A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum (original) (raw)
Related papers
Pythium insidiosum: An overview
Veterinary Microbiology, 2010
Pythium insidiosum is an oomycete pathogenic in mammals. The infection occurs mainly in tropical and subtropical areas, particularly in horses, dogs and humans. Infection is acquired through small wounds via contact with water that contains motile zoospores or other propagules (zoospores or hyphae). The disease, though described as emerging has in fact already been described since 1884. Depending on the site of entry, infection can lead to different forms of pythiosis i.e. a cutaneous, vascular, ocular, gastrointestinal and a systemic form, which is rarely seen. The infection is not contagious; no animal-animal or animalhuman transmission has been reported so far. Therapy includes radical surgery, antifungal drugs, immunotherapy or a combination of these therapies. The prevention to contract the disease in endemic areas is difficult. Avoiding stagnant waters could be of help, although the presence of P. insidiosum on grass and soil in enzootic areas renders this practice useless.
Pythium insidiosum sp. nov., the etiologic agent of pythiosis
Journal of Clinical Microbiology, 1987
Pythium insidiosum sp. nov., the etiologic agent of pythiosis, a cosmopolitan disease of horses, cattle, and dogs, is described and illustrated. Pythiosis (5) is a cosmopolitan granulomatous disease of horses, cattle, and dogs (10, 16, 17, 18) that is caused by a long-unnamed "phycomycete." (The term pythiosis was proposed in 1980 by Chandler et al. [5] as a more appropriate name for the equine disease variously referred to as bursatii, Florida horse leeches, granular dermatitis, hyphomycosis destruens equi, phycomycosis, phycomycotic granuloma, and swamp cancer.) It is probable that the first reports referring to this disease were those of Smith (20) and Drouin (9), who observed the mycelial nature of the etiologic agent. Although the organism could be cultured, it could not be identified, as it did not sporulate. de Haan and Hoogkamer (7) gave an extensive description of several cases of diseased horses in Indonesia and named the disease hyphomycosis destruens. This name was extended by de Haan (6) to hyphomycosis destruens equi. In a publication by Bridges and Emmons (4), the etiologic agent was called Hyphomyces destruens. It was not clear from that publication whether the authors intended to introduce a new binomial or only a provisional name they were anticipating to validate in the future (see reference 21, article 34.1b). The binomial was proposed without a Latin description (21, article 36), and the nomenclatural type was not designated for H. destruens (21, article 37). The binomial H. destruens, therefore, was in direct violation of articles 34.1b, 36, and 37 of the International Code of Botanical Nomenclature (21). Bridges and Emmons considered H. destruens to be a phycomycete (zygomycete) on the basis of its morphology in equine tissue as well as its broad, branched, sparsely septate to coenocytic, nonsporulating mycelium in cultures. They could not induce sporulation when H. destruens was grown on a wide variety of media. They speculated that the fungus they had studied "may be a species of Mortierella." Austwick and Copland (1) reported that isolates recovered from horses afflicted with swamp cancer in Papua, New Guinea, formed biflagellate zoospores. Zoospore formation occurred when the isolates, grown on Sabouraud dextrose agar, were transferred to a sterilized aqueous medium of rotten maize silage. They concluded that H. destruens was a phycomycete belonging to the Pythiaceae in the Peronosporales and that it could be included in the genus Pythium Pringsheim. These investigators also stated that "Further work is in progress to establish whether it is a recognized or new species." However, additional work on the identity of this oomycete of the kingdom Protoctista was not published.
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.
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.
Mycological Research, 1994
Polyclonal antibodies were prepared against crude cell wall fractions of a Pythium isolate with filamentous non-inflated sporangia, associated with root rot of tomato in soilless cultures. In ELISA tests, the antiserum showed sensitivity and specificity against original antigens and those from closely related isolates with filamentous sporangia (such as Pythium aquatile and Pythium coloratum). The antiserum reacted weakly with mycelial preparations of several fungi commonly found on tomato roots, such as Fusarium spp. or Penicillium spp. An immunocytochemical staining test was developed in order to study the colonization by filamentous noninflated sporangial isolates of Pythium in the presence of other fungi in soilless cultures.
Clinical and Vaccine Immunology, 2009
Studies by Western blot analyses have shown that antibodies in the sera of host species infected by Pythium insidiosum recognized several prominent proteins expressed by this fungus-like pathogen. Although these studies have utilized sera from infected patients and relevant local strains of P. insidiosum, the results are difficult to compare because of the lack of method standardization. In an effort to resolve this issue, we have utilized standardized methodologies to evaluate six P. insidiosum strains from Asia and the Americas and 15 serum samples from cattle, cats, dogs, horses, and humans with pythiosis from the same geographical regions. Our data show that the antibodies present in these sera recognize a wide variety of unique P. insidiosum immunogenic proteins. Although some of the prominent proteins in this study have been previously reported, several others have yet to be described. For instance, a ∼28-kDa-molecular-mass antigen was detected by the antibodies in all serum s...
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.
Journal of Medical Microbiology, 2015
Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-b-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a realtime (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1610 24 ng, respectively. In conclusion, the RT-PCR assay retained 100 % sensitivity and 100 % specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum.
Morphology, taxonomy, and phylogenetic analysis of a new species of Pythium isolated from France
Fungal …, 2008
During the course of investigation on pythiaceous fungi occurring in the burgundian vineyards, a new species of Pythium has been isolated. This oomycete is characterised by its non-proliferating and non sporulating type of sporangia (hyphal bodies), smooth walled oogonia that have hypogynous, monoclinous or at times diclinous antheridia, and smooth walled oospores that can have up to 3 per oogonia. The oomycete produces appressoria from which sexual structures may originate which is a rare feature for the genus. Sequence analyses of its ITS regions of rDNA show a close relationship with P. debaryanum and P. violae but has its own distinguishing characteristics. Morphological and molecular features of this isolate justify its description as a new species: Pythium viniferum. When grown together with Botrytis cinerea, the causal agent of the grey mould disease of grapevine, this oomycete shows a pronounced antagonism and suppresses its growth. Since it is not pathogenic to the grapevine it can be used as a bio-control agent. Morphology, antagonism with Botrytis cinerea, and the phylogenetic position of the new species are discussed here.