title: A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.
abstract: 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 [kappa = 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.
authors.affiliation: DDL Diagnostic Laboratory, Fonteijnenburghlaan 7, 2275 CX Voorburg, The Netherlands.
authors.forename: Koen D
authors.initials: KD
authors.lastname: Quint
authors.forename: Leen-Jan
authors.initials: LJ
authors.lastname: van Doorn
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kleter
authors.forename: Maurits N C
authors.initials: MN
authors.lastname: de Koning
authors.forename: Henk A M
authors.initials: HA
authors.lastname: van den Munckhof
authors.forename: Servaas A
authors.initials: SA
authors.lastname: Morre
authors.forename: Bram
authors.initials: B
authors.lastname: ter Harmsel
authors.forename: Elisabete
authors.initials: E
authors.lastname: Weiderpass
authors.forename: Gonneke
authors.initials: G
authors.lastname: Harbers
authors.forename: Willem J G
authors.initials: WJ
authors.lastname: Melchers
authors.forename: Wim G V
authors.initials: WG
authors.lastname: Quint
mesh_terms: D015373:Bacterial Typing Techniques; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D005838:Genotype; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D014626:Vaginal Smears
chemical_list: D004269:DNA, Bacterial