Utility of quantitative loop mediated isothermal amplification (qLAMP) assay for the diagnosis of invasive pneumococcal disease (IPD).

PURPOSE: Quantitative LAMP (qLAMP) assay is one of the recent and emerging diagnostic tests for infectious diseases. Only a few studies exist comparing this assay with quantitative real-time PCR (qPCR) for the diagnosis of invasive pneumococcal disease (IPD). AIM: To compare the diagnostic performance of qLAMP assay with qPCR targeting autolysin gene for the diagnosis of invasive pneumococcal disease. METHODS: Ninety six blood samples and 73 CSF samples from patients clinically suspected with community acquired pneumonia and acute meningitis were tested by qPCR and qLAMP assays using previously published primers and protocols. The qPCR was considered as the gold standard test and the diagnostic performance was assessed by calculating sensitivity, specificity, positive and negative predictive values, and kappa coefficient for the level of agreement between the tests. Chi-squared/Fisher exact test was used to compare categorical variables (positive/negative). RESULTS: Thirty two blood samples and 22 CSF samples were positive by qPCR while 24 and 20 samples were positive by qLAMP assay respectively. The sensitivity of qLAMP assay was only 86.4% and 75% when tested on CSF and blood samples respectively. However, the qLAMP assay was in substantial to almost perfect agreement when compared with qPCR. The results were statistically significant in both sample types (P < 0.001). CONCLUSIONS: The performance of qLAMP assay can vary based on the specimen type. It has very high specificity and had substantial to almost perfect agreement, and thus may be an alternative to qPCR for the diagnosis of IPD.

Structure Elucidation and Interaction Dynamics of MefA-MsrD Efflux Proteins in Streptococcus pneumoniae: Impact on Macrolide Susceptibility.

Macrolides are empirically used to treat bacterial community-acquired pneumonia (CAP). Streptococcus pneumoniae, being the major pathogen responsible for bacterial CAP with high mortality rates, express MefA-MsrD efflux pumps to hinder macrolide susceptibility. Despite its importance, the structural features of the efflux-protein complex and its impact on macrolide susceptibility have not yet been elucidated explicitly. Therefore, in the present study, combining homology, threading, and dynamics approaches, MefA and MsrD proteins in pathogenic S. pneumoniae were modeled. Both membrane (lipid-bilayer) and cytoplasmic (aqueous) environments were considered to simulate the MefA and MsrD proteins in their ideal cellular conditions followed by dynamics analyses. The simulated MefA structure represented a typical major facilitator superfamily protein structure with 13 transmembrane helices. MefA-MsrD interaction via clustering-based docking revealed low-energy conformers with stable intermolecular interactions. The higher clinical MIC value of azithromycin over erythromycin was reflected upon erythromycin eliciting stronger interactions (dissociation constant or ki = ∼52 µM) with the cytoplasmic ATP-binding MsrD than azithromycin (ki = ∼112 µM). The strong (binding energy = -132.1 ± 9.5 kcal/mol) and highly stable (root-mean-square fluctuation < 1.0 Å) physical association between MefA with MsrD was validated and was found to be unaffected by the antibiotic binding. Higher propensity of the macrolides to interact with MsrD than MefA established the importance of the former in macrolide susceptibility. Ours is probably the first report on the structural arrangements in the MefA-MsrD efflux complex and the macrolide susceptibility in S. pneumoniae. This study provides a novel lead for experimental explorations and efflux-pump inhibitor designs.

Variable Virulence Genes in Clinical Isolates of Burkholderia pseudomallei : Impact on Disease Severity and Outcome in Melioidosis.

Objectives To isolate Burkholderia pseudomallei from clinical specimens and study the association of virulence genes with clinical manifestations and outcome in patients with melioidosis. Materials and Methods Burkholderia pseudomallei isolates obtained from melioidosis cases diagnosed during 2018 to 2021 were identified using VITEK 2 system and confirmed by polymerase chain reaction (PCR) targeting a Type III secretion system gene cluster. Multiplex PCR was performed to detect the genotypes of lipopolysaccharide (LPS) namely A, B, and B2, and singleplex PCR was performed to detect the presence of the Burkholderia intracellular motility gene ( BimA ) and filamentous hemagglutinin gene ( fhaB3 ). Statistical Analysis Chi-square/Fisher's exact tests were performed to study the association between various clinical manifestations and outcome and different virulence genes. The results were expressed as unadjusted odds ratios with 95% confidence intervals. Results Sixty-seven isolates were available for characterization. BimA Bm and BimA Bp were observed among 82 and 18% of the isolates, respectively. Both sepsis and mortality were significantly associated with BimA Bm . Majority of the isolates had fhaB3 (97%). Most of the isolates showed the presence of LPS A gene (65.7%) followed by LPS B gene (6%), while LPS B2 was not detected. Nineteen isolates could not be assigned to any LPS genotypes. Conclusion Among the virulence genes studied, only BimA Bm was significantly associated with sepsis and mortality. More than a quarter (28.3%) of the isolates could not be assigned to any LPS genotypes, hinting at a greater genetic diversity in our isolates.

Validation of pneumococcal iron acquisition (piaA) gene for accurate identification of Streptococcus pneumoniae.

PURPOSE: The pneumococcal iron acquisition (piaA) gene is found to be highly specific and hence proposed as a diagnostic marker for identification of pneumococci. The objective of the present study was to evaluate the piaA gene as a genetic marker for the identification of pneumococci. METHODS: Twenty isolates were initially sequenced for lytA gene using published primers. PiaA-PCR (piaA polymerase chain reaction) was performed using in-house primers and protocol. Based on the sensitivity and specificity results, a final sample of 30 pneumococcal isolates and 11 non-pneumococcal isolates confirmed with lytA- sequencing were selected. Statistical analyses were performed using OpenEpi v3.01 and GraphPad Quickcalc at P < 0.05 as the level of statistical significance. RESULTS: Of the initial 20 samples tested, piaA PCR was positive in only 71.43% (10/14) of the pneumococcal isolates but was 100% specific (0/6 non-pneumococcal isolates) P = 0.011. When the PCR was performed on 41 samples, the sensitivity increased to 73.33% (95% of confidence interval [CI] = 55.55-85.82) and specificity remained the same P < 0.001. The level of agreement between the PCR and lytA-sequencing was found to be moderate (κ = 0.694; 95% CI = 0.432-0.955). CONCLUSIONS: PiaA-PCR can be used as a specific marker for the identification of pneumococcus, though it is less sensitive. As the level of agreement was moderate, further analyses on a large number of samples can give conclusive evidence for its use as a diagnostic marker for pneumococcus.