Simultaneous Detection of Neisseria gonorrhoeae and Fluoroquinolone Resistance Mutations to Enable Rapid Prescription of Oral Antibiotics.

BACKGROUND: Absence of rapid antimicrobial resistance testing of Neisseria gonorrhoeae (Ng) hinders personalized antibiotic treatment. To enable rapid ciprofloxacin prescription, a real-time polymerase chain reaction (PCR) for simultaneous detection of Ng and fluoroquinolone resistance-associated gyrA-S91F mutation was evaluated. METHODS: Analytical NG quantitative PCR kit (NYtor BV) performance was assessed on 50 Ng transcription-mediated amplification (TMA)-negative and 100 Ng TMA-positive samples. To assess clinical use, 200 samples were prospectively analyzed, in parallel to routine diagnostic tests. Also, 50 urine, 50 anal, 50 pharyngeal, and 50 vaginal Ng TMA-positive samples were retrospectively analyzed. To assess if patients carried strains with different ciprofloxacin sensitivity at different anatomical locations, 50 urine/anal or vaginal/anal sample pairs collected during a single visit were analyzed. RESULTS: The NG quantitative PCR kit showed 97% sensitivity and 100% specificity for Ng detection and 92% sensitivity and 99% specificity for gyrA-S91F detection. Relative to TMA results, 85% Ng detection sensitivity and 99% specificity were found. Regarding the 200 prospectively analyzed clinical samples, 13 were Ng positive, of which 10 were also tested for antibiotic susceptibility by culture. The kit showed concordance for GyrA-S91F detection in 9 of 10 samples. Ng was detected in 96% and 94% of vaginal and urine TMA-positive samples, in 84% of anal samples and only in 22% of pharyngeal samples. Discordant ciprofloxacin sensitivity was found for 2 of 26 characterized urine/anal sample pairs. CONCLUSION: The NG quantitative polymerase chain reaction (qPCR) kit can be implemented in diagnostic testing for vaginal, urine, and anal Ng TMA-positive samples to enable rapid prescription of oral ciprofloxacin.

Ceftriaxone Reduced Susceptible Neisseria gonorrhoeae in the Netherlands, 2009 to 2017: From PenA Mosaicism to A501T/V Nonmosaicism.

OBJECTIVES: To compare molecular and epidemiological differences between ceftriaxone-reduced susceptible (CRO-RS) and ceftriaxone-susceptible (CRO-S) N. gonorrhoeae (Ng) and to study the genetic relatedness of CRO-RS isolates. METHODS: Demographic and clinical data and samples for cultures were routinely collected from gonorrhoea patients visiting the Amsterdam STI clinic in 2009 to 2017. Ng multiantigen sequence typing (NG-MAST) and penA types were compared between CRO-RS and CRO-S Ng (frequency matched on year of isolation and sexual risk group). Minimum spanning trees were produced based on multilocus variable number of tandem repeats analysis for Ng (NG-MLVA) genotypes. RESULTS: We selected 174 CRO-RS isolates (minimum inhibitory concentration, ≥0.064 mg/L) and 174 CRO-S isolates (minimum inhibitory concentration, ≤0.016 mg/L). Demographic and clinical characteristics of patients were overall comparable between those infected with CRO-RS Ng and CRO-S Ng. However, CRO-RS isolates were more often collected from the pharyngeal site (odds ratios [OR], 3.64; P < 0.001), and patients with CRO-RS Ng were less often human immunodeficiency virus (HIV) and syphilis positive (OR, 0.63; P = 0.041 and OR, 0.58; P = 0.028, respectively). We identified 12 clusters based on NG-MLVA genotypes, including 3 large (>25 isolates) clusters predominantly containing CRO-RS isolates. Those from cluster 1 (n = 32) were mostly from 2009 to 2012 (n = 24; 75.0%), with a mosaic penA XXXIV pattern (n = 27; 84.4%) and belonging to NG-MAST genogroup G1407 (n = 24; 75.0%). Isolates from cluster 2 (n = 29) were mostly from 2013 to 2015 (n = 24; 82.7%), had a nonmosaic penA IX + A501T mutation (n = 22; 75.9%) and NG-MAST G2400 (n = 14; 48.3%). Most isolates from cluster 3 (n = 37) were from 2015 to 2017 (n = 26; 70.2%), had a nonmosaic penA IV + A501V mutation (n = 24; 64.9%) and NG-MAST G2318 (n = 22; 59.5%). CONCLUSIONS: We observed a shift in the predominant penA (from mosaic toward nonmosaic plus A501T/V mutation), NG-MAST and NG-MLVA types among CRO-RS Ng over time. This indicates a successive spread of different CRO-RS Ng clones.

Molecular epidemiology of Neisseria gonorrhoeae strains circulating in Indonesia using multi-locus variable number tandem repeat analysis (MLVA) and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) techniques.

BACKGROUND: Control of gonorrhea in resource-limited countries, such as Indonesia, is mostly unsuccessful. Examining Neisseria gonorrhoeae (Ng) transmission networks using strain typing might help prioritizing public health interventions. METHODS: In 2014, urogenital Ng strains were isolated from clients of sexually transmitted infection clinics in three Indonesian cities. Strains were typed using Multiple-Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) and Ng Multi-Antigen Sequence Typing (NG-MAST) at the Public Health Service, Amsterdam, the Netherlands, and compared to Dutch strains collected from 2012 to 2015. Minimum spanning trees (MSTs) were constructed using MLVA profiles incorporating demographics and NG-MAST genogroups. A cluster was defined as ≥5 strains differing in ≤1 VNTR locus. The concordance between MLVA and NG-MAST was examined with the adjusted Wallace coefficients (AW). RESULTS: We collected a total of 78 Indonesian strains from Yogyakarta (n = 44), Jakarta (n = 25), and Denpasar (n = 9). Seven MLVA clusters and 16 non-clustered strains were identified. No cluster was specific for any geographic area, risk group or age group. Combined with 119 contemporary Dutch strains, 8 MLVA clusters were identified, being four clusters of Indonesian strains, two of Dutch strains, and two of both Indonesian and Dutch strains. Indonesian strains (79.5%) were more often clustered compared to Dutch strains (24.3%). The most prevalent NG-MAST genogroups among Indonesian strains was G1407 (51.3%) and among Dutch strains was G2992 (19.3%). In Indonesian strains, the AW [95% confidence interval] for MLVA to NG-MAST was 0.07 [0.00-0.27] and for NG-MAST to MLVA was 0.03 [0.00-0.12]. CONCLUSION: Indonesian Ng strains are more often clustered than Dutch strains, but show no relation with geographical area, risk group, or age group, suggesting a more clonal Ng epidemic in Indonesia. Some similar Ng strains circulate in both Indonesia and the Netherlands.

A Case-Control Study of Molecular Epidemiology in Relation to Azithromycin Resistance in Neisseria gonorrhoeae Isolates Collected in Amsterdam, the Netherlands, between 2008 and 2015.

Neisseria gonorrhoeae resistance to ceftriaxone and azithromycin is increasing, which threatens the recommended dual therapy. We used molecular epidemiology to identify N. gonorrhoeae clusters and associations with azithromycin resistance in Amsterdam, the Netherlands. N. gonorrhoeae isolates (n = 143) were selected from patients visiting the Amsterdam STI Outpatient Clinic from January 2008 through September 2015. We included all 69 azithromycin-resistant isolates (MIC ≥ 2.0 mg/liter) and 74 frequency-matched susceptible controls (MIC ≤ 0.25 mg/liter). The methods used were 23S rRNA and mtrR sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), N. gonorrhoeae multilocus variable-number tandem-repeat analysis (NG-MLVA), and a specific PCR to detect mosaic penA genes. A hierarchical cluster analysis of NG-MLVA related to resistance and epidemiological characteristics was performed. Azithromycin-resistant isolates had C2611T mutations in 23S rRNA (n = 62, 89.9%, P < 0.001) and were NG-MAST genogroup G2992 (P < 0.001), G5108 (P < 0.001), or G359 (P = 0.02) significantly more often than susceptible isolates and were more often part of NG-MLVA clusters (P < 0.001). Two resistant isolates (2.9%) had A2059G mutations, and five (7.3%) had wild-type 23S rRNA. No association between mtrR mutations and azithromycin resistance was found. Twenty-four isolates, including 10 azithromycin-resistant isolates, showed reduced susceptibility to extended-spectrum cephalosporins. Of these, five contained a penA mosaic gene. Four of the five NG-MLVA clusters contained resistant and susceptible isolates. Two clusters consisting mainly of resistant isolates included strains from men who have sex with men and from heterosexual males and females. The co-occurrence of resistant and susceptible strains in NG-MLVA clusters and the frequent occurrence of resistant strains outside of clusters suggest that azithromycin resistance develops independently from the background genome.

Molecular epidemiology of Neisseria gonorrhoeae in Amsterdam, The Netherlands, shows distinct heterosexual and homosexual networks.

Molecular typing, added to epidemiological data, can better identify transmission patterns of gonorrhea in Western countries, where the incidence has recently been rising. From September 2002 to September 2003, patients with a laboratory-confirmed diagnosis of gonorrhea at the Clinic for Sexually Transmitted Infections in Amsterdam, The Netherlands, were subjected to a questionnaire pertaining to sexual risk behavior and sexual partners in the 6 months prior to the diagnosis. The Neisseria gonorrhoeae isolates were all genotyped using PCR-restriction fragment length polymorphism of the porin and opacity genes. All patients with a completed questionnaire and genotyped isolates were included in the study. We obtained 885 N. gonorrhoeae isolates from 696 patients that revealed 88 clusters and 46 unique genotypes. Patients infected at multiple anatomical sites with one or more strains and patients infected several times during the study period were shown to pursue high-risk sexual behavior and were considered core groups. There were 11 clusters of > or =20 patients; in seven clusters, 81% to 100% of patients were men who have sex with men (MSM), three clusters contained 87 to 100% heterosexual men and women, and one cluster was formed by equal proportions of MSM and heterosexual male and female patients. However, the various clusters differed in characteristics such as types of coinfections, numbers of sexual partners, Internet use to seek sexual partners, and locations of sexual encounters. Molecular epidemiology of gonococcal isolates in Amsterdam revealed core groups and clusters of MSM and heterosexual patients that probably indicate distinct transmission networks.

Practical implementation of a multiplex PCR for acute respiratory tract infections in children.

Molecular testing for acute respiratory infections (ARIs) has documented value but limited implementation due to questions that typically slow the acceptance of new tests. This study sought to address these questions and achieve implementation. Rhinovirus was added to a nested multiplex PCR (M-PCR), increasing its diagnostic yield. Over one winter, three hospital pediatric departments used the M-PCR to complement their direct fluorescent-antibody assay (DFA) for respiratory syncytial virus (RSV). Clinicians recorded "pretest probability estimates" (using continuous scales for various pathogen groups) for comparison with test results; treatments and test turnaround times were also recorded. Transnasal and throat swabs, with or without nasopharyngeal aspirate (NPA), were M-PCR tested. NPA-containing sample sets found to be RSV positive by DFA were not further tested. Single PCR for human metapneumovirus (hMPV) was performed retrospectively. Of 178 ARI episodes representing 172 patients, NPA was included in 97 sample sets; 54 (56%) were determined to be RSV positive. The other NPA-containing sample sets (n = 43) yielded 27 findings (63%), and the swab-only sets (n = 81) yielded 47 findings (58%); rhinovirus was found most often. Testing for hMPV yielded seven positive results. M-PCR median turnaround times were 4 days in swab-only samples and 5 days with NPA. Antibiotics were prescribed in 50 episodes, at rates similar for RSV and rhinovirus. Pretest probability estimates of a viral cause were lower in episodes caused by rhinovirus than in episodes caused by RSV. The hospitals continued to use M-PCR for NPA-containing samples found to be RSV negative by DFA. Test implementation is more likely with higher diagnostic yield and a protocol that reflects day-to-day clinical and laboratory operations.