A Single Amino Acid Substitution in Elongation Factor G Can Confer Low-Level Gentamicin Resistance in Neisseria gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.

Gentamicin Susceptibility in Neisseria gonorrhoeae and Treatment Outcomes for Urogenital Gonorrhea After 25 Years of Sustained Gentamicin Use in Malawi.

BACKGROUND: Gentamicin has been used for the treatment of gonorrhea in Malawi since 1993. However, declining clinical cure rates have been suspected. We evaluated current Neisseria gonorrhoeae susceptibility to gentamicin in vitro and clinically. METHODS: Men with acute urethritis were recruited at the Bwaila District Hospital STI Clinic in Lilongwe, Malawi, between January 2017 and August 2019. All men provided urethral swabs for etiological testing at enrollment and test of cure (TOC), 1 week later, using Gram-stained microscopy and culture. We used Etest to determine minimum inhibitory concentrations (MICs) of gentamicin, azithromycin, cefixime, ceftriaxone, ciprofloxacin, and spectinomycin; disc diffusion for tetracycline susceptibility; and whole-genome sequencing (WGS) to verify/refute treatment failure. RESULTS: Among 183 N. gonorrhoeae culture-positive men enrolled, 151 (82.5%) had a swab taken for TOC. Of these 151 men, 16 (10.6%) had a positive culture at TOC. One hundred forty-one baseline isolates were tested for gentamicin susceptibility using Etest: 2 (1.4%), MIC = 2 µg/mL; 111 (78.7%), MIC = 4 µg/mL; and 28 (19.9%), MIC = 8 µg/mL. All isolates were susceptible to azithromycin, cefixime, ceftriaxone, and spectinomycin, whereas 63.1% had intermediate susceptibility or resistance to ciprofloxacin. Almost all (96.1%) isolates were resistant to tetracycline. All examined isolates cultured at TOC (n = 13) had gentamicin MICs ≤8 µg/mL. Ten men had pretreatment and posttreatment isolates examined by whole-genome sequencing; 2 (20%) were verified new infections (4119 and 1272 single-nucleotide polymorphisms), whereas 8 (80%) were confirmed treatment failures (0-1 single-nucleotide polymorphism). CONCLUSIONS: Gentamicin MICs poorly predict gonorrhea treatment outcome with gentamicin, and treatment failures are verified with gonococcal strains with in vitro susceptibility to gentamicin. The first-line treatment of gonorrhea in Malawi should be reassessed.

Preferences for Pre-exposure Prophylaxis Service Delivery Among Female Sex Workers in Malawi: A Discrete Choice Experiment.

Female sex workers (FSW) in Malawi have among the highest HIV prevalence estimates worldwide. Daily oral pre-exposure prophylaxis (PrEP) is an effective HIV prevention method, yet preferences for PrEP delivery among FSW are lacking. Eight focus group discussions, a literature review, and cognitive interviews were conducted to identify modifiable PrEP delivery attributes and inform discrete-choice experiment (DCE) development for FSW in Lilongwe. Enrolled FSW received an interviewer-assisted DCE. Data were analyzed using mixed logit regression. Dispensing location was most preferred, followed by the provision of additional services. Women preferred receiving PrEP at family planning clinics or non-governmental organization run drop-in centers. Cervical cancer screening was the most preferred additional service, while pregnancy testing and partner risk reduction counseling were less valued. This study was the first study to examine PrEP delivery preferences in Malawi using DCE-a powerful elicitation tool to apply to other key populations at risk for HIV.

The association between substance use and sub-optimal HIV treatment engagement among HIV-infected female sex workers in Lilongwe, Malawi.

Female sex workers (FSW) have a high prevalence of substance use and HIV, but the impact of substance use on HIV treatment engagement is not well established. We evaluated the association between alcohol and marijuana use and sub-optimal HIV treatment engagement outcomes among HIV-infected FSW in Lilongwe, Malawi. We enroled FSW using venue-based recruitment into a cross-sectional evaluation assessing substance use and HIV treatment engagement. Seropositive FSW, identified through HIV rapid testing, received rapid CD4 count and viral load testing. We used Poisson regression with robust variance estimates to ascertain associations of alcohol and marijuana use with sub-optimal HIV treatment outcomes: (1) lack of ART use among previously diagnosed, ART-eligible FSW and (2) viral nonsuppression among FSW on ART. Of previously diagnosed, ART-eligible FSW (n = 96), 29% were not using ART. Patterns of hazardous drinking were identified in 30%, harmful drinking in 10%, and alcohol dependence in 12%. ART-eligible FSW with harmful drinking or alcohol dependency were 1.9 (95% CI: 1.0, 3.8) times as likely to not use ART compared to FSW without harmful or dependent drinking. Among those on ART, 14% were virally nonsuppressed. The prevalence ratio for viral nonsuppression was 2.0 (95% CI: 0.6, 6.5) for harmful drinkers and alcohol-dependent FSW. Over 30% of ART-eligible FSW reported using marijuana. Marijuana-using FSW were 1.9 (95% CI: 0.8, 4.6) times as likely to not use ART compared to FSW who were not using marijuana. Given the high prevalence of alcohol use and its association with lack of ART use, ART uptake and alcohol reduction strategies should be tailored for alcohol-using FSW in Malawi.