1. Introduction
Sexually transmitted infections (STIs) represent a public health problem, due to the morbidity of acute infections and the risk of serious sequelae [1] . The World Health Organization estimated that 450 million people are victims of STI per year worldwide, among which the number of patients suffering from gonorrhea accounts for more than 78 million [1] . Gonorrhoea is the second most common bacterial STIs in the world [2] . The causative agent: Neisseria gonorrhoeae is an obligate human pathogen which infects the mucosa of the lower genital tract [2] . 75 million gonorrhea cases are recorded each year worldwide, concerning 19 per 1000 females and 24 per 1000 males [3] . In England, gonorrhea is the second most common bacterial STI after chlamydia [3] . In 2014, 34,958 cases of gonorrhea were reported through routine surveillance by public Health England (PHE) [4] . The burden is heavier in Africa and it is continuously evolving [3] [5] . A study conducted in Cameroon by Chanyi et al. from 2010 to 2015 in the southwest region showed that the prevalence of gonorrhoea was increasing over the years till 12.9% in 2015 [6] .
Gonorrhoeae is more often asymptomatic especially in women (approximately 50% of cases), thereby leading to undiagnosed and untreated gonorrhoea [7] . Less biased studies’ findings revealed that 70% to 90% of women, carrying Neisseria gonorrhoeae, are asymptomatic [8] [9] . However, when it is symptomatic, signs such as cervicitis with purulent leucorrhoea, or even vulvar irritation are frequently observed [10] . In men, symptoms often include painful urination, abnormal urethral discharge, and swelling of the testicles [10] . Untreated gonorrhoea can progress to disseminated gonococcal infection, pelvic inflammatory disease with long-term sequelae such as chronic pelvic pain, tissue scarring, ectopic pregnancy and infertility [11] . In addition, it can be transmitted from mother to newborn during delivery, causing conjunctivitis in the newborn, and even blindness if the infection is not detected and treated promptly and accordingly [11] .
Gonorrhoea has become a public health priority due to the emergence of strains resistant to ceftriaxone/cefixime, penicillin and azithromycin worldwide between 2007 and 2018 [12] . In 2020, the WHO included gonococcus resistant to third-generation cephalosporins and fluoroquinolones on the list of priority bacteria to be monitored. To escape the toxicity of these antibiotics, Neisseria gonorrhoeae has developed several resistance mechanisms such as chromosomal mutations and the production of some enzymes (β-lactamases) [13] . Thus, the emergence of resistant phenotypes within this bacterium can have many consequences on human health, such as therapeutic failures, which contribute to increase morbidity and mortality rates, as well as high expenditure [14] .
A recent study in France showed that the proportions of N. gonorrhoeae isolates resistant to penicillin, tetracycline and ciprofloxacin were respectively 13%, 56% and 42% [15] . Another study carried out in China from 2014 to 2015 showed that the proportions of N. gonorrhoeae isolates resistant to tetracycline, ciprofloxacin and penicillin were respectively 81.7%, 100% and 73.3%. This latter study also showed that the proportion of penicillinase producing N. gonorrhoeae was 39.7% [16] . More recently, isolates of Neisseria gonorrhoeae resistant to several antibiotics have been identified and included penicillin, azithromycin, ciprofloxacine and extended spectrum cephalosporins, such as ceftriaxone and cefixime [17] . Moreover, another study conducted in Cameroon from 2012 to 2018 showed high proportions of resistant N. gonorrhoeae to ciprofloxacin (64.4%) [18] . The resistance to ciprofloxacin in this latter study had increased significantly from 15% in 2012 to 79.5% in 2018 [18] .
For long time, penicillinase-type β-lactamase producing Neisseria gonorrhoeae has been evolving. It has been recently found related to co-resistance, concerning various classes of antibiotics. Moreover, genes encoding for penicillinases being plasmid mediated, have readily spread throughout the world, thereby by challenging the global public health [3] . In 1984, a study realized in Central African Republic on 460 males showed that the prevalence of penicillinase-type β-lactamase producing Neisseria gonorrhoeae was 1.5% [3] . More recently, a study conducted in South Africa showed a prevalence of 60% [5] . In Cameroon, a study showed that the period prevalence (2012-2018) was 81% [18] . However, penicillinase-type β-lactamase producing Neisseria gonorrhoeae remains insufficiently described in Cameroon. This current research hence aimed to describe the epidemiology of penicillinase-type β-lactamase producing Neisseria gonorrhoeae in patients presenting with genital disorders in two health-facilities in Yaounde, Cameroon.
2. Material and Method
2.1. Study Design
A cross-sectional descriptive and analytical study was carried out from the 2nd May 2022 to the 2nd October 2022 in two hospital laboratories of Yaounde: namely Gyneco-Obstetrics and Pediatric Hospital of Yaounde’s (HGOPY) laboratory and the Saint Martin of Porres Dominican Hospital’s laboratory.
2.2. Sampling Population and Data Collection
This study included patients of both male and female gender who came to the laboratories of the concerned health facilities for cervical-vaginal/urethral samples and who consented. The minimum sample size was calculated using the Lorentz formula, including the prevalence of N. gonorrhoeae infection in Africa (5%) [3] as follows:
Cervical-vaginal/urethral secretions consecutively collected from them were subjected to N. gonorrhoeae screening. Data relative to demographics and potential risk factors to penicillinase-type β-lactamase producing Neisseria gonorrhoeae carriage were collected using a questionnaire designed in the two national languages (English and French). The questionnaire was constituted of closed ended questions and was pretested prior to the study, toward patients consulting the laboratories of a healthfacility in Bangangte (“Clinique Universitaire des Montagnes”) for cervical-vaginal/urethral samples (Appendix).
2.3. Sample Analysis
The samples were inoculated on Chocolate agar [prepared from Columbia agar (Titan Biotech Ltd., India) and sheep blood] + polyvitex (LiofilchemSrl, Italy) for urethral samples, and on Chocolate agar + polyvitex + VCN (LiofilchemSrl, Italy) for cervical & vaginal samples. After inoculation, the media were placed together with a CO2 DC-100 device in a jar ([CO2]: 5% - 10%), and incubated at 37˚C for 24 to 48 h. Identification of isolates and detection of penicillinases were carried out using the API NH micro gallery (Biomerieux, France) and subsequently tested for antimicrobial susceptibility.
2.4. Antimicrobial Susceptibility Testing
The antimicrobial susceptibility testing was carried out using the Kirby Bauer disk diffusion method [19] on Chocolate agar + polyvitex. The following antibiotics (Rapid Labs, England): amoxicillin (30 µg), amoxicillin + clavulanic acid (20 - 10 µg), cefixime (30 µg), penicillin G (10 µg), ceftriaxone (30 µg), tetracycline (30 µg), nalixidic acid (30 µg), ciprofloxacin (5 µg), azithromycin (15 µg), gentamycin (10 µg), spectinomycin (100 µg) [20] ; were tested. The incubation was done at 37˚C with 5% - 10% of CO2. This assay was performed according to the 2022 recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2022) [20] .
The confirmation of penicillinase production was carried out using thedouble disk synergy testing according to the EUCAST recommendations [20] . The production of penicillinase was shown when the inhibition zone around penicillin antibiotic disk was enhanced on the side of the clavulanate-containing disk, resulting in a characteristically shaped zone referred to as a “champagne cork” on one hand, and a reduction in the inhibition diameters around tetracycline and ciprofloxacin disks on the other hand.
The Minimal Inhibitory Concentrations (MIC) of Azithromycin, Ceftriaxone, Ciprofloxacin, and Penicillin G toward penicillinase type β-lactamase producing Neisseria gonorrhoeae isolates were determined using E-Test strips (Biomerieux, France).
2.5. Statistical Analysis
Statistical analysis was carried out using Epi-Info 7 software. The confidence interval was calculated when necessary. Fisher-Exact test helped to compare proportions and logistic regression helped to identify the predictive factors of participants’ carriage of penicillinase type β-lactamase producing Neisseria gonorrhoeae. p ≤ 0.05 was considered statistically significant.
2.6. Ethical Consideration
The study was approved by the Institutional Ethics Committee of Université des Montagnes (N˚2022/173/UdM/PR/CEAQ) and the Institutional Committee for Research on Human Health of HGOPY (Authorization N˚350/CIERSG/DM/2022). Informed consent was obtained from all participants.
3. Results
3.1. Socio-Demographics of Participants
Table 1 presents the distribution of participants according to their socio-demographic characteristics.
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Table 1. Socio-demographic characteristics of participants.
Table 1 shows that, out of the 198 participants sampled, 100 (51.51%) were <30 years old, 130 (65.66%) were females, 108 were married (53.03%), 132 had a superior level of education (66.67%) and 56 were students (28.28%).
3.2. Prevalence of Neisseria gonorrhoeae Carriage
Table 2 shows that the prevalence of Neisseria gonorrhoeae carriage among participants was 8.08%. The prevalence of Neisseria gonorrhoeae carriage among participants of the male gender (14.71%) was significantly higher as compared to the prevalence among participants of the female gender (4.62%).
3.3. Prevalence of Penicillinase Type β-Lactamases Producing Neisseria gonorrhoeae
Figure 1 shows that the prevalence of penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates was 81.25%.
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Table 2. Distribution of Neisseria gonorrhoeae infection according to socio-demographic characteristics.
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Figure 1. Prevalence of penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates.
3.4. Resistance Prevalence of Penicillinase-Like β-Lactamases Producing Neisseria gonorrhoeae Isolates to Tested Antibiotics
Figure 2 illustrates the co-resistance levels of penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates to Macrolides, Aminoside, Cycline, and Quinolones.
Penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates showed 100% co-resistance to ciprofloxacin, 92.31% to nalidixic acid and 84.62% to azithromycin (Figure 2).
3.5. Minimum Inhibitory Concentrations of 4 Antibiotics Tested on Penicillinase-Like β-Lactamases Producing Neisseria gonorrhoeae Isolates
Figure 3 shows that 2 mg/L, 6 mg/L, 2 mg/L, and 0.094 mg/L were respectively the MICs of azithromycin, ciprofloxacin, penicillin G and ceftriaxone for respectively 25%, 18.75%,18.75%, and 18.75% of penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates.
3.6. Analysis of Risk Factors Related to Penicillinase-Like β-Lactamases Producing Neisseria gonorrhoeae
Table 3 shows that a history of STIs (OR = 5.00; p = 0.00), unprotected sexual practices (OR = 4.98; p = 0.00) and hospitalization within the past 3 months (OR = 4.62; p = 0.05) were statistically associated to carriage of penicillinase-type β-lactamases producing Neisseria gonorrhoeae.
Furthermore, Table 4 shows that the adjustment of these risk factors highlighted both history of STIs (adjusted OR = 5.18; p = 0.01) and unprotected sexual practices (adjusted OR = 4.72; p = 0.01) as significant risk factors to penicillinase-type β-lactamases producing Neisseria gonorrhoeae carriage. Obviously, the odds of having a history of STI among participants was 5.18 folds higher
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Figure 2. Co-resistance prevalence of penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates to non-β-lactam antibiotics.
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Figure 3. Frequencies of minimum inhibitory concentrations of azithromycin, ceftriaxone, ciprofloxacin and penicillin G tested on penicillinase-like β-lactamases producing Neisseria gonorrhoeae isolates.
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Table 3. Univariate analysis of risk factors related to penicillinase-type β-lactamases producing Neisseria gonorrhoeae carriage.
OR. Odds Ratio, CI. Confidence Interval.
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Table 4. Multivariate analysis of risk factors related to penicillinase-type β-lactamases producing Neisseria gonorrhoeae carriage.
for those contaminated with penicillinase-type β-lactamases producing Neisseria gonorrhoeae as compared to non-contaminated ones. Likewise, the odds of practicing unprotected sex were 4.72 folds higher for participants contaminated with penicillinase-type β-lactamases producing Neisseria gonorrhoeae as compared to non-contaminated participants.
4. Discussion
The objective of this study was to determine the prevalence and risk factors of penicillinase type β-lactamase-producing Neisseria gonorrheae among patients consulting for genital infections in two health-facilities in Yaounde.
The most represented age group was that of <30 years with a proportion of 50.51%, thereby highlighting the fact that younger people were more frequently visiting the concerned health facilities for gynecological consultations during the study period. Regarding gender, women were approximately twice as represented (65.66%) as men did (34.34%). This is because cervico-vaginal tests were more requested by prescribers for diagnosis of various diseases, whereas men’s urethral sample tests were requested only for suspicious gonorrhoea cases.
The Neisseria gonorrhoeae genital carriage was quite prevalent: 8.08%. This finding is lower than that of Chuan et al who obtained a prevalence of 40% in China [21] . This difference can be explained by the therapeutic routes in the Cameroonian context, where most infected patients rely firstly on traditional or self-treatments in communities before consulting health facilities after the persistence of the infections [22] [23] .
The distribution of Neisseria gonorrhoeae infection according to socio-demographic characteristics showed that the percentage of Neisseria gonorrhoeae contamination was significantly higher in men (14.71%) than in women (4.62%). This can be explained by the fact that in men, the incubation period of this bacterium is shorter (2 to 6 days). Thus, after the third day of infection, men will begin to present symptoms, and will therefore seek for care, whereas in women, the infection is asymptomatic in more than 50% of cases, and the diagnosis is therefore delayed [7] .
A high prevalence of penicillinases producing Neisseria gonorrhoeae (81.25%) was obtained during this study. This result is higher than that of Teke et al. who reported a prevalence of 65% of penicillinases producing Neisseria gonorrhoeae [24] . This is an evidence of the emergence of penicillinases producing Neisseria gonorrhoeae upon time which can be attributed to the fact that penicillin has been used as a first-line drug for gonorrhea treatment [18] . Penicillin usage has probably induced the selection of penicillinases producing Neisseria gonorrhoeae strains through adaptation [25] .
The qualitative antimicrobial susceptibility testing on penicillinases producing Neisseria gonorrhoeae showed high co-resistance rates to ciprofloxacin (100%), nalidixic acid (92.31%) and azithromycin (84.62%). These findings are similar to those of Mabonga et al, who also obtained high levels of co-resistance toward these antibiotics [25] . Indeed, ciprofloxacin and azithromycin have been used earlier as first-line treatment for gonorrheaand are readily sold in streets and thus, accessible to the community at affordable prices [22] . Additionally, the findings of Kularatne et al. have shown a considerable increase in the co-resistance prevalence of Neisseria gonorrhoeae to ciprofloxacin, from 25% in 2008 to 69% in 2017 in South Africa. This illustrates the overuse, misuse or non-standard use of ciprofloxacin [26] . It was on this basis that quinolones have been excluded from the list of antibiotics recommended for gonorrhea therapy [23] .
This study showed a high MICs for azithromycin (2 mg/L, 25%), ciprofloxacin (6 mg/L, 18.75%), and penicillin G (2 mg/L, 18.75%), toward penicillinase type β-lactamase producing Neisseria gonorrhoeae isolates. This further highlights the co-resistance suspicion of penicillinases producing Neisseria gonorrhoeae strains to these antibiotics. Similarly, high MICs have been observed for ceftriaxone (6 mg/L, 12.5%; 8 mg/L, 6.25%; 24 mg/L, 12.5%). These findings are in line with those of Crucitti et al., who also observed high MICs for ceftriaxone (1 and 1.5 mg/L) [18] . This raises the concern that ceftriaxone has to be sparingly monitored in order to mitigate Neisseria gonorrhoeae strains resistant to this antibiotic.
Multivariate logistic regression analysis showed that, having a history of STIs and practicing unprotected sexual intercourse are risk factors to the carriage of penicillinase-type β-lactamases producing Neisseria gonorrhoeae. This can be explained by the fact that persistent gonorrhea (due to its resistant nature) significantly increases the risk of transmission of other STIs such as HIV, because of its invasive nature, leading to lesions and thereby promoting or facilitating the transmission of other sexually transmissible pathogens [27] . Also, unprotected sexual practices due to the absence of a physical barrier procured by condoms, increase the chances of transmission of penicillinase-type β-lactamases producing Neisseria gonorrhoeae. Accordingly, these findings highlight the fact that the presence of history of STIs and the practice of unprotected sex among patients consulting for genital disorders, independently exacerbate the chances of being contaminated with penicillinase-type β-lactamases producing Neisseria gonorrhoeae. This can serve as basis for decision making, thereby constituting a tool to mitigate this phenomenon.
This study had nevertheless two main limitations. The first one is the detection of penicillinase production through classical bacteriological techniques, which have relatively lower sensitivity and specificity as compared to molecular techniques. This can hence contribute to an over-estimation or under-estimation of our principal measurement, i.e. prevalence of penicillinase-type β-lactamases producing Neisseria gonorrhoeae. The second one is the non-representative population included in this study and likely represented by patients of low socio-economic level and visiting only two health facilities. This calls for more researches to study accurately and globally penicillinase-type β-lactamases producing Neisseria gonorrhoeae, in order to guide effective preventive actions.
5. Conclusion
This present study revealed a high prevalence of penicillinase-type β-lactamases producing Neisseria gonorrhoeae. This latter eventually showed high proportions of co-resistance, mainly to quinolones and macrolides. The factors identified to increase the risk of contamination with penicillinase-type β-lactamases producing Neisseria gonorrhoeae were unprotected sexual intercourse practices and having a history of Sexually Transmitted infections. Ultimately, this research indicates a worrisome emergency of resistant Neisseria gonorrhoeae. Hence, identified risk factors can constitute a basic outlook to tackle resistant Neisseria gonorrhoeae, and therefore sustain antibiotic stewardship.
Consent for Publication
All authors consented for publication.
Availability of Data and Material
All data are available upon request.
Authors’ Contributions
Conceptualization: CID; Project administration: CID and BDPT; Resources: CID, LND, and BDPT; Methodology: CID, CSN, CT, BDPT and LND, Funding acquisition: CID, LND, and BDPT; Investigation: LND, CSN, JMT, BKM, FW, GFN, TVKK and HKT; Software: CID and CSN; Formal analysis: CSN and WYD; Visualization: CID, LND, CSN, FW, WYD and BDTP; Writing-original draft: LND; Writing-review & editing: CID, CSN, FW, and BDTP; Validation: BDTP, NAM and CID; Supervision: BDTP and NAM; Data curation: CID.
Acknowledgements
The authors hereby thank the directors and staff of the Saint Martin of Porres Dominican Hospital, the Yaounde Gynaeco-Obstetric and Paediatric Hospital and the Public Health National Laboratory for their financial and material support. The authors are also grateful to the data collectors and participants in the study.
Appendix
Questionnaire
What is your age? What is your sex? What was your highest educational level? What is your marital status? What is your actual occupation? Do you always protect yourselves during sexual intercourse? Have you got a sexually transmissible infection in the past? Have you been hospitalized for the past three months? Do you always rely on a health professional to buy drugs when you are ill? Do you sometimes take personal initiatives in the choice of drugs you consume when you feel ill? If yes, are antibiotics included in some of those drugs? Have you taken other treatments before coming to the health facility?