Novel Duplex Polymerase Chain Reaction for the Rapid Detection of Pythium insidiosum Directly From Corneal Specimens of Patients With Ocular Pythiosis

Abstract

Purpose: To standardize a novel duplex polymerase chain reaction (PCR) targeting 18S rRNA gene and internal transcribed spacer region for the identification of Pythium insidiosum isolates and also to detect P. insidiosum genome directly from corneal specimens of patients with suspected ocular pythiosis. Methods: A total of 42 nonsporulating molds culturally and morphologically resembling suspected unidentified fungal isolates (corneal buttons 33 and corneal scrapings 9) and 14 clinical specimens (corneal buttons 7 and corneal scrapings 7) clinically suspected to be ocular pythiosis were included in the present study. Standardization of uniplex PCRs and duplex PCRs targeting 18S rRNA gene and internal transcribed spacer region and further application of the standardized PCRs on both clinical isolates and clinical specimens suspected to have fungal keratitis. The sensitivity and specificity of the standardized duplex PCR were calculated using Medcal.net software. Results: The standardized uniplex and duplex PCRs were found specific for the detection of only P. insidiosum DNA, and the analytical sensitivities of the primers were 1.36 Zg. Of the 14 clinical specimens analyzed, 13 were positive in both corneal specimens and their respective P. insidiosum isolates. The specificity of the novel duplex PCR was 100% when applied on corneal specimens and clinical isolates, but the sensitivity was 92.8% (13/14) and 100% (42/42), respectively, for the clinical specimens and fungal isolates from suspected ocular pythiosis patients included in the study. Conclusions: The novel duplex PCR developed in this study will aid in rapid identification of P. insidiosum clinical isolates and clinical specimens from suspected ocular pythiosis specimens, which in turn will help the ophthalmologists to initiate appropriate treatment.

Diagnostic armamentarium of infectious keratitis: A comprehensive review

“…A summary of the recent literature (i.e. studies published after year 2010) on the use of PCR in IK is provided in Table 3 [ [74] , [75] , [76] , [77] , [78] , [79] , [80] , [81] , [82] , [83] , [84] , [85] , [86] , [87] , [88] , [89] , [90] , [91] , [92] , [93] ].…”

“…Compared to 18S rRNA, ITS has been shown to exhibit higher species-level resolution in fungi, serving as a primary fungal barcode marker [ 95 ]. The overall sensitivity and specificity of PCR in diagnosing fungal keratitis are 57–91% and 79%, respectively [ [79] , [80] , [81] , [82] , [83] ]. Zhao et al [ 83 ] demonstrated that PCR utilizing ITS1 and ITS4 primers, which amplify ITS1 and ITS2 sequences respectively, had a significantly higher detection rate for fungal keratitis compared to conventional culture method (85% vs. 35%).…”

History and Perspective of Immunotherapy for Pythiosis

“…Patients with pythiosis usually manifest with clinical features associated with an infection of the skin [11,23,48,49], artery [13,50,51], eye [47,[52][53][54], gastrointestinal tract [28,[55][56][57], or another internal organ [5,12,[58][59][60]. Diagnostic methods have been established for pythiosis [61], such as culture identification [62][63][64], histopathological examination [65][66][67][68], serological assays [69][70][71][72][73][74][75], molecular techniques [76][77][78][79][80][81][82], and proteomic analysis [83,84].…”

Review of methods and antimicrobial agents for susceptibility testing against Pythium insidiosum

“…Early diagnosis and prompt treatment are critical factors to determine the favorable outcome of an affected individual. The diagnosis of pythiosis relies on clinical presentation and laboratory investigations [10,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68]. The use of conventional antifungal drugs is usually ineffective against P. insidiosum due to the lack of a drug-target ergosterol biosynthesis pathway [31,69].…”