Genetic Typing of the Porin Protein of Neisseria gonorrhoeae from Clinical Noncultured Samples for Strain Characterization and Identification of Mixed Gonococcal Infections

Abstract

Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplificationbased diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Molecular Epidemiology of Neisseria gonorrhoeae in Amsterdam, The Netherlands, Shows Distinct Heterosexual and Homosexual Networks

“…Lynn et al directly amplified clinical samples from 20 culture-positive women from Baltimore, Md., stored under different conditions. Those authors found that 40% of isolates from the paired cervical swab and wick samples and culture samples differed in strain type per patient, when the por gene was characterized with DNA-DNA hybridization (9). Repetition of the genotyping process with several strains in our study, however, showed that the por-opa RFLP patterns were reproducible, yielding only one strain per sampled anatomical location.…”

por Variable-Region Typing by DNA Probe Hybridization Is Broadly Applicable to Epidemiologic Studies of Neisseria gonorrhoeae

“…One of the goals of molecular typing is to allow for the characterization of strains directly from nucleic acid amplification test samples. We have identified mixed infections in direct clinical specimens by using por VR typing (22), and in the present study, unrecognized coinfection with more than one gonococcal strain may have contributed to the overall 9.3% (4 of 43 pairs) discrepancy observed in partners where only cultivated strains were tested rather than infected secretions. Identifying the presence of and distinguishing mixed infections in direct clinical samples may be difficult with sequence-based typing.…”

Molecular Surveillance of Clinical Neisseria gonorrhoeae Isolates in Russia

“…Multiresistant isolates (i.e., isolates resistant to at least two antibiotics) are indicated by thick lines. (16,17) for a broad spectrum of clinical samples are more convenient than sequencing.…”