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

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.

• You define acronyms in the methods section, however that comes after the results, so I would recomme'nd also defining the acronyms in the results section to avoid confusion • Line 108 -I am confused by the number in the brackets next to the name of the primers, e.g (opa-rev ( 01)).You should refer to table 1 here to avoid confusion.
• Line 161 -Can you please explain this sentence: " For each assay one false positive and four false negative test results could be determined."• Line 173 -"In 4,5 % of chlamydial ..." There is a typo • Section 2.4 -I am a bit confused about the clinical samples tested.A total of 4,298 samples were screened.Of this, 1,914 were additionally tested by NG culture.Then, on line 177, you mention 1,914 culture negative samples, is this a mistake?Also, on line 178 you mention extra-genital specimens, are these part of the 4298 samples?I think some clarity is needed in this section.
• Line 201, you speak of another study, was this part of this study?Or unpublished data?I think clarification is needed.
• Line 294 -how was the standard created?I think more detail is needed for this section.
Reviewer #2 (Comments for the Author): This is a well written analysis of a new laboratory developed test designed to identify Chlamydia trachomatis and Neisseria gonorrhoeae, in addition the assay is able to identify CT serovars associated with lymphogranuloma venereum.

Major revisions:
As matrix of the specimen type is important to indicate there should be more emphasis put on the different specimen types collected via swab.These specimen types should be evaluated independently as different specimen types and body fluids associated with those sites can differently impact extraction and amplification of the target.Example: ocular swabs and rectal swabs should be evaluated separately with adequate positive and negative samples from each sample type.Refer to CLSI and validation requirements for LDTs.Was a cutoff Ct value defined for your assay for each target as the result of this study?For Neisseria please define the criteria of a positive and negative value.Since 2 targets are involved do both need to be positive or one positive at a low Ct value (if so what is the cutoff).This is not clear.
Minor revisions: Line 60: Capitalize "World Health Organization" Line 66-70: multiple grammatical issues.Line 105-106: reword for clarity.I think you mean that there is not a high risk of formation of primer-dimers; the sentence does not currently read as such.Line115: define dcp Line 120-123: The swabs should not be lumped together for calculating the LLOD unless all of the swabs were obtained from the same body site.Different specimen sample types are likely to have different LLODs dependent upon body fluid composition at site.See major revision comment.Line169-170: Write out the dates.Do not use abbreviations.Line169: Specify how many samples came from each sample type/body site Line 195: add spp after N. Line211: Please clarify if oropharyngeal specimens are hard to eradicate or hard to diagnose because of diagnostic assay limitations.If difficult to treat, please provide a reference.

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Reviewer #1 (Comments for the Author):
This is an excellent study describing the development of a sensitive Neisseria gonorrhoeae and Chlamydia trachomatis multiplex assay.Given that this assay was created from previously published primers/probes, I believe the work done by the authors to validate this assay was thorough.I just have a few comments/questions about the results.

Response:
We would like to thank the reviewer for the positive comments and time spend on reviewing the paper.The comments and corrections proposed by the reviewer certainly improved the manuscript.

•
Line 91 -"my harbor fragments of" spelling error

Response:
The spelling error was corrected (see line 91).
• Line 96 -grammar error "The aim of this study was, to compile a multiplex"

Response:
The sentence was changed accordingly (see line 96).
• You define acronyms in the methods section, however that comes after the results, so I would recomme'nd also defining the acronyms in the results section to avoid confusion

Response:
We fully agree with the reviewer on this point.The acronyms are now introduced in the first section they are mentioned in.
• Line 108 -I am confused by the number in the brackets next to the name of the primers, e.g (opa-rev (01)).You should refer to table 1 here to avoid confusion.
Response: For clarity, oligonucleotide names have been changes throughout the manuscript and in table 1 (e.g. from opa-rev (01) to opa-rev_01).Further, we added a note specifying that the oligonucleotide names in the manuscript match the names in table 1 (see lines 109 and 110).
• Line 161 -Can you please explain this sentence: " For each assay one false positive and four false negative test results could be determined." Response: For both pathogens detected by the UC_CTNG assay, one false positive and four false negatives were identified when comparing test results to the comparator assay (CE-IVD, CTNG assay, Roche).False positive results were defined as UC_CTNG positive and CE-IVD negative, while false negative results were defined as UC_CTNG negative and CE-IVD positive.For this analysis, both Neisseria gonorrhoeae targets (opa and porA) must be reactive with the new UC_CTNG assay for the overall result to be considered Neisseria gonorrhoeae positive.
We share the reviewer's opinion that more clarity is needed in this section.The manuscript has been adapted to be more precise and a reference to figure 2 has been added (see lines 176 -179).

Response:
The typing error was corrected (see line 195).
• Section 2.4 -I am a bit confused about the clinical samples tested.A total of 4,298 samples were screened.Of this, 1,914 were additionally tested by Neisseria gonorrhoeae culture.Then, on line 177, you mention 1,914 culture negative samples, is this a mistake?Also, on line 178 you mention extragenital specimens, are these part of the 4298 samples?I think some clarity is needed in this section.

Response:
We thank the reviewer for bringing this mistake to our attention.Indeed, the numbers were incorrect.We screened 1,962/4,298 samples additionally by culture (not 1,914/4,298 as mentioned in the manuscript) of which 62 samples were positive for both the opa and the porA target with the UC_CTNG assay.The numbers have been changes accordingly and the sentence was rephrased for clarity (see lines 186 and 198-200).
Moreover, we have revised the statement regarding the extra-genital samples analyzed to specify that we are referring to the analysis of the oropharyngeal swabs that were screened as part of the evaluation of the assay performance in routine conditions (see lines 200 and 201).
• Line 201, you speak of another study, was this part of this study?Or unpublished data?I think clarification is needed.
Response: Again, we thank the reviewer for pointing out that more clarification is needed.We added the information that the retrospective analysis of the 4,298 clinical samples was part of the evaluation of the clinical performance of the new UC_CTNG assay of this study (see line 222).
• Line 294 -how was the standard created?I think more detail is needed for this section.
Response: Clinical samples that tested highly positive for Chlamydia trachomatis serovars L1-L3 (LGV strain) and Neisseria gonorrhoeae using our in-house LDT were selected.If necessary, the clinical samples were diluted to reach a final volume of 2 ml and aliquots containing 500 µl each were stored at -20° C. Next, one aliquot each was thawed and tested in a serial dilution series (10-fold, 3 steps) on a digital PCR Platform (Qiagen, Hilden, Germany) according to the manufacture's instructions.A negative control was included for each target.Nucleic acid extraction was performed on the QiaSymphony platform (Qiagen, Hilden, Germany).The same primers and probes as described in the study were used for amplification of the pmpH (Chlamydia trachomatis) and the opa gene (Neisseria gonorrhoeae).Determination of lower limit of detection and linearity were based on the average concentration as reported by the respective digital PCR software in digital copies/ml of the clinical samples.For each experiment a new separate aliquot was used to assure that the same conditions applied for all tests.
It is important to note, that for each pathogen detected with the UC_CTNG assay only one target was quantified.However, the opa target (Neisseria gonorrhoeae) is a multi-copy gene whereas the porA target is only a single-copy target.We thank the reviewer for the pointing out that more detail was needed about the quantification of the standards, as we feel that this clarification improved the manuscript (see updated methods section lines 328 -335 and legend of table 2).

Reviewer #2 (Comments for the Author):
This is a well written analysis of a new laboratory developed test designed to identify Chlamydia trachomatis and Neisseria gonorrhoeae, in addition the assay is able to identify CT serovars associated with lymphogranuloma venereum.

Response:
We appreciate the reviewer's careful evaluation of the manuscript and their helpful and detailed feedback, which has enhanced the quality of the paper.

Major revisions:
• As matrix of the specimen type is important to indicate there should be more emphasis put on the different specimen types collected via swab.These specimen types should be evaluated independently as different specimen types and body fluids associated with those sites can differently impact extraction and amplification of the target.Example: ocular swabs and rectal swabs should be evaluated separately with adequate positive and negative samples from each sample type.Refer to CLSI and validation requirements for LDTs.

Response:
We agree with the reviewer that different matrixes can impact extraction and amplification.Therefore, careful evaluation and validation is required especially concerning different clinically relevant matrixes before using newly established assays in routine diagnostics.
To address this topic, we have performed further experiments to verify that lower limits of detection (LLoDs) are similar in swabs taken from different body sites.For this purpose, we have collected swabs from the most clinically relevant body sites at our center (oropharyngeal, anal/rectal, cervical/vaginal, urethral/penile and ocular).All swabs used for these verification experiments were collected and stored in eSwab medium (Copan).3-4 matrix-pools per specimen type each containing 9-12 ml were created and tested for Chlamydia trachomatis and Neisseria gonorrhoeae by qPCR.Only non-reactive pools were included for further verification experiments.All matrix-pools were diluted 1:2 with Cobas PCR media (CPM, Roche).In addition to the matrix-pools also eSwab media diluted with CPM (1:2) was tested in the same manner to establish the technical performance without the presence of possible PCR inhibitors from human specimens.Next, dilution series from the same clinical standards used to determine LLoDs in eSwab and urine were used.The standards were stored in aliquots at -20° C (freeze-thawcycles: 0).Serial dilutions were designed to start well above the beforehand established LLoDs in eSwab and urine.The highest applied concentration was 800 dcp/ml and the lowest 25 dcp/ml.All concentrations were tested in technical replicas of 8.
LLoDs were determined to be similar compared to those established in eSwab media for oropharyngeal and ocular swabs and slightly higher for anal/rectal and urethral/penile swabs.However, cervical/vaginal swabs performed significantly worse compared to all other matrixes tested.This could be partly due to mucus that is present in cervical and vaginal swabs and known to interfere with PCR performance.Still, all internal control test results were valid for each dilution tested (for all tested matrixes).The detailed results of the verification experiments were added as a new supplementary table (supplementary table 1 and an explanation was added to the manuscript (see lines 126-133 and 343-349).Further, the results are enclosed in this point-topoint response at the end of this comment.
To provide a more comprehensive overview of the 4,298 clinical samples that were tested as part of this study under routine laboratory conditions, we have added a new supplementary table showing a detailed analysis of all swabs that were tested during the respective seven-month long period.The table lists all swabs tested according to the body sites where the swabs were collected and the respective number of Chlamydia trachomatis and Neisseria gonorrhoeae positive samples.
Another important point is to ensure a high quality of newly established LDTs, is to prevent falsenegative test result due to PCR inhibition.To this end, we chose the option "positive target confirmation with IC (internal control)" for the UC_CTNG assay.The IC is thus automatically spiked-in before extraction, thereby acting as a full process control.If the IC is not detected in the beforehand selected range (between ct min.and max), the PCR test result is marked as invalid.
Moreover, we would like to point out that regulations and guidelines on requirements for the use of LDTs for routine diagnostic vary depending on the different respective regulatory bodies as well as from country to country.Therefore, it is important to note, that the in our study described establishment and validation of the UC_CTNG assay warrants further verification experiments if use of the assay for diagnostics purposes is planned, especially concerning the different matrixes that the assay is going to be used for.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

performance in swabs collected from different body sites ocular swabs eSwab medium (Copan) urethral/penile swabs cervical/vaginal swabs oropharyngeal swabs anal/rectal swabs Chlamydia trachomatis Neisseria gonorrhoeae
This point was also added in discussion (see line 255-261).