Vol_32_3_2024

Le Infezioni in Medicina, n. 3, 352-362, 2024

doi: 10.53854/liim-3203-9

ORIGINAL ARTICLES

Promoting molecular diagnostic equity: assessing in-house real-time PCR for Neisseria gonorrhoeae in anal samples from MSM recruited in an outpatient setting in Morocco

Rokaya Aitlhaj-Mhand1,2, Aicha Qasmaoui4, Bahija Bellaji3, Chaimae Remz1, Reda Charof4, Rachid El Jaoudi5, Hanaa Abdelmoumen2, Amina Hançali3, Hicham Oumzil5

1Virology Department, National Institute of Hygiene, Rabat, Morocco;

2Microbiology and Molecular Biology Team, Research Center for Plant and Microbial Biotechnology, Biodiversity and Environment, Faculty of Sciences, Mohammed V University in Rabat, Rabat, Morocco;

3STIs Reference Laboratory, Medical Bacteriology Department, National Institute of Hygiene, Rabat, Morocco;

4Epidemic Diseases Laboratory, Medical Bacteriology Department, National Institute of Hygiene, Rabat, Morocco;

5Medical Biotechnology Pedagogy and Research Unit, School of Medicine and Pharmacy, Mohammed V University in Rabat, Morocco

Article received 30 May 2024, accepted 26 July 2024

Corresponding author

Rokaya Aitlhaj-Mhand

E-mail: rokayaaitlhaj@gmail.com

SummaRY

Objectives: Gonorrhea is a prevalent sexually transmitted infection among men who have sex with men (MSM). In Morocco, the basic laboratory diagnosis of Neisseria gonorrhoeae (NG) is based on microscopy and, in some settings, on culture. However, no nucleic acid amplification test (NAAT) has been implemented for routine diagnosis of gonorrhoeae.

The aim of this study is to assess the effectiveness of an in-house real-time PCR test for detecting N. gonorrhoeae DNA in anal swabs samples collected during an Integrated Behavioral and Biological survey.

Patients and methods: Samples from 245 MSM, recruited using a Respondent Driven Sampling, were collected and tested for NG infection using GeneXpert CT/NG assay (Cepheid, USA). An In-House real-time PCR technique targeting the pseudo gene porA was developed and used for a parallel investigation of the same infection. The reliability of the in-house RT-PCR was validated through tests of reproducibility, repeatability, limit of detection, and cross-reactivity with other bacteria. The intrinsic performance characteristics of the qRT-PCR were assessed, namely, the sensitivity, the specificity, the positive predictive value (PPV), and the negative predictive value (NPV). The GeneXpert CT/NG assay was adopted as a reference method.

Results: For N. gonorrhoeae detection, the in-house real-time PCR assay showed a sensitivity and specificity of 80% and 100%, respectively. The PPV of the assay was 100% and the NPV was 97.3%.

Conclusion: The in-house real-time PCR assay has high specificity and sensitivity, and it emerges as a promising approach for detecting N. gonorrhoeae in clinical specimens, particularly in decentralized settings such as regional laboratories.

Keywords: Neisseria gonorrhoeae, real time PCR, porA pseudogene, Morocco.

INTRODUCTION

Neisseria gonorrhoeae (NG) is one of the most common sexually transmitted infections (STIs), that affects people of both sexes, with worldwide around 82.4 million new infections among adults and adolescents (15-49 years old) each year according to World Health Organization (WHO) estimations [1]. It can infect the pharynx, rectal, and urogenital sites, and most often results in urethritis [2]. Men who have sex with men (MSM) are more likely to develop rectal and pharyngeal gonorrhea, which is typically asymptomatic [2]. N. gonorrhoeae can also facilitate the transmission and acquisition of the human immunodeficiency virus (HIV), and is frequently associated with other STIs such as syphilis, monkeypox, and human papillomavirus (HPV) [3-6].

Laboratory diagnosis of gonococcal infection is based on direct detection of the pathogen in urogenital, anorectal, pharyngeal or conjunctival swab specimens or in primary urine [7]. Previously, culture was regarded as the “gold standard” for identifying N. gonorrhoeae, and in some settings, it is still the sole diagnostic approach accessible due to its low cost [3]. In addition, it has been touted as a highly sensitive and specific approach for the detection of gonorrhoeae in urogenital specimens, and enables for antimicrobial susceptibility testing [7, 8]. However, the main drawbacks of culture are that it takes a long time (results typically take more than 48 hours), requires adequate growth conditions, and is less sensitive for pharyngeal and rectal samples [3, 8].

Due to the limitations of culture, nucleic acid amplification tests (NAATs) are currently the preferred method for detecting NG. In fact, sample collection is non-invasive and identification of viable organisms is not necessary, which allows for less restrictive transport and storage procedures [9, 10]. In addition, NAAT are considered to be the most sensitive for the diagnosis of extragenital NG infection and are thus recommended for laboratory diagnosis of rectal or pharyngeal infections [11]. Therefore, in resource-limited situations, a number of in-house NAATs have been and are used as confirmatory or diagnostic algorithms [12, 13]. The success of this tests depends on two main aspects: the proper selection of the target gene and the use of specific primers amplifying the conserved region of the target genes [14]. For N. gonorrhoeae, the porA pseudogene, opa, gyrA (encoding the A subunit of DNA gyrase), cppB (encoding the B protein of the cryptic plasmid), and the methyltransferase genes of N. gonorrhoeae, are the most targeted genes by in-house NAAT [15]. The porA N. gonorrhoeae pseudogene is sufficiently distinct from the porA gene of Neisseria meningitidis and exhibits high conservation, low genetic polymorphism, and absence in commensal Neisseria species [16].

In Morocco, based on recent analysis using the spectrum-STIs model, the overall annual incidence of STIs estimation is around 800,000, with a prevalence of N. gonorrhoeae of 0.37% among women and 0.32% among men [17]. Even though, there is a limited data of real prevalence within the Moroccan context.

After being recommended by the WHO at global level, Morocco has embraced the syndromic approach as a nationwide strategy for addressing STIs 1998 [18]. Despite the primary objective of this approach being to interrupt the transmission chain of STIs through rapid and effective diagnosis, without the need for complex laboratory tests such as microscopy and, in certain instances, culture, several issues emerged; particularly the development of resistance to antibiotics [19-22].

As a matter of fact, the non- adherence to medication prescription, the wild use antibiotics, has resulted in a situation where routine laboratory culture-based isolation of NG has become challenging, if not unfeasible, which subsequently led to inadequate treatment and promoted the spread of infection [21].

Moreover, considering that MSMs constitute a key population in the dynamics of STI transmission in Morocco, the establishment of an efficient, affordable, and sustainable molecular screening system becomes urgent. Furthermore, during the COVID-19 period, health laboratories have undergone infrastructure upgrades and are now equipped to conduct molecular biology tests for diagnostic or investigative purposes in the context of public health programs

To address this challenge, the current study aims specifically to evaluate the performance of an in-house real-time PCR assay targeting the porA pseudogene for the detection of N. gonorrhoeae DNA. This assessment aims to establish it suitability as a routine diagnostic tool, serving as an adjunct technique in health laboratories to make broad-spectrum testing accessible and to address supply limitations due to the high cost of the GeneXpert test.

PATIENTS AND METHODS

Participants Recruitment and sampling

From November 2020 to January 2021, a total of 245 MSM were recruited through an RDS (respondent-driven sampling) methodology in the framework of an Integrated Behavioral and Biological survey. Eligible participants were aged of 18 or older, living in Agadir for the last 6 months, and report having experienced anal intercourse with a man during the previous six-month period prior to interview; nationality was not considered.

Recruitment processing and sampling details were previously reported [23].

Laboratory molecular testing

The assessment the NG infection was performed on anal swab using the Xpert® CT/NG Kit (Cepheid, USA) as the primary tested and adopted as the gold standard method. In a subsequent phase, the in-house qRT-PCR was employed as an alternative method.

- Xpert® CT/NG test

Each specimen was processed and analyzed according to the manufacturer’s instructions. Briefly, a total of 500 µL of liquid from anorectal swabs was loaded in the single-use disposable GeneXpert® cartridge. Within less than 90 minutes, the results were obtained and confirmed as either positive or negative by the computer.

- Optimization of the In-House assay

Prior the use of the in-house real-time PCR assay, an optimization process was used to validate the reliability of the assay and to enhance its sensitivity and specificity.

The assay optimization was conducted on Genomic DNA extracted from cultured bacteria. Extraction was performed using QIAamp DNA mini kit (Qiagen Sciences Inc., USA) according to the manufacturer’s instructions. The quality of the obtained extracts was evaluated using the Nanodrop spectrophotometer (NanoDropOnne, Thermo Fisher scientific, VerinaRd, USA) and stored at -20°c until use.

a) In-house real-time PCR assay for the detection of N. gonorrhoeae infection and Internal control Human RNase P

The in-house real-time PCR assay primers and probe target the N. gonorrhoeae porA pseudogene. The forward and reverse primer sequences were 5’-CCGGAACTGGTTTCATCTGATT-3’(100nM) and 5’-GTTTCAGCGGCAGCATTCA-3’ (200 nM), respectively. The sequence for the TaqMan probe for the porA pseudogene was 5’-FAM-CGTGAAAGTAGCAGGCGTATAGGCGGACTT-BHQ1-3’(200nM) labelled with FAM 6- carboxy fluorescein [24].

A second real-time PCR targeting the human RNAse P gene was used to assess the extraction of adequate amplifiable DNA and absence of PCR inhibitory substances in the extracted DNA [25]. The forward and reverse primers for Internal control Human RNase P were 5’-CCAAGTGTGAGGGCTGAAAAG-3’ and 5’-TGTTGTGGCTGATGAACTATAAAAGG-3’(80nM) respectively and the probe sequence was 5’-CCCCAGTCTCTGTCAGCACTCCCTTC-3’ (80 nM) labelled with VIC.

Each PCR contained 4 µl of extracted DNA and 16 µl of Master Mix containing 10µl TaqMan Universal® Master Mix (Applied Biosystems, Foster City, California), 2 µl of each primer, 1µl of the probe and 1µl of nuclease free water.

PCR amplification and detection was performed in QuantStudio 5 Real-Time PCR (Applied Biosystems) using the following thermal cycling protocol: 2 min at 50°C, followed by 10 min at 95°C, 40 cycles of 15s at 95°C and 1min at 60°C.

b) Limit of detection (LoD)

To assess the detection limit of each PCR assay, DNA extracted from N. gonorrhoeae strain ATCC 49926 was produced in tenfold dilutions (from 10-1 to 10-9). Serial dilutions were prepared in triplicate.

c) PCR efficiency

The PCR efficiency was determined using a standard curve generated from the same dilution series as before. The efficiency was calculated according to the following formula: E=10−1/slope−1.

d) Inter-assay and intra-assay variability

Inter-assay and intra-assay variability were assessed to determine the repeatability and reproducibility of the real-time PCR assay. Repeatability was measured by assessing the variation in cycle threshold (Ct) values of three different concentrations of ATCC strains (high 10-2, moderate10-4 and low10-6) each tested six times within a single assay (intra-assay). Meanwhile, reproducibility was assessed by analyzing the fluctuations in Ct values of the three concentrations (10-3, 10-5, 10-7); each consisting of six replicates and repeated five times in separate PCR assays (inter-assay).

e) Cross-reaction over other microorganisms

The potential cross-reaction of the Real time PCR assay was examined using 26 bacterial strains (See Table 1) selected from laboratory bacterial stocks and chosen for their phylogenetically close links to N. gonorrhoeae or their presence in the extragenital tract. In addition, Testing for N. gonorrhoeae infection was carried out on anal swabs tested previously by GeneXpert [23].

Table 1 - Bacteria used in specificity test for the PCR assay of N. gonorrhoeae.

Statistical analysis

Performance parameters of the in-house real-time PCR assay, including sensitivity, specificity, Positive predictive value, and Negative predictive value for the detection of N. gonorrhoeae in anal specimens were estimated by comparing the results of the in-house real-time assay with those of the GeneXpert CT/NG. To analyze the data, Microsoft Excel and Jamovi were used.

Ethical Statement

Informed consent was obtained from all participants. The study protocol, questionnaires, and consent forms for the target groups, was approved by Casablanca Biomedical Research Ethics Committee at the Faculty of Medicine and Pharmacy of Casablanca at the Hassan II University. All procedures conducted in this study adhered to the ethical standards set by the institutional and/or national research committees, in line with the 1964 Helsinki Declaration and its subsequent amendments or comparable ethical guidelines.

RESULTS

Performance characteristics of PCR assays

- Analytical limit of detection (LOD) and PCR efficiency

The detection limit of PCR assay was determined as the lowest concentration returning a positive reaction. The porA pseudogene-based PCR was found to have a limit of detection of 0.6 pg N. gonorrhoeae DNA (Figure 1). A positive result was defined as a Ct value of less than 38. The 10-fold dilutions showed good linearity, determined by an r-squared (r^2) value of 0.99, indicating a consistent linear relationship across the dilution series. Through this optimization test, we see a PCR efficiency of 94%, indicates that the majority of our target DNA is being amplified accurately with each cycle (Figure 2).

Figure 1 - Amplification curves generated from dilutions series of N. gonorrhoeae DNA (ATCC strain).

Figure 2 - Standard curve based on the dilution series of Neisseria gonorrhoeae ATCC. The x-axis represents Ct values, while the y-axis represents quantity of the input DNA. The PCR efficiency, calculated from the slope (-3.461), is determined to be 94.509%, with a margin of error of 0.076.

- Analytical repeatability and reproducibility

The PCR profile curves generated from the repeatability test demonstrated consistent amplification across the three different concentrations (Figure 3).

To assess the repeatability and reproducibility of the real-time PCR assay, mean, standard deviation (SD), and coefficient of variation (CV) were calculated using cycle threshold values (Table 2). The analysis revealed CVs below 3% for both assay variability, with values ranging from 0.73% to 2.81% in intra-assay and from 2.01% to 2.86% in Inter-assay variability. The low standard deviation (SD) values for both assay suggest minimal variation or scatter around the mean, indicating good repeatability and reproducibility of the assay.

Figure 3 - Amplification curves generated from dilution series of three different concentrations of ATCC strains (high, moderate and low), replicate sex times within a single assay (intra-assay).

Table 2 - Intra and inter-assay variability.

- Analytical cross reactivity

DNA extracts from all N. gonorrhoeae strains examined were PCR positive, but none of the non- Neisseria gonococcal species, genital commensal bacterial strains, and yeast strains, were positive (See Figure 4).

Figure 4 - Analytical cross reactivity of N. gonorrhoeae with non- Neisseria gonococcal species, genital commensal bacterial strains, and yeast strains.

Performance of PCR assay in patient samples

The performance characteristics (sensitivity, specificity, PPV, and NPV) of the in-house real-time PCR assay were evaluated in comparison to GeneXpert.

Out of 245 MSM, 24 were positive by both GeneXpert, and porA pseudogene. Also, six samples tested positive for N. gonorrhoeae with the GeneXpert assay, but negative with the in-house real time PCR. The assay showed a sensitivity of 80%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 97.28% (See Table 3).

Table 3 - Performance of PCR assay in patient samples.

DISCUSSION

In recent years, the approach to gonorrhea diagnosis has evolved considerably, as culture, previously considered the “gold standard” for the identification of N. gonorrhoeae, has been replaced by NAATs, which are now routinely used for the detection of N. gonorrhoeae DNA in clinical specimens. Indeed, culture is a challenging technique and depends on various factors such as the method of specimen collection, the medium and the conditions of specimen transport, as well as requires ideal growth conditions and takes time to yield results [8, 9]. Additionally, the sensitivity of culture in pharyngeal and rectal specimens is much lower, whereas NAATs have been shown to be more sensitive for the detection of extragenital gonorrhea [3, 11].

In Morocco, the diagnostic strategy for Neisseria infections traditionally relied on culture and more often the syndromic approach, which was introduced by the World Health Organization and has had a profound impact on managing sexually transmitted infections STIs. This approach, implemented in 1998 and extended to all Health and Basic Services Establishments by 2000, sought to revolutionize STI management by facilitating swift and efficient diagnosis without the requirement for intricate laboratory procedures [18]. Prioritizing the syndromic approach aimed primarily at disrupting the transmission chain of STIs, including Neisseria infections, with the ultimate goal of enhancing the country’s capacity to control and prevent the spread of these diseases. However, this strategy may encounter challenges. Without an accurate diagnosis of the causative pathogen, it can result in inadequate treatment, fostering inappropriate antibiotic use, and thereby fueling the emergence of drug resistance [19, 20]. This issue is further complicated by coinfections with other STIs such as Mycoplasma genitalium, which increasingly exhibit resistance to macrolides - a common treatment for such infections [26]. What’s more, initiating antibiotic treatment without prior identification can compromise the ability to culture and identify the causative agent if initial treatment proves ineffective, hampering efforts to adjust therapy appropriately [21, 22].

Therefore, the use of PCR represents a major advance. This molecular diagnostic technique has revolutionized the management of STIs, enabling rapid and specific identification of infectious agents directly from their genetic material. Unlike the symptom-based approach, PCR facilitates the prescription of targeted, effective treatment, reducing the risk of antibiotic resistance and improving therapeutic outcomes for patients [27, 28].

In this context, our study is the first national research of its kind to evaluate a NAAT for the routine diagnosis of N. gonorrhoeae. By using in-house real-time PCR, a 102 bp fragment of the conserved region of the por A pseudogene was selected for amplification and detection.

The evaluation of PCR test demonstrated remarkable sensitivity, detecting N. gonorrhoeae DNA at concentrations as low as 1 pg/µL, underlining its potential for accurate diagnosis, even in cases where the presence of pathogens is insignificant. As for the PCR efficiency obtained in our assay, it showed efficient amplification (the amount of DNA doubling with each cycle), making our PCR assay reaction consistent and reliable. Nevertheless, the value of 94% may indicate a slight deviation from optimal conditions.

The outcomes of our real-time PCR assay’s assessment on repeatability and reproducibility demonstrate promise. In fact, CV values, below 3% for both intra-assay and inter-assay variability, indicate minimal variability in the cycle threshold values across and between experiments. Furthermore, the low SD values observed corroborate these results, suggesting minimal scatter of data points around the mean. In summary, these findings suggest that the PCR assay displays outstanding repeatability and reproducibility, bolstering confidence in the accuracy of the assay outcomes.

In addition, the porA pseudogene was found to be extremely specific, as all y gonococcal strains studied tested positive for this NAAT, and neither of the nongonococcal or non-Neisseria species tested positive. The porA pseudogene-based assay’s excellent specificity was consistent with previous research that found this gene to be highly conserved among different N. gonorrhoeae subtypes [29]. Further research has demonstrated that the porA is a good target for NG diagnosis [29, 30].

To further evaluate the sensitivities and specificities in clinical samples, 245 anal swabs previously assayed by GeneXpert were selected [23]. The in-house real time PCR assay provided 80% sensitivity and 100% specificity compared with Xpert CT/NG.

Comparing our findings with various studies reveals a consistent detection of N. gonorrhoeae using different sample types, qRT-PCR methods, and targeted genes over several years (See Table 4). Notably, approaches focusing on the porA pseudogene have shown high sensitivity and specificity rates, with one study achieving 100% for both parameters [31, 32]. Moreover, employing PCR assays targeting different genes has also shown significant sensitivity and specificity. Indeed, targeting the cytosine-specific DNA methyltransferase gene, penA gene and gyrA gene of N. gonorrhoeae has resulted in high levels of both sensitivity and specificity [33-35. Regarding studies using urethral and endocervical specimens, notable sensitivity and specificity were achieved in targeting the opa gene based on nucleic acid amplification tests [36].

Table 4 - Sensitivity and specificity of N. gonorrhoeae detection across studies: A comparison of specimen types, qRT-PCR Methods, and targeted genes.

As for other molecular diagnostic methods, such as loop-mediated isothermal amplification (LAMP), it exhibited excellent sensitivity but showed lower specificity at 85.7% [37].

Although studies typically show high sensitivity and specificity when targeting various genes for N. gonorrhoeae detection, such as those previously mentioned, some gene targets, like 16S rRNA in other research, produced lower sensitivity [38].

Using the in-house real-time PCR test has some advantages over the GeneXpert. The cost of the commercial test, including cartridges, is higher than our in-house PCR test. In addition, our PCR assay takes about 1h 30 minutes, therefore, for 46 clinical samples, the total test time including extraction is 2 hours and 30 minutes, whereas the GeneXpert system takes 90 minutes per sample.

Although the porA pseudogene-based assay in this study was found to be specific, false-negative findings could emerge from searching for a single specific gene that can be obtained from a commensal strain, particularly in low-prevalence circumstances [39]. However, by targeting two genes simultaneously, multiplex PCR can reduce the possibility of false-negative results. In a previous work, a duplex PCR targeting the gonococcal porA pseudogene and opa multicopy genes was found to be superior to a monoplex real-time PCR test [40].

Several constraints are present within this study. A limited number of samples were tested by PCR assay, as well as the analytical specificity test was only performed on a small panel of bacteria and yiest strains that have close phylogenetic links with N. gonorrhoeae or that are present in the extragenital tract.

The discrepant results between the in-house real-time PCR and the GeneXpert can be attributed to several factors. Specifically, it is noteworthy that the samples underwent prolonged storage at a temperature of -20°C and were subjected to at least one freeze-thaw cycle. These circumstances have the potential to induce degradation of the pathogens present.

Lastly, the current study demonstrates the suitability of the employed real-time PCR method for assessing gonorrhoeae infection. Furthermore, there exists potential for the expansion of this approach into a multiplex PCR assay, capable of detecting additional sexually transmitted pathogens like Trichomonas vaginalis, Chlamydia trachomatis and Mycoplasma genitalium. This avenue could serve as a promising direction for future research endeavor.

CONCLUSIONS

Our research has showcased that the in-house real-time PCR assay for detecting N. gonorrhoeae in extragenital samples boasts high sensitivity and specificity. This makes it well-suited for routine implementation in central as well as regional laboratories.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Global fund to fight HIV, Tuberculosis and Malaria (grant code: MAR-C-MOH).

Conflicts of interest

None.

Funding

This work was supported by the Global fund to fight HIV, Tuberculosis and Malaria (grant code: MAR-C-MOH).

Acknowledgements

We would like to thank the National Institute of Hygiene, the Global Fund to Fight HIV/AIDS (GFATM), the Joint United Nations Program on HIV/AIDS (UNAIDS) and the Ministry of Health. Also, a special acknowledgement for NGOS and ALCS.

Informed consent statement

Informed consent was obtained from all subjects involved in the study.

Authors’ contribution

HO, AH, designed the study and implemented the study. AQ, BB collected the data. Laboratory investigations performed by RA, BB, AQ. Validation of analysis was done by HO AH HA. RA HO wrote the first draft of the manuscript. All authors critically revised the manuscript for important intellectual content and gave final approval of the version to be published.

RA and HO have full access to all of the data and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.

REFERENCES

[1] New partnership to combat antimicrobial resistance in gonorrhoea 2021. Available from: https://www.who.int/news/item/08-12-2021-new-partnership-to-combat-antimicrobial-resistance-in-gonorrhoea. [accessed August 11, 2022].

[2] Unemo M, Seifert HS, Hook EW 3rd, Hawkes S, Ndowa F, Dillon JR. Gonorrhoea. Nat Rev Dis Primers. 2019; 5(1): 79. Published 2019 Nov 21. doi: 10.1038/s41572-019-0128-6.

[3] Quillin SJ, Seifert HS. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol. 2018; 16(4): 226-240. doi: 10.1038/nrmicro.2017.169.

[4] Rob F, Jůzlová K, Kružicová Z, et al. Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae co-infections among patients with newly diagnosed syphilis: a single-centre, cross-sectional study. Cent Eur J Public Health. 2019; 27(4): 285-291. doi: 10.21101/cejph.a5142.

[5] Ciccarese G, Di Biagio A, Bruzzone B, et al. Monkeypox outbreak in Genoa, Italy: Clinical, laboratory, histopathologic features, management, and outcome of the infected patients. J Med Virol. 2023; 95(2): e28560. doi: 10.1002/jmv.28560.

[6] de Abreu AL, Malaguti N, Souza RP, et al. Association of human papillomavirus, Neisseria gonorrhoeae and Chlamydia trachomatis co-infections on the risk of high-grade squamous intraepithelial cervical lesion. Am J Cancer Res. 2016; 6(6): 1371-1383.

[7] Meyer T, Buder S. The laboratory diagnosis of Neisseria gonorrhoeae: current testing and future demands. Pathogens. 2020; 9(2): 91. Published 2020 Jan 31. doi: 10.3390/pathogens9020091.

[8] Clinical manifestations and diagnosis of Neisseria gonorrhoeae infection in adults and adolescents - UpToDate 2019. Available at: https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-neisseria-gonorrhoeae-infection-in-adults-and-adolescents. [accessed March 13, 2021].

[9] Unemo M, Ballard R, Ison C, et al. Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus. World Health Organization; 2013.

[10] Tabrizi SN, Unemo M, Limnios AE, et al. Evaluation of six commercial nucleic acid amplification tests for detection of Neisseria gonorrhoeae and other Neisseria species. J Clin Microbiol. 2011; 49(10): 3610-3615. doi: 10.1128/JCM.01217-11.

[11] Cornelisse VJ, Chow EP, Huffam S, et al. Increased detection of pharyngeal and rectal gonorrhea in men who have sex with men after transition from culture to nucleic acid amplification testing. Sex Transm Dis. 2017; 44(2): 114-117. doi: 10.1097/OLQ.0000000000000553

[12] Kulkarni S, Bala M, Risbud A. Performance of tests for identification of Neisseria gonorrhoeae. Indian J Med Res. 2015; 141(6): 833-835. doi: 10.4103/0971-5916.160721.

[13] Venter JME, Mahlangu PM, Müller EE, et al. Comparison of an in-house real-time duplex PCR assay with commercial HOLOGIC® APTIMA assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urine and extra-genital specimens. BMC Infect Dis. 2019; 19(1): 6. Published 2019 Jan 3. doi: 10.1186/s12879-018-3629-0.

[14] Centers for Disease Control and Prevention. Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae - 2014. MMWR Recomm Rep. 2014; 63(RR-02): 1-19.

[15] Whiley DM, Tapsall JW, Sloots TP. Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge. J Mol Diagn. 2006; 8(1): 3-15. doi: 10.2353/jmoldx.2006.05004

[16] Whiley DM, Anderson TP, Barratt K, et al. Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target. Pathology. 2006; 38(5): 445-448. doi: 10.1080/00313020600928253.

[17] El-Kettani A, Mahiané G, Bennani A, et al. Trends in adult Chlamydia and Gonorrhea prevalence, incidence and urethral discharge case reporting in Morocco over 1995-2015-estimates using the spectrum-sexually transmitted infection model. Sex Transm Dis. 2017; 44(9): 557-564. doi: 10.1097/OLQ.0000000000000647.

[18] Hançali A, Ndowa F, Bellaji B, et al. Antimicrobial resistance monitoring in Neisseria gonorrhoeae and strategic use of funds from the Global Fund to set up a systematic Moroccan gonococcal antimicrobial surveillance programme. Sex Transm Infect. 2013; 89 Suppl 4:iv24-iv27. doi: 10.1136/sextrans-2013-051166.

[19] Karim S, Bouchikhi C, Banani A, et al. Bacterial sexually transmitted infections and syndromic approach: a study conducted on women at Moroccan University Hospital. Germs. 2021; 11(4): 544-553. doi: 10.18683/germs.2021.1289.

[20] Broom A, Peterie M, Ridge D, et al. Sex, drugs and superbugs: the rise of drug resistant STIs. SSM - Qual Res Health. 2023; 4: 100310. https://doi.org/10.1016/j.ssmqr.2023.100310.

[21] Verma R, Sood S. Gonorrhoea diagnostics: An update. Indian J Med Microbiol. 2016; 34(2): 139-145. doi: 10.4103/0255-0857.180278.

[22] Sarenje KL, Ngalamika O, Maimbolwa MC, Siame A, Munsaka SM, Kwenda G. Antimicrobial resistance of Neisseria gonorrhoeae isolated from patients attending sexually transmitted infection clinics in Urban Hospitals, Lusaka, Zambia. BMC Infect Dis. 2022; 22(1): 688. doi: 10.1186/s12879-022-07674-y.

[23] Aitlhaj-Mhand R, Bellaji B, Jennane S, et al. Assessment of Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis prevalence using a molecular Point of Care: Findings from a respondent driven sampling study among MSM. Infez Med. 2023; 31(2): 234-242. doi: 10.53854/liim-3102-12.

[24] Hjelmevoll SO, Olsen ME, Sollid JU, Haaheim H, Unemo M, Skogen V. A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene. J Mol Diagn. 2006; 8(5): 574-581. doi: 10.2353/jmoldx.2006.060024.

[25] Tatti KM, Sparks KN, Boney KO, Tondella ML. Novel multitarget real-time PCR assay for rapid detection of Bordetella species in clinical specimens. J Clin Microbiol. 2011; 49(12): 4059-4066. doi: 10.1128/JCM.00601-11.

[26] Lam PPH, Nguyen NH, Nguyen TTT, Trinh NB, Luong BA. Mycoplasma genitalium prevalence, co-infection and macrolide resistance-associated mutations in Southern Vietnam. Infez Med. 2024; 32(2): 222-230. doi: 10.53854/liim-3202-10.

[27] Mitchev N, Singh R, Ramsuran V, et al. Assessment of Antibiotic resistance and efflux pump gene expression in neisseria gonorrhoeae isolates from south africa by quantitative real-time pcr and regression analysis. Int J Microbiol. 2022; 2022: 7318325. doi: 10.1155/2022/7318325.

[28] de Korne-Elenbaas J, Pol A, Vet J, Dierdorp M, van Dam AP, Bruisten SM. Simultaneous detection of Neisseria gonorrhoeae and fluoroquinolone resistance mutations to enable rapid prescription of oral antibiotics. Sex Transm Dis. 2020; 47(4): 238-242. doi: 10.1097/OLQ.0000000000001141.

[29] Unemo M, Norlén O, Fredlund H. The porA pseudogene of Neisseria gonorrhoeae--low level of genetic polymorphism and a few, mainly identical, inactivating mutations. APMIS. 2005; 113(6): 410-419. doi: 10.1111/j.1600-0463.2005.apm_206.x.

[30] Hjelmevoll SO, Olsen ME, Sollid JU, et al. Clinical validation of a real-time polymerase chain reaction detection of Neisseria gonorrheae porA pseudogene versus culture techniques. Sex Transm Dis. 2008; 35(5): 517-520. doi: 10.1097/OLQ.0b013e3181644bc9.

[31] Whiley DM, Buda PJ, Bayliss J, Cover L, Bates J, Sloots TP. A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the porA pseudogene. Eur J Clin Microbiol Infect Dis. 2004; 23(9): 705-710. doi: 10.1007/s10096-004-1170-0.

[32] Alexander S, Coelho da Silva F, Manuel R, Varma R, Ison C. Evaluation of strategies for confirming Neisseria gonorrhoeae nucleic acid amplification tests. J Med Microbiol. 2011; 60(Pt 7): 909-912. doi: 10.1099/jmm.0.028563-0.

[33] Venter JME, Mahlangu PM, Müller EE, et al. Comparison of an in-house real-time duplex PCR assay with commercial HOLOGIC® APTIMA assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urine and extra-genital specimens. BMC Infect Dis. 2019; 19(1): 6. doi: 10.1186/s12879-018-3629-0.

[34] Sethi S, Saini G, Sreenivasan P, et al. Performance evaluation of loop-mediated isothermal amplification, polymerase chain reaction and real-time polymerase chain reaction methods to detect Neisseria gonorrhoeae among symptomatic patients from India. Int J STD AIDS. Published online May 9, 2024. doi: 10.1177/09564624241252185.

[35] Perera SR, Khan NH, Martin I, et al. Multiplex Real-Time PCR Assay for simultaneous identification of Neisseria gonorrhoeae and its ciprofloxacin susceptibility status. J Clin Microbiol. 2017; 55(11): 3201-3209. doi: 10.1128/JCM.00855-17.

[36] Verma R, Sood S, Bala M, et al. Evaluation of an opa gene-based nucleic acid amplification test for detection of Neisseria gonorrhoeae in urogenital samples in North India. Epidemiol Infect. 2012; 140(11): 2110-2116. doi: 10.1017/S0950268811002883.

[37] Liu ML, Xia Y, Wu XZ, Huang JQ, Guo XG. Loop-mediated isothermal amplification of Neisseria gonorrhoeae porA pseudogene: a rapid and reliable method to detect gonorrhea. AMB Express. 2017; 7(1): 48. doi: 10.1186/s13568-017-0349-6.

[38] Oree G, Naicker M, Maise HC, Tinarwo P, Ramsuran V, Abbai NS. Comparison of methods for the detection of Neisseria gonorrhoeae from South African women attending antenatal care. Int J STD AIDS. 2021; 32(5): 396-402. doi: 10.1177/0956462420971439.

[39] Priyadarshi K, Prakash P, Rani A, Singh SK. Multiplex nested polymerase chain reaction targeting multiple genes for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens. Indian J Sex Transm Dis AIDS. 2019; 40(2): 152-158. doi: 10.4103/ijstd.IJSTD_73_18.

[40] Goire N, Nissen MD, LeCornec GM, Sloots TP, Whiley DM. A duplex Neisseria gonorrhoeae real-time polymerase chain reaction assay targeting the gonococcal porA pseudogene and multicopy opa genes. Diagn Microbiol Infect Dis. 2008; 61(1): 6-12. doi: 10.1016/j.diagmicrobio.2007.12.007.