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Background: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health challenge. Limitations to AMR surveillance reporting, alongside reduction in culture-based susceptibility testing, has resulted in a need for rapid diagnostics and strain detection. We investigated Nanopore sequencing time, and depth, to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina sequencing., Methods: N. gonorrhoeae strains collected from a London sexual health clinic were cultured and sequenced with MiSeq and MinION sequencing platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions (37 resistance-associated markers). Accuracy at varying MinION sequencing depths was determined through retrospective time-stamped read analysis., Results: Of 22 MinION-MiSeq pairs reaching sufficient sequencing depth, agreement of variant call positions passing quality control criteria was 185/185 (100%; 95% confidence interval [CI], 98.0%-100.0%), 502/503 (99.8%; 95% CI, 98.9%-99.9%), and 564/565 (99.8%; 95% CI, 99.0%-100.0%) at 10x, 30x, and 40x MinION depth, respectively. Isolates identified as closely related by MiSeq, within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified via MinION., Conclusions: Nanopore sequencing shows utility as a rapid surveillance tool, identifying closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median time of 29 minutes. This highlights its potential for tracking local transmission and AMR markers., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types., Methods: NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection., Results: NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%-98.3%) and 100% (95% CI 94.7%-100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%-100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%-100%), 100% (95% CI 87.2%-100%) and 100% (95% CI 82.4%-100%), respectively., Conclusions: Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for surveillance., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: To describe the pharmacokinetics of darunavir in pregnant HIV-infected women in the third trimester and post-partum., Patients and Methods: This was a non-randomized, open-label, multicentre, Phase IV study in HIV-infected pregnant women recruited from HIV treatment centres in Europe. HIV-infected pregnant women treated with darunavir (800/100 mg once daily or 600/100 mg twice daily) as part of their combination ART were included. Pharmacokinetic curves were recorded in the third trimester and post-partum. A cord blood sample and maternal sample were collected. The study is registered at ClinicalTrials.gov under number NCT00825929., Results: Twenty-four women were included in the analysis [darunavir/ritonavir: 600/100 mg twice daily (n=6); 800/100 mg once daily (n=17); and 600/100 mg once daily (n=1)]. Geometric mean ratios of third trimester versus post-partum (90% CI) were 0.78 (0.60-1.00) for total darunavir AUC0-tau after 600/100 mg twice-daily dosing and 0.67 (0.56-0.82) for total darunavir AUC0-tau after 800/100 mg once-daily dosing. The unbound fraction of darunavir was not different during pregnancy (12%) compared with post-partum (10%). The median (range) ratio of darunavir cord blood/maternal blood was 0.13 (0.08-0.35). Viral load close to delivery was <300 copies/mL in all but two patients. All children were tested HIV-negative and no congenital abnormalities were reported., Conclusions: Darunavir AUC and Cmax were substantially decreased in pregnancy for both darunavir/ritonavir regimens. This decrease in exposure did not result in mother-to-child transmission. For antiretroviral-naive patients, who are adherent, take darunavir with food and are not using concomitant medication reducing darunavir concentrations, 800/100 mg of darunavir/ritonavir once daily is adequate in pregnancy. For all other patients 600/100 mg of darunavir/ritonavir twice daily is recommended during pregnancy., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background:  Empirical antibiotic therapy for nongonococcal urethritis (NGU) and cervicitis is aimed at Chlamydia trachomatis, but Mycoplasma genitalium, which also commonly causes undiagnosed NGU, necessitates treatment with macrolides or fluoroquinolones rather than doxycycline, the preferred chlamydia treatment. Prevalence of M. genitalium and associated genotypic markers of macrolide and fluoroquinolone resistance among men symptomatic of urethritis were investigated. Genetic diversity of M. genitalium populations was determined to infer whether findings were applicable beyond our setting., Methods:  Mycoplasma genitalium and other NGU pathogens were detected using nucleic acid amplification methods, and DNA sequencing was used to detect genotypic resistance markers of macrolide and fluoroquinolone antibiotics in 23S ribosomal RNA, gyrA, gyrB, and parC genes. MG191 single-nucleotide polymorphism typing and MG309 variable number tandem analysis were combined to assign a dual locus sequence type (DLST) to each positive sample., Results:  Among 217 men, M. genitalium prevalence was 16.7% (95% confidence interval [CI], 9.5%-24.0%) and C. trachomatis prevalence was 14.7% (95% CI, 7.8%-21.6%) in NGU cases. Nine of 22 (41%; 95% CI, 20%-62%) patients with M. genitalium were infected with DLSTs possessing genotypic macrolide resistance and 1 patient was infected with a DLST having genotypic fluoroquinolone resistance. Typing assigned M. genitalium DLSTs to 2 major clusters, broadly distributed among previously typed international strains. Genotypic macrolide resistance was spread within these 2 clusters., Conclusions:  Mycoplasma genitalium is a frequent undiagnosed cause of NGU in this population with rates of macrolide resistance higher than those previously documented. Current guidelines for routine testing and empirical treatment of NGU should be modified to reduce treatment failure of NGU and the development of further resistance.

Objectives: To determine differences in CYP2B6 loss of function (LoF) single nucleotide polymorphisms (SNPs) and haplotypes between Zimbabweans and Ugandans, and within Ugandan populations (Bantu and Nilotic)., Methods: Genetic epidemiological study enrolling adult black African Ugandan and Zimbabwean patients attending a UK HIV-1 clinic, irrespective of antiretroviral therapy status. Genomic DNA was extracted from whole blood and the presence of CYP2B6 alleles was determined by direct sequencing of all nine exons of the CYP2B6 gene. Blood was also collected, where appropriate, for determination of efavirenz concentrations. Frequency of SNPs in all patients and LoF haplotype frequencies were calculated. The relationship between the number of LoF haplotype alleles possessed and efavirenz trough concentration (ETC) was determined., Results: Thirty-six Zimbabweans and 74 Ugandans (58 Bantu and 16 Nilotic) were recruited. The definite haplotypes determined were *6, *18, *20 and *27 as LoF and *4 as gain of function. Among those with definite genotypes, the frequency of LoF alleles was 65% [95% confidence interval (95% CI): 51-80] of Zimbabweans versus 22% (95% CI: 12-31) of Ugandan Bantus (P = 10(-6)) and versus 39% (95% CI: 14-64) of Ugandan Nilotics (P = 0.09). Among the 19 patients with definite genotype and with available ETCs, log ETCs were associated with a greater number of LoF haplotype alleles [848 ng/mL (n = 12), 1069 ng/mL (n = 4) and 1813 ng/mL (n = 3) for 0, 1 or 2 LoF haplotypes, respectively (P = 0.016)]., Conclusions: Among Zimbabweans, LoF haplotypes constitute the majority of CYP2B6 alleles and are significantly higher in prevalence compared with Ugandans. Frequencies of LoF haplotypes and SNPs in Ugandan Nilotics appear to lie between those of Zimbabweans and Ugandan Bantus. These findings may have relevance to pharmacokinetics and dosing of efavirenz in African populations.