We conducted a matched case-control study to investigate N. meningitidis urethritis and risk factors in men seeking treatment for urinary discharge at Ho Chi Minh City Hospital of Dermato-Venereology (HHDV; Ho Chi Minh City, Vietnam). Cases of N. meningitidis urethritis were confirmed by either real-time PCR or culture of urethral discharge. Controls were matched to case-patients by age range and sexual orientation (Appendix). During September 2019-December 2020, we recruited 19 case-patients and 76 controls from HHDV (Appendix Figure 1). We collected information on sociodemographic factors, sexual behaviors, and medical history by face-to-face interviews and from medical records (Appendix). The HHDV institutional review board approved the study.
We identified N. meningitidis by using bacterial culture and real-time PCR targeting the sodC gene (6) and determined serogroups by using latex agglutination and real-time PCR. We performed antimicrobial susceptibility testing according to Clinical and Laboratory Standards Institute guidelines (7). We conducted whole-genome sequencing, then analyzed multilocus sequence types (MLST) in PubMLST (https://pubmlst.org/organisms/neisseria-spp). We used BEAST (http://beast.community) to estimate the time of bacterial arrival in Vietnam and to conduct antimicrobial-resistance typing (Appendix). We performed conditional logistic regression to assess risk factors for US NmNG urethritis clade by using Stata 14 (StataCorp LLC, https://www.stata.com). We used a log likelihood-ratio test to select the best-fitting model (Appendix).
The mean age of case-patients was lower than that of controls (26.9 vs. 27.8 years). Condom use was low in both case-patients and controls before pyuria developed (5.3% of case-patients, 2.7% of controls). More case-patients than controls lived with male partners (42.1% vs. 13.7%) and had sex with foreign-born persons (15.8% vs. 1.3%). Multivariate analysis results showed that persons living with male partners (adjusted odds ratio [aOR] 14.41, 95% CI 1.01-204.62) and having sex with foreign-born persons (aOR 26.78, 95% CI 1.03-697.82) were more likely to contract US NmNG urethritis (Table 1). Moreover, most (79%) case-patients reported sex with male partners. Persons having oral or vaginal sex with female partners in the past 12 months were less likely to have US NmNG urethritis (aOR 0.13, 95% CI 0.02-0.87). Those findings suggest that the US NmNG outbreak was concentrated within the MSM population.
Among 19 case-patients, 7 were co-infected with >1 other pathogen: 2 (11%) cases of syphilis, 4 (21%) cases of chlamydia, and 1 (0.1%) case involving both ureaplasma and mycoplasma. Ten (83%) case-patients without co-infections and 6 (86%) with co-infections experienced >1 symptom. Pyuria was reported in 2 (29%) co-infected case-patients, and dysuria was reported in 10 (83%) case-patients without co-infections and 3 (43%) with co-infections (Appendix Table 1). Most participants had not received meningococcal vaccines, nor recalled being vaccinated against N. meningitidis (Table 2). The prevalence of HIV, syphilis, and chlamydia infections was higher among case-patients compared to controls but not statistically significant, whereas gonorrhea was only found in controls (98.7%) (Table 2). Multivariate analysis showed that those who had US NmNG urethritis were less likely to report burning sensations during urination (odds ratio [OR] 0.08, 95% CI 0.01-0.46) and more likely to delay seeking treatment (OR 16.0, 95% CI 2.0-127.54) (Table 2).
In this study, uncomplicated gonorrhea was treated with a single 500-mg intramuscular dose of ceftriaxone, followed by either a 7-day course of doxycycline (100 mg 2×/day) or a single 1,000-mg dose of azithromycin. In Vietnam, gonococci isolated in 2011 and during 2015-2016 increasingly resisted antimicrobial drugs except for ceftriaxone, spectinomycin, and azithromycin (8). Moreover, 98.3% of N. gonorrhoeae isolates were ciprofloxacin-resistant (9). In a national survey conducted in Vietnam, 30% of persons reported purchasing antibiotics primarily for addressing symptoms, including genitourinary manifestations, and 81.7% did so without a prescription; ciprofloxacin was among the top 5 antimicrobial drugs acquired (10). In another study among MSM in Vietnam, 64% reported ever taking antibiotics without a prescription (11).
The US NmNG urethritis clade in our study displayed intermediate susceptibility to penicillin, with MIC values ranging from 0.125-0.38 mg/L. Nine of the 19 isolates demonstrated resistance to ciprofloxacin (MIC 0.19-3.0 µg/mL). MLST analysis revealed that the isolates belonged to the sequence type 11 complex (Appendix Table 2). A phylogenetic tree displayed the isolates from Vietnam and the United Kingdom forming a monophyletic clade with those from Ohio, USA, one of the 2 US NmNG urethritis clades (12) (Appendix Figure 2). BEAST analysis estimated that the time of most recent common ancestor of Vietnam and UK isolates appeared between 2016 and 2018 (median 2017.3; 95% high posterior density interval 2016.4-2018.1), with a Bayesian posterior probability of 1.0 (Figure 1; Appendix Figure 3).
All isolates carried the penA_316 allele and point mutations that reduced their susceptibility to penicillin (13). Nine ciprofloxacin-resistant isolates exhibited 2 new alleles in gyrA, assigned as gyrA_381 (n = 8), which had dual mutations at T91F and D95A, and gyrA_382, which had monomutation at T91I. We used RDP4 (https://rdp4.software.informer.com) to analyze the full 2,751-bp length and found that gyrA_381 received a fragment containing a mutation from gonococci (Figure 2). Our study revealed that isolates containing mutations at both T91F and D95A in the gyrA gene displayed a high level of resistance to ciprofloxacin, similar to that found in N. gonorrhoeae (14). Moreover, isolate 20158 had a mutation at S87R of parC and the gyrA_381 allele had an elevated MIC of 3 µg/mL.
The emergence of ciprofloxacin-resistant N. meningitidis US NmNG urethritis clade in Vietnam is a major concern, especially considering ciprofloxacin resistance is rare in the United Kingdom and United States (4,5). A previous study observed that strains with MICs >0.064 mg/L were correlated with alterations in gyrA (15). Hence, when specimens cannot be cultured, gyrA-sequencing can be particularly useful in predicting susceptibility of ciproloxacin.