred in the last 4 years of the 18-year study interval. Clinical presentations fell into 4 groups: cutaneous/subcutaneous cases (5% of cases), vascular cases (59%), ocular cases (33%), and disseminated cases (3%). Almost all patients with cutaneous/subcutaneous, vascular, and disseminated pythiosis (85%) had underlying thalassemia-hemoglobinopathy syndrome. Most ocular cases (84%) were associated with no underlying disease. A majority of the patients were male (71%), were aged 20-60 years (86%), and reported an agricultural occupation (75%). Regarding treatment outcomes, all patients with disseminated infection died; 78% of patients with vascular disease required limb amputation, and 40% of these patients died; and 79% of patients with ocular pythiosis required enucleation/evisceration. Here, we report, to our knowledge, the largest case study of human pythiosis. The disease has high rates of morbidity and mortality. Early diagnosis and effective treatment are urgently needed to improve clinical outcomes. Because P. insidiosum is distributed worldwide and can infect healthy individuals, an awareness of human pythiosis should be promoted in Thailand and in other countries.

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Hermann Voglmayr, Johann Greilhuber (1998)

Genome size was determined, by nuclear Feulgen staining and image analysis, in 46 accessions of 31 species of Peronosporales (Oomycota), including important plant pathogens such as Bremia lactucae, Plasmopara viticola, Pseudoperonospora cubensis, and Pseudoperonospora humuli. The 1C DNA contents ranged from 0.046 (45. 6 Mb) to 0.163 pg (159.9 Mb). This is 0.041- to 0.144-fold that of Glycine max (soybean, 1C = 1.134 pg), which was used as an internal standard for genome size determination. The linearity of Feulgen absorbance photometry method over this range was demonstrated by calibration of Aspergillus species (1C = 31-38 Mb) against Glycine, which revealed differences of less than 6% compared to the published CHEF data. The low coefficients of variation (usually between 5 and 10%), repeatability of the results, and compatibility with CHEF data prove the resolution power of Feulgen image analysis. The applicability and limitations of Feulgen photometry are discussed in relation to other methods of genome size determination (CHEF gel electrophoresis, reassociation kinetics, genomic reconstruction) that have been previously applied to Oomycota. Copyright 1998 Academic Press.

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Author and article information

Contributors

: +66-043202086 , +66-043202086 , chupra@kku.ac.th

Journal

Journal ID (nlm-ta): Mycopathologia

Journal ID (iso-abbrev): Mycopathologia

Title: Mycopathologia

Publisher: Springer Netherlands (Dordrecht )

ISSN (Print): 0301-486X

ISSN (Electronic): 1573-0832

Publication date (Electronic): 15 August 2013

Publication date PMC-release: 15 August 2013

Publication date (Print): 2013

Volume: 176

Pages: 369-376

Affiliations

[ ]Faculty of Graduated School, Khon Kaen University, Khon Kaen, Thailand

[ ]Microbiology Laboratory, Faculty of Medicine Srinagarind Hospital, Khon Kaen University, Khon Kaen, Thailand

[ ]Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

[ ]Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic

[ ]Centre for Research and Development of Medical Diagnostic Laboratories, Department of Microbiology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002 Thailand

Article

Publisher ID: 9695

DOI: 10.1007/s11046-013-9695-3

PMC ID: 3843749

PubMed ID: 23948967

SO-VID: 53d4d9fe-834a-4915-95b9-5a0f2a3e0f22

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.