Detection of the Oomycete Pythium insidiosum by Real-Time PCR Targeting the Gene Coding for Exo-1,3-β-Glucanase (original) (raw)
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Evaluation for the Clinical Diagnosis of Pythium insidiosum Using a Single-Tube Nested PCR
Mycopathologia, 2013
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.
Asian Biomedicine, 2014
Background: Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease. Diagnosis of pythiosis is difficult and often delayed. Early diagnosis can lead to prompt treatment, and therefore a better prognosis for patients with pythiosis. Molecular diagnostic techniques are useful if microbiological and immunological assays are not available, or in cases of suspected pythiosis that test negative by other methods. So far, PCR identification of P. insidiosum has been largely relied on amplification of the rDNA region. Objective: To evaluate the diagnostic performance of Dx3 and Dx4 primers specific for a putative gene for exo- 1,3-β-glucanase (PinsEXO1), which encodes a specific immunogen of P. insidiosum, for rapid single-round PCR identification of P. insidiosum, in comparison with the previously-reported rDNA-specific primers, ITSpy1 and ITSpy2. Materials and Methods: Genomic DNA (gDNA) from 35 P. insidiosum isolates and 48 control organisms were prep...
Journal of Clinical Microbiology, 2004
Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.
Ciência Rural
ABSTRACT: We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecu...
Pythium insidiosum: morphological and molecular identification of Brazilian isolates
Pesquisa Veterinária Brasileira, 2012
Pesq. Vet. Bras. 32 :619-622, julho 2012 619 RESUMO.-[Pythium insidiosum: identiϐicação morfológica e molecular de isolados brasileiros.] Pythium insidiosum é um oomiceto pertencente ao Reino Stramenopila e agente etiológico da pitiose, uma doença infecciosa com riscos de morte. A pitiose é caracterizada pelo desenvolvimento de lesões crônicas sobre os tecidos cutâneos, subcutâneas, intestinal e ósseo em humanos e muitas espécies de animais. A identiϐicação de P. insidiosum é importante, a ϐim de se obter um diagnóstico rápido e deϐinitivo, bem como um tratamento eϐicaz. Este estudo relata a identiϐicação de 54 isolados de P. insidiosum de cavalos, cães e ovelhas que apresentavam lesões compatíveis e suspeita clínicas de pitiose, provenientes de diferentes regiões do Brasil, através ABSTRACT.-Azevedo M.I., Pereira D.I.B., Botton S.A., Costa M.M., Mahl C.D., Alves S.A. & Santurio J.M. 2012. Pythium insidiosum: Morphological and molecular identiϐication of Brazilian isolates. Pesquisa Veterinária Brasileira 32 :619-622. Laboratório de Pesquisas Micológicas, Pythium insidiosum is an oomycete belonging to the kingdom Stramenipila and it is the etiologic agent of pythiosis. Pythiosis is a life-threatening infectious disease characterized by the development of chronic lesions on cutaneous and subcutaneous, intestinal, and bone tissues in humans and many species of animals. The identiϐication of P. insidiosum is important in order to implement a rapid and deϐinitive diagnosis and an effective treatment. This study reports the identiϐication of 54 isolates of P. insidiosum of horses, dogs and sheep that presented suspicious clinical lesions of pythiosis from different regions in Brazil, by using morphological and molecular assays. Throughout the PCR it was possible to conϐirm the identity of all Brazilian isolates as being P. insidiosum.
Medical Mycology, 2017
Pythiosis is caused by Pythium insidiosum, a fungus-like microbe belonging to the kingdom Stramenopila. Its diagnosis is challenging due to clinical and histopathological similarities with the fungal microbes that cause mucormycosis and entomophthoramycosis. In addition, the proper identification of P. insidiosum in the clinical laboratory is difficult. We have developed a rapid and accurate, species-specific identification method using a thermophilic helicase DNA amplification (tHDA) technique, to differentiate this pathogen from closely related pathogenic fungi. Sixty-seven fungal isolates, including 39 of P. insidiosum, were evaluated. A 91 base-pair (bp) DNA fragment was consistently amplified using a COX2 primer. The limiting concentrations of the one-and two-step tHDA protocols were 100 picograms (1.74 × 10 2 copies) and 100 femtograms (1.74 × 10 −1 copies), respectively. The CviKI-1 enzyme in restriction fragment length polymorphism (RFLP) with the 91 bp amplicons accurately separated P. insidiosum from other fungal species. The data suggest that this tHDA-RFLP assay is a rapid and accurate test for the identification of P. insidiosum. The potential use of the assay directly in clinical samples is also discussed.
Journal of Fungi
Pythium insidiosum causes pythiosis, a fatal infectious disease of humans and animals worldwide. Prompt diagnosis and treatment are essential to improve the clinical outcome of pythiosis. Diagnosis of P. insidiosum relies on immunological, molecular, and proteomic assays. The main treatment of pythiosis aims to surgically remove all affected tissue to prevent recurrent infection. Due to the marked increase in case reports, pythiosis has become a public health concern. Thailand is an endemic area of human pythiosis. To obtain a complete picture of how the pathogen circulates in the environment, we surveyed the presence of P. insidiosum in urban (Bangkok) and rural areas of Thailand. We employed the hair-baiting technique to screen for P. insidiosum in 500 water samples. Twenty-seven culture-positive samples were identified as P. insidiosum by multiplex PCR, multi-DNA barcode (rDNA, cox1, cox2), and mass spectrometric analyses. These environmental strains of P. insidiosum fell into Cl...