Published online before print September 14, 2007

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Journal of Molecular Diagnostics 2007, Vol. 9, No. 5
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2007.070011

A Highly Sensitive, Multiplex Broad-Spectrum PCR-DNA-Enzyme Immunoassay and Reverse Hybridization Assay for Rapid Detection and Identification of Chlamydia trachomatis Serovars

Koen D. Quint^*, Leen-Jan van Doorn^*, Bernhard Kleter^*, Maurits N.C. de Koning^*, Henk A.M. van den Munckhof^*, Servaas A. Morre, Bram ter Harmsel, Elisabete Weiderpass^¶||, Gonneke Harbers^**, Willem J.G. Melchers^** and Wim G.V. Quint^*

From the DDL Diagnostic Laboratory, * Voorburg, The Netherlands; the Departments of Pathology and Internal Medicine, VU University Medical Center, Amsterdam, The Netherlands; the Department of Medical Microbiology, Academic Hospital Maastricht, Maastricht, The Netherlands; Reinier de Graaf Groep, Delft, The Netherlands; the Cancer Registry of Norway, ^¶ Oslo, Norway; the Department of Medical Epidemiology and Biostatistics, ^|| Karolinska Institutet, Stockholm, Sweden; and the Department of Medical Microbiology, ** Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [ = 0.95 (95% confidence interval = 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common. Thus, multiplex broad-spectrum PCR in combination with DEIA and RHA permits highly sensitive and specific detection and identification of Ct serovars.

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