Abstract
ObjectivesChloramphenicol is a broad-spectrum antimicrobial widely available in sub-Saharan Africa. With susceptibility re-emerging among Enterobacteriaceae in Blantyre, Malawi, we designed and evaluated a new high-resolution melt (HRM) RT-PCR assay, ChloS-HRM, to identify chloramphenicol-susceptible infections in a hospital setting.MethodsSeventy-two previously whole-genome sequenced isolates of Escherichia coli and Klebsiella pneumoniae from the Queen Elizabeth Central Hospital, Malawi, were subjected to determination of chloramphenicol MICs. Primers were designed to detect 18 chloramphenicol resistance genes that produce seven distinct peaks correlating with different gene groups (catA1, catA2, catA3, catB2, catB group 3, cmlA and floR) following HRM analysis. ChloS-HRM results were compared with MIC and WGS results.ResultsChloS-HRM correctly identified 15 of 17 phenotypically susceptible isolates and 54 of 55 resistant isolates, giving an accuracy of 88% in identifying susceptibility and 98% in identifying resistance. WGS identified 16 of 17 susceptible and 54 of 55 resistant isolates, giving an accuracy of 94% in identifying susceptibility and 98% in identifying resistance. The single false-susceptible result had no detectable gene by ChloS-HRM or WGS. Compared with WGS, ChloS-HRM had 100% sensitivity and specificity for catA (catA1–3), cmlA and floR, and 96% specificity for catB; sensitivity could not be estimated due to the lack of catB in the clinical sample collection. The overall agreement between MIC and HRM was 96% and between MIC and WGS it was 97%.ConclusionsChloS-HRM could support antimicrobial stewardship in enabling de-escalation from third-generation cephalosporins by identifying chloramphenicol-susceptible infections. This would be valuable in areas with chloramphenicol-susceptible MDR and XDR Enterobacteriaceae.
Highlights
The greatest burden of antimicrobial resistance (AMR) is in the developing world where severe bacterial infection is an important cause of morbidity and mortality[1] and third-generation cephalosporins are frequently both first- and last-line antimicrobials
AMR prevalence data from sub-Saharan Africa are limited, available data consistently reveal that AMR is increasing, in the Enterobacteriaceae,[2] with resistance to thirdgeneration cephalosporins encountered in up to 46% of isolates depending on the setting.[3]
We report here the design of an RT-PCR assay to detect chloramphenicol susceptibility in E. coli and Klebsiella spp. employing highresolution melt (HRM) analysis, which enables a high degree of multiplexing, without expensive fluorescent probes.[22]
Summary
AMR prevalence data from sub-Saharan Africa are limited, available data consistently reveal that AMR is increasing, in the Enterobacteriaceae,[2] with resistance to thirdgeneration cephalosporins encountered in up to 46% of isolates depending on the setting.[3] Between 1996 and 2016 in Malawi 68.3% of Gram-negative infections were resistant to first-line drugs amoxicillin or penicillin, chloramphenicol and co-trimoxazole compared with just 6.6% of Gram-positive infections.[4]. With the current paucity of new drugs in the pipeline,[5] reimplementing older ‘forgotten’ drugs is a potential solution, in resource-limited settings.[6] Many of these drugs, such as chloramphenicol, were introduced in the 1940s–70s and increasing resistance has led to their restriction or removal from clinical use.[7] In their absence susceptibility has returned, increasing their utility in treating susceptible infections.[8,9] This is important with the increasing prevalence of ESBLs and carbapenemases reducing the effectiveness of first-line agents.
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