Background Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. Method In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel. Results All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45–99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. Conclusion This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.
Cardiomyopathy (CMP) constitutes a diverse group of myocardium diseases affecting the pumping ability of the heart. Genetic predisposition is among the major factors affecting the development of CMP. Globally, there are over 100 genes in autosomal and mitochondrial DNA (mtDNA) that have been reported to be associated with the pathogenesis of CMP. However, most of the genetic studies have been conducted in Western countries, with limited data being available for the Asian population. Therefore, this study aims to investigate the mutation spectrum in the mitochondrial genome of 145 CMP patients in Malaysia. Long-range PCR was employed to amplify the entire mtDNA, and whole mitochondrial genome sequencing was conducted on the MiSeq platform. Raw data was quality checked, mapped, and aligned to the revised Cambridge Reference Sequence (rCRS). Variants were named, annotated, and filtered. The sequencing revealed 1,077 variants, including 18 novel and 17 CMP and/or mitochondrial disease-associated variants after filtering. In-silico predictions suggested that three of the novel variants (m.8573G>C, m.11916T>A and m.11918T>G) in this study are potentially pathogenic. Two confirmed pathogenic variants (m.1555A>G and m.11778G>A) were also found in the CMP patients. The findings of this study shed light on the distribution of mitochondrial mutations in Malaysian CMP patients. Further functional studies are required to elucidate the role of these variants in the development of CMP.
Cardiomyopathy comprises a diverse group of diseases affecting the myocardium. The genetic composition is one of the major disease-defining factors of cardiomyopathies, with globally more than 100 genes implicated in this pathogenesis. Most genetic studies were performed in the Western populations, with only limited data available for the Asian populations. In this study, 152 cardiomyopathy patients (104 dilated cardiomyopathy and 48 hypertrophic cardiomyopathy) were recruited. A total of 20 genetic mutations previously reported in Caucasian populations in MYBPC3, MYH7, TNNT2 and TPM1 genes were examined via Tetra Primer Amplification-Refractory Mutation System Polymerase Chain Reaction approach. Of all subjects, only one patient with hypertrophic cardiomyopathy was found as heterozygous carrier of a point mutation (c.1208G>A) in the MYH7 gene. The remaining 19 mutations were not observed among the cardiomyopathy patients in this study. Our finding suggests a different genetic architecture of the Malaysian cardiomyopathy patients compared to the Caucasian populations. Therefore, a more comprehensive mutation study on the Malaysian cardiomyopathy patients is essential for better illustration of the genetic causes of cardiomyopathy in Malaysia.
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