Development of a SimpleProbe real-Time PCR Assay for rapid detection and identification of the US novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC)

Abstract

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_N-mUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.

Sexual behavior shapes male genitourinary microbiome composition

“…A genital examination and a urethral Gram stain were performed to evaluate for urethral discharge, Gram-negative intracellular diplococci, and the number of polymorphonuclear leukocytes per high-power field (PMN/HPF). Participants were excluded if they tested positive for Chlamydia trachomatis , Mycoplasma genitalium , Neisseria gonorrhoeae , Trichomonas vaginalis , or urethritis clade Neisseria meningitidis US_NmUC strains, 104 had urethral discharge, exhibited >1 PMN/HPF on their Gram stain smear, reported antibiotic use in the last month, or reported urethral symptoms. This study was approved by the Indiana University-Purdue University-Indianapolis (IUPUI) Institutional Review Board and the Marion County Public Health Department.…”