Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo.

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056 T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.

Improving the clinical recognition, prognosis, and treatment of melioidosis through epidemiology and clinical findings: The Sabah perspective.

INTRODUCTION: Melioidosis is a deadly endemic disease in northern Australia and Southeast Asia, including Sabah, Malaysia, which is caused by the bacterium Burkholderia pseudomallei. It contributes to high fatality rates, mainly due to misdiagnosis leading to the wrong treatment being administered to the patients. Local epidemiology and data on clinical features could assist clinicians during diagnosis and treatment. However, these details are still scarce, particularly in Sabah. METHODS: A retrospective study of 246 culture-confirmed melioidosis cases in Queen Elizabeth Hospital, Sabah, Malaysia was performed between 2016 and 2018. The epidemiological data and clinical and laboratory findings were extracted and analysed. RESULTS: The annual incidence of culture-confirmed melioidosis cases was estimated to be 4.97 per 100,000 people. The mean age of the patients was 50±15 years. Males and members of the Kadazan-Dusun ethnic group accounted for the majority of the melioidosis cases. The odds ratio analysis indicated that bacteraemic melioidosis in this region was significantly associated with fever (76%), and patients having at least one underlying illness (43%), including diabetes mellitus (32%). Sixty-eight patients (28%) succumbed to melioidosis. Contrary to what is known regarding factors that promote bacteraemic melioidosis, neither patients with fever nor patients with at least one comorbid disease, including diabetes mellitus, were significantly associated with death from melioidosis. There was no statistically significant difference between patients without comorbidities (24, 27%) and those with at least one comorbid disease (26, 25%), including diabetes mellitus (18, 23%). The odds ratios indicate that melioidosis mortality in this region is related to patients showing respiratory organ-associated symptoms (29%), bacteraemia (30%), and septic shock (47%). Burkholderia pseudomallei isolates in this study were highly susceptible to ceftazidime (100%), imipenem (100%), and trimethoprim-sulfamethoxazole (98%). CONCLUSIONS: Information obtained from this study can be used by clinicians to recognise individuals with the highest risk of acquiring melioidosis, estimate an accurate prognosis, and provide effective treatment for melioidosis patients to reduce death from melioidosis.

True-positive reflex threshold value for HCV antibody screening test.

INTRODUCTION: The use of a signal-to-cut-off ratio has been recommended by the Centre for Disease Control and Prevention to determine the need for further validation using a supplemental test. In this study, we aimed to determine the optimal true-positive signal-to-cut-off ratio for the ABBOTT ARCHITECT i2000SR immunoassay (Abbott Laboratories, Illinois, USA), using the Serodia® HCV particle agglutination (HCV-PA) assay (Fujirebio Inc, Tokyo, Japan) as the reference test for anti-HCV screening. METHODOLOGY: We analysed a total of 13,240 specimens using the ARCHITECT i2000SR immunoassay and subsequently subjected all the reactive specimens with a signal-to-cut-off ratio ≥ 1.00 (n = 267) to the Serodia® HCV-PA reference assay. Receiver operating characteristic (ROC) curve analysis was carried out and performance characteristics for each signal-to-cut-off ratio were determined. The selected signal-to-cut-off ratio value was then assessed using a line immunoassay (LIA) test. RESULTS: ROC curve analysis determined that the optimal signal-to-cut-off ratio was 5.05, which gave the highest Youden's Index (J) value of 0.89, with a sensitivity of 93.1% (88.9-97.2), a specificity of 96.0% (92.4-99.4), a positive predictive value of 96.4% (93.3-99.5), and a negative predictive value of 92.2% (87.5-96.8). Validation of the optimal S/Co value using the LIA test yielded an accuracy of 91.8%, with sensitivity and specificity values of 92.0% and 91.7%, respectively. CONCLUSIONS: The optimal signal-to-cut-off ratio value for the ARCHITECT i2000SR immunoassay, which was determined using HCV-PA assay as the reference test and validated using a HCV-LIA assay, showed high sensitivity and specificity, and may be used in routine anti-HCV screening.