Concentrated specimen smear microscopy utilising a polymer membrane sandwich filtration vessel for the detection of acid-fast bacilli in health facilities in Sabah, East Malaysia

A simple, ready-to-use concentrated specimen smear microscopy method employing a nanometer silicon polyvinylidene fluoride (PVDF) polymer membrane sandwich filtration vessel to concentrate acid-fast bacilli (AFB) in samples (SFV-CSSM, Hunan-Tech New Medical System Co. Ltd. China) was compared with direct sputum smear microscopy (DSSM) to determine its performance using culture on modified Ogawa agar as reference. The results for 4114 clinical samples collected from health facilities in Sabah were interpreted with reference to culture results, sample collection-transportation conditions and clinical data including responses to anti-TB drug treatment. The SFV-CSSM showed higher sensitivity than DSSM (79.4% versus 60.5%) and less background interference. Its ability to detect low levels of AFB at an affordable cost makes it an excellent tool for the screening of pauci-bacillary samples as well as for active case finding in TB control programs

Molecular Identification of Nontuberculous Mycobacteria from Clinical Sources by hsp65 PRA and Sequence Analysis

Identification of nontuberculous mycobacteria (NTM) species is becoming increasingly important as differences in antimicrobial susceptibility of these microorganisms may affect treatment options. In this study, PCR-RFLP analysis (PRA) of the gene encoding 65-kD heat shock protein (hsp65) was used for identification of 70 clinical NTM isolates obtained from two mycobacteriology laboratories in Malaysia. Approximately one third of the isolates were identified as M. fortuitum. A total of 24.3% and 10% of the isolates were identified as M. abscessus and M. avium complex respectively. Other isolates identified were M. kansasii, M. marinum, M. simiae, M. parascrofulaceum, M. szulgai, and M. gordonae. Six clinical isolates were untypable by this approach. Sequence analysis of the 439-bp region of the hsp65 gene and a 564-bp region of the 16S rRNA gene revealed high degrees (97%–100%) of sequence similarity of these isolates with “M. insubricum”, M. terrae, M. senegalense/M. conceptionense and M. houstonense in the public sequence databases. This study reported the identification of mycobacteria species which are considered rare or new to this region. As NTM are found widely in the environment, the clinical significance of the isolates in this study is yet to be determined

Tigecycline resistance may be associated with dysregulated response to stress in Mycobacterium abscessus

Previously, we characterized 7C, a laboratory-derived tigecycline-resistant mutant of Mycobacterium abscessus ATCC 19977, and found that the resistance was conferred by a mutation in MAB_3542c, which encodes an RshAlike protein. In M. tuberculosis, RshA is an anti-sigma factor that negatively regulates the SigH-dependent heat/ oxidative stress response. We hypothesized that this mutation in 7C might dysregulate the stress response which has been generally linked to antibiotic resistance. In this study, we tested this hypothesis by subjecting 7C to transcriptomic dissection using RNA sequencing. We found an over-expression of genes encoding the SigH ortholog, chaperones and oxidoreductases. In line with these findings, 7C demonstrated better survival against heat shock when compared to the wild-type ATCC 19977. Another interesting observation from the RNA-Seq analysis was the down-regulation of ribosomal protein-encoding genes. This highlights the possibility of ribosomal conformation changes which could negatively affect the binding of tigecycline to its target, leading to phenotypic resistance. We also demonstrated that transient resistance to tigecycline could be induced in the ATCC 19977 by elevated temperature. Taken together, these findings suggest that dysregulated stress response may be associated with tigecycline resistance in M. abscessus

A mutation in anti-sigma factor MAB_3542c may be responsible for tigecycline resistance in Mycobacterium abscessus

In this study, we characterized 7C, a spontaneous mutant selected from tigecycline-susceptible Mycobacterium abscessus ATCC 19977. Whole-genome sequencing (WGS) was used to identify possible resistance determinants in this mutant. Compared to the wild-type, 7C demonstrated resistance to tigecycline as well as cross-resistance to imipenem, and had a slightly retarded growth rate. WGS and subsequent biological verifications showed that these phenotypes were caused by a point mutation in MAB_3542c, which encodes an RshA-like protein. In Mycobacterium tuberculosis, RshA is an anti-sigma factor that negatively regulates the heat/oxidative stress response mechanisms. The MAB_3542c mutation may represent a novel determinant of tigecycline resistance. We hypothesize that this mutation may dysregulate the stress-response pathways which have been shown to be linked to antibiotic resistance in previous studies

Tigecycline resistance may be associated with dysregulated response to stress in Mycobacterium abscessus

Previously, we characterized 7C, a laboratory-derived tigecycline-resistant mutant of Mycobacterium abscessus ATCC 19977, and found that the resistance was conferred by a mutation in MAB_3542c, which encodes an RshA-like protein. In M. tuberculosis, RshA is an anti-sigma factor that negatively regulates the SigH-dependent heat/oxidative stress response. We hypothesized that this mutation in 7C might dysregulate the stress response which has been generally linked to antibiotic resistance. In this study, we tested this hypothesis by subjecting 7C to transcriptomic dissection using RNA sequencing. We found an over-expression of genes encoding the SigH ortholog, chaperones and oxidoreductases. In line with these findings, 7C demonstrated better survival against heat shock when compared to the wild-type ATCC 19977. Another interesting observation from the RNA-Seq analysis was the down-regulation of ribosomal protein-encoding genes. This highlights the possibility of ribosomal conformation changes which could negatively affect the binding of tigecycline to its target, leading to phenotypic resistance. We also demonstrated that transient resistance to tigecycline could be induced in the ATCC 19977 by elevated temperature. Taken together, these findings suggest that dysregulated stress response may be associated with tigecycline resistance in M. abscessus

The use of the amplification refractory mutation system (arms) in the detection of rare beta-thalassemia mutations in the Malays and Chinese in Malaysia

Molecular characterization and prenatal diagnosis for beta-thalassemia can be carried out using the Amplification Refractory Mutation System (ARMS). The ARMS is a rapid and direct molecular technique in which beta-thalassemia mutations are visualized immediately after DNA amplification by gel electrophoresis. In the University of Malaya Medical Center, molecular characterization and prenatal diagnosis for beta-thalassemia is carried out using ARMS for about 96 of the Chinese and 84.6 of the Malay patients. The remaining 4 and 15.4 of the uncharacterized mutations in the Chinese and Malay patients respectively are detected using DNA sequencing. DNA sequencing is an accurate technique but it is more time-consuming and expensive compared with the ARMS. The ARMS for the rare Chinese beta-mutations at position -29 (A-->G) and the ATG-->AGG base substitution at the initiator codon for translation in the beta-gene was developed. In the Malays, ARMS was optimized for the beta-mutations at codon 8/9 (+G), Cap (+1) (A-->C) and the AATAAA-->AATAGA base substitution in the polyadenylation region of the beta-gene. The ARMS protocols were developed by optimization of the parameters for DNA amplification to ensure sensitivity, specificity and reproducibility. ARMS primers (sequences and concentration), magnesium chloride concentration, Taq DNA polymerase and PCR cycling parameters were optimized for the specific amplification of each rare beta-thalassemia mutation. The newly-developed ARMS for the 5 rare beta-thalassemia mutations in the Chinese and Malays in Malaysia will allow for more rapid and cost-effective molecular characterization and prenatal diagnosis for beta-thalassemia in Malaysia

Data_Sheet_1_Anxiety and coping strategies during the COVID-19 pandemic: A cross-sectional study of staff and students from a tertiary education center in Malaysia.PDF

AimWe examined the anxiety levels and coping strategies among staff and students of a tertiary educational institution during the COVID-19 pandemic and determined the association between anxiety level and coping strategies.MethodThrough an online survey, we used Coronavirus Anxiety Scale (CAS) to measure the level of anxiety associated with the COVID-19 crisis and Brief Coping Orientation to Problems Experienced (COPE) to assess the coping responses adopted to handle stressful life events. Coping strategies were classified as adaptive and maladaptive, for which the aggregate sores were calculated. Multiple linear regression was used to determine the predictors of anxiety adjusted for potentially confounding variables. Results from 434 participants were available for analysis.ResultsThe mean score (SD) of the CAS was 1.1 (1.8). The mean scores of adaptive and maladaptive coping strategies were 35.69 and 19.28, respectively. Multiple linear regression revealed that maladaptive coping [Adjusted B coefficient = 4.106, p-value ConclusionMaladaptive coping and presence of comorbidities were the predictors of coronavirus anxiety. The apparent lack of anxiety in relation to COVID-19 and movement restriction is reflective of the reported high level of satisfaction with the support and services provided during the COVID-19 outbreak in Malaysia. Adaptive coping strategies were adopted more frequently than maladaptive. Nevertheless, public education on positive coping strategies and anxiety management may be still be relevant to provide mental health support to address the needs of the general population.</p