Analytical and clinical validation of a multiplex PCR assay for detection of
Neisseria gonorrhoeae
and
Chlamydia trachomatis
including simultaneous LGV serotyping on an automated high-throughput PCR system
Authors: Pflüger Lisa Sophie , Nörz Dominik , Grunwald Moritz , Pfefferle Susanne , Giersch Katja , Christner Martin , Weber Beatrice , Aepfelbacher Martin , Rohde Holger , Lütgehetmann Marc 
Published: February 2024

ABSTRACT

For effective infection control measures for
Chlamydia trachomatis
(
CT
) and
Neisseria gonorrhoeae
(
NG
), a reliable tool for screening and diagnosis is essential. Here, we aimed to establish and validate a multiplex PCR assay on an automated system using a dual-target approach for the detection of
CT/NG
and differentiation between lymphogranuloma venereum (LGV) and non-LGV from genital and extra-genital specimens. Published primer/probe sets (
CT
: pmpH, cryptic plasmid;
NG
: porA, opa) were modified for the cobas 5800/6800/8800. Standards quantified by digital PCR were used to determine linearity and lower limit of detection (LLoD; eSwab, urine). For clinical validation, prospective samples (
n
= 319) were compared with a CE-marked
in vitro
diagnostics (CE-IVD) assay. LLoDs ranged from 21.8 to 244 digital copies (dcp)/mL and 10.8 to 277 dcp/mL in swab and urine, respectively. A simple linear regression analysis yielded slopes ranging from −4.338 to −2.834 and Pearson correlation coefficients from 0.956 to 0.994. Inter- and intra-run variability was <0.5 and <1 cycle threshold (ct), respectively. No cross-reactivity was observed (
n
= 42). Clinical validation showed a sensitivity of 94.74% (95% confidence interval (CI): 87.23%–97.93%) and 95.51% (95% CI: 89.01%–98.24%), a specificity of 99.59% (95% CI: 97.71%–99.98%) and 99.57% (95% CI: 97.58%–99.98%), positive predictive values of 89.91% (estimated prevalence: 3.7%; 95% CI: 80.91%–95.6%) and 88.61% (estimated prevalence: 3.4%; 95% CI: 80.18%–94.34%), and negative predictive values of 99.81% (95% CI: 98.14%–100%) and 99.85% (95% CI: 98.14%–100%) for the detection of
CT
and
NG
, respectively. In conclusion, we established a dual-target, internally controlled PCR on an automated system for the detectiwon of
CT/NG
from genital and extra-genital specimens. Depending on local regulations, the assay can be used as a screening or a confirmatory/typing assay.

IMPORTANCE

Chlamydia trachomatis
(
CT
) and
Neisseria gonorrhoeae
(
NG
) represent a major global health burden, with the World Health Organization estimating that >128 million and >82 million people, respectively, were newly infected in 2020. For effective infection control measures, a reliable tool for sensitive diagnosis and screening of
CT/NG
is essential. We established a multiplex PCR assay for the detection of
CT/NG
and simultaneous discrimination between lymphogranuloma venereum (LGV) and non-LGV strains, which has been validated for genital and extra-genital specimens on a fully automated system. To increase assay sensitivity, a dual-target approach has been chosen for both pathogens. This strategy reduces false-positive results in oropharyngeal swabs due to the detection of commensal
N
. species that may harbor
NG
DNA fragments targeted in the PCR due to horizontal gene transmission following previous infection. In sum, the established assay provides a powerful tool for use as either a screening/diagnostic or a typing/confirmatory assay.