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

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.

Pythium keratitis in South India: Incidence, clinical profile, management, and treatment recommendation

“…In the laboratory, the combination of the classic colony morphology along with the zoospore production as well as molecular tools for the identification of Pythium will help in the identification and confirmation. [1926272829] Although this organism grows readily on common laboratory medium such as blood agar and potato dextrose agar, it might not grow from the early specimens, such as the first corneal scraping, and direct microscopy examination of the smear also may be negative. Although the clinical features are a strong indicator, laboratory confirmation is also very much needed to accurately classify as Pythium keratitis .…”

Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum

“…The LAMP interpretation is based on the presence of an amplicon, regardless of what the target gene is used. The resulting interpretation of other amplification methods relies not only on the presence of an amplicon, but also a more specific feature, such as melting temperature (i.e., real-time PCR) and molecular size (i.e., conventional, nested, and multiplex PCR) of the amplicon (Grooters and Gee, 2002;Thongsri et al, 2013;Keeratijarut et al, 2014Keeratijarut et al, , 2015Rujirawat et al, 2017). Further analysis by RFLP assay (Figure 5) could facilitate more accurate detection of a LAMP product (Notomi et al, 2000).…”

“…PCR amplification of the rDNA sequence by the universal fungal primers, followed by DNA sequencing and BLAST search against the GenBank database, have been used for the definitive identification of P. insidiosum for decades (Badenoch et al, 2001). The multistep procedure, limited efficiency, and long turnaround time of such sequence homology-based analysis can be overcome by using the P. insidiosum-specific primers, alternative target genes (i.e., exo1 and cox2), and advanced DNA amplification techniques, such as nested PCR (Grooters and Gee, 2002;Thongsri et al, 2013), singleround species-specific PCR (Vanittanakom et al, 2004;Keeratijarut et al, 2014), real-time PCR (Keeratijarut et al, 2015;Worasilchai et al, 2018b), thermophilic helicase DNA amplification (Worasilchai et al, 2018a), and multiplex PCR (Rujirawat et al, 2017;Weiblen et al, 2019;Kulandai et al, 2020). A primary concern regarding the molecular diagnosis for pythiosis is the lack of costly equipment in remote areas, where the disease is prevalent.…”

Recent update in diagnosis and treatment of human pythiosis

“…Therefore, a new set of P. insidiosum-specific primers (ITSpy1 and ITSpy2) was used in a simplified one round-PCR reaction (Vanittanakom et al, 2004) that was able to detect all four Thai strains tested. Thongsri et al reported a single-tube nested PCR using a different set of primers (CPL6, CPR8, YTL1, and YTR1) for specific detection of P. insidiosum (Thongsri et al, 2013). More recently, Rujirawat et al developed a multiplex PCR assay, using four primers (ITS1, Ra, R2, and R3) to target several single nucleotide polymorphisms of rDNA (Rujirawat et al, 2017).…”