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

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

Journal of Clinical Microbiology, January 2005, p. 368-375, Vol. 43, No. 1

Freyja Lynn,¹ Marcia M. Hobbs,² Jonathan M. Zenilman,³ Frieda M. T. F. Behets,² Kathleen Van Damme,² Andry Rasamindrakotroka,⁴ and Margaret C. Bash^1,5*

Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research,¹ Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda,⁵ Division of Allergies and Infectious Disease, Johns Hopkins University School of Medicine, Baltimore, Maryland,³ Department of Medicine, University of North Carolina, Chapel Hill, North Carolina,² Ministry of Health, Antananarivo, Madagascar⁴

Received 23 June 2004/ Returned for modification 13 August 2004/ Accepted 8 September 2004

            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 amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.