High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens

Nireshni Mitchev; Ravesh Singh; Veron Ramsuran; Arshad Ismail; Mushal Allam; Stanford Kwenda; Florah Mnyameni; Nigel Garrett; Khine Swe Swe-Han; Abraham J Niehaus; Koleka P Mlisana

doi:10.1155/2022/9094328

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens

Int J Microbiol. 2022 Jan 18:2022:9094328. doi: 10.1155/2022/9094328. eCollection 2022.

Authors

Nireshni Mitchev¹, Ravesh Singh^{1

2}, Veron Ramsuran¹, Arshad Ismail³, Mushal Allam^{3

4}, Stanford Kwenda³, Florah Mnyameni³, Nigel Garrett^{5

6}, Khine Swe Swe-Han^{1

2}, Abraham J Niehaus¹, Koleka P Mlisana^{1

5

7}

Affiliations

¹ School of Laboratory Medicine and Medical Sciences, University of KwaZulu Natal (UKZN), Durban, South Africa.
² National Health Laboratory Service, Durban, South Africa.
³ Sequencing Core Facility, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa.
⁴ Department of Genetics and Genomics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE.
⁵ Centre for the AIDS Programme of Research in South Africa, Durban, South Africa.
⁶ School of Nursing and Public Health, UKZN, Durban, South Africa.
⁷ National Health Laboratory Service, Johannesburg, South Africa.

Abstract

Background: Antimicrobial resistance is limiting treatment options for Neisseria gonorrhoeae infections. To aid or replace culture and the syndromic management approach, molecular assays are required for antimicrobial susceptibility testing to guide appropriate and rapid treatment.

Objective: We aimed to detect single-nucleotide polymorphisms and plasmids associated with antimicrobial resistance from N. gonorrhoeae isolates from a clinic population in South Africa, using real-time PCR as a rapid test for AMR detection.

Methods: N. gonorrhoeae isolates, from female and male patients presenting for care at a sexually transmitted infections clinic in Durban, South Africa, were analysed using phenotypic and genotypic methods for identification and antibiotic susceptibility testing (AST). Real-time PCR and high-resolution melting analysis were used to detect porA pseudogene (species-specific marker) and resistance-associated targets. Whole-genome sequencing was used as the gold standard for the presence of point mutations.

Results: The real-time porA pseudogene assay identified all N. gonorrhoeae-positive isolates and specimens. Concordance between molecular detection (real-time PCR and HRM) and resistance phenotype was ≥92% for bla _TEM (HLR penicillin), rpsJ_V57M (tetracycline), tetM (tetracycline), and gyrA_S91F (ciprofloxacin). Resistance determinants 16SrRNA_C1192U (spectinomycin), mtrR_G45D (azithromycin), and penA_D545S, penA_mosaic (cefixime/ceftriaxone) correlated with the WHO control isolates.

Conclusions: Eight resistance-associated targets correlated with phenotypic culture results. The porA pseudogene reliably detected N. gonorrhoeae. Larger cohorts are required to validate the utility of these targets as a convenient culture-free diagnostic tool, to guide STI management in a South African population.