Development of a C.trachomatis amplification, detection and genotypings assay

By Koen Quint

DDL diagnostics laboratory

Leiden University

Email:k.d.quint@umail.leidenuniv.nl

Outline

• Introduction

• Ct amplification assay

• Ct detection assay

• Ct genotypings assay

• Conclusions

• Ct cofactor study

Introduction

• In the ’70 culture systems were the golden standard.

• Serology was also used to distinguish between acute and chronic infections.

• EIA assays were a first quick alternative for culture.

• DNA-probes and Nucleid Acid Amplification Tests (NAAT) have a high sensitivity relative to culture.

• The second generation NAATs are developed to improve the specificity (cross-reaction, contamination etc.) and sensitivity (the Swedish variant).

The different serovars of C. trachomatis display diverse biological activity.

• Serovar A, B/Ba and C are commonly associated with an ocular disease, trachoma. Serovars B and C are rarely detected in the urogenital tract.

• Serovars D/Da, E, F, G/Ga, H, I/Ia, J and K are common in the urogenital tract and can sometimes be detected in the respiratory tract or eye of newborns.

• Serovars L1, L2/L2a, and L3 are mainly detected in the inguinal lymph nodes and the rectum, and may cause lymphogranuloma venereum.

Development of a Ct-Amplification, Ct-Detection and Ct-Genotyping assay

• The Ct-Amplification assay comprises a Ct-multiplex-broad-spectrum PCR primer mix with multiple forward and reverse primers.

• The Ct-Detection assay comprises a DNA enzyme immuno assay (DEIA) with a mix of conserved probes.

• The Ct-Genotyping assay is based on the reverse hybridisation methodology allowing the simultaneous identification of multiple C. trachomatis serovars in a single hybridization step.

Produced :Labo Biomedical Products BV, Rijswijk, The Netherlands

Ct amplification step

Ct detection step

Ct genotyping step

Cervical scrape DNA isolations

CT-negative CT-positive

Detection in microtiterplate hybridization assay

CT genotypes

Algorithm in Detection and Genotyping of Ct

0

Ct amplification step with multiplex broad-spectrum PCR

CS1

VS1 VS2 VS3 VS4

CS2 CS3 CS4 CS5

950 bp

Bacterial chromosome, ompI

160 bp

Cryptic plasmid

89 bp

Probe region

Probe region

Phylogenetic tree of 160 bp amplicon of C. trachomatis

Group B

Group C

Interm. group

Ct Detection Assay

• DNA enzyme immuno assay for screening

• Mix of conserved probes (based on the cryptic plasmid amplicon as well as omp1 amplicon)

• Within 5 hours for 93 results (+3 controls)

• More sensitive then an agarose gel

Comparison Ct-Dt assay with Cobas Taqman (Roche)

Quint K et al, J. Mol. Diagn. 2007 vol. 9 p.631

Comparison Ct-Dt assay with Hybrid Capture2 (Digene)

Quint K et al. J. Clin. Microbiol. 2007 vol 45 p3986

Strip

DNA-probe

BiotinStreptavidin

Alkaline phosphatase Substrate

Purple precipitate

PCR-amplified target

Principle of reverse hybridization analysis The Ct-Genotyping assay

Outline and specificity of the Ct-genotyping assay

Clinical examples of multiple infections

Quint K et al, J. Mol. Diagn. 2007 vol. 9 p.631

Serovar distribution for 4 different countries

Conclusion

• The amplification assay will generate two amplicons (one based on cryptic plasmid and one based on the omp1 gene)

• The Ct-detection test has the same sensitivity as the COBAS TaqMan and detects significant more Ct infections compared with the HC2 test.

• The Ct- genotyping test is specific for all available (in the genebank) serovars. Multiple infections within the same serogroup need to be sequenced

• The Ct-genotyping test is a quick method for serovar distribution studies.