Development of Loop-mediated Isothermal Amplification (lamp) for detection of macrolide-resistant genes in Streptococcus Pneumoniae

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Kamuzu University of Health Sciences
Antibiotic resistance is fast becoming a major health problem, which makes it difficult to treat several infections, including Streptococcus pneumoniae infection. Increasing access to antibiotics, presumptive treatment especially in low-resource settings, and self-medication are some of the factors contributing to the rise in antibiotic resistant pathogens. Therefore, a robust surveillance platform for emergence of drug-resistant pathogens is a public health imperative. Diagnostic tools that could help with early detection, monitoring of resistance patterns and determining the prevalence of pathogens and pathogen resistance are urgently needed. In this study we have addressed the latter need by developing a method for the detection of macrolide resistant Streptococcus pneumoniae using loop-mediated isothermal amplification (LAMP) assay. The LAMP assay has increased specificity and sensitivity with a reduced time frame of detection. Rational: There is a growing need to develop diagnostic tools in early detection of antimicrobial resistance and real time surveillance. We aimed to develop a LAMP method for the rapid detection of antimicrobial resistant S. pneumoniae that could help in monitoring macrolide resistance in resource limited areas. Methods: A laboratory method, development study was conducted to develop a LAMP assay for the detection of macrolide resistance genes in S. pneumoniae from Blantyre, Malawi. S. pneumoniae isolates were obtained from an on-going community-based pneumococcal carriage surveillance project. Nasopharyngeal swabs were collected from children below the age of five in urban Blantyre. Isolates’ resistance profiles including erythromycin and azithromycin resistance were determined using phenotypic methods at Malawi-Liverpool-Wellcome Trust (MLW) laboratories. Whole genome sequencing (WGS) was done at the University of Oxford, United Kingdom. LAMP primers for the detection of macrolide resistance genes mefA and ermB were designed from two sequenced S. pneumoniae isolates genomes using Premier Biosoft software. The cut-off for amplification and the sensitivity and specificity of the LAMP primers was determined using the Receiver operating characteristic curve (ROC) Antimicrobial resistance phenotyping on culture was used as the gold standard test. Resistance results turn-around time was calculated for both culture and the LAMP assay. Results: 79 S. pneumoniae isolates were analyzed on the LAMP assay and results were compared to conventional culture method. Primers designed to detect azithromycin, mefA had sensitivity = 97.1% and specificity = 100%, at 95% confidence interval 0.952 to 1.000; while for erythromycin, ermB had sensitivity = 97.1% and specificity = 95.8%, at 95% confidence interval 0.452 to 0.701. LAMP assay amplified both azithromycin and erythromycin resistance genes within 30 minutes of the run. Furthermore, detection time for both ermB and mefA primers including DNA isolation and LAMP assay took <3 hours versus the conventional culture methods antimicrobial sensitivity testing (AST) which ranges from 24 to 48 hours to generate results. Conclusion: The study successfully developed and evaluated the performance of the LAMP assay for mefA and ermB gene in S. pneumoniae isolates. LAMP assay sensitivity and specificity performance for detection of mefA was excellent being a classifier of 0.98 the area under the Receiver Operating Curve (ROC). While for ermB it was an average classier with 0.57 area under ROC when both compared to conventional AST methods. The turnaround time for LAMP assay from pure isolate was less than 3 hours including nucleic acid isolation and detection compared to culture AST, which ranges from 24 to 48 hours. The study developed LAMP assay that can be used in the detection of bacterial resistance and provide results in timely fashion. Further evaluation of performance of the LAMP assays is recommended.