Prevalence of genital mycoplasmas and co-occurrence with Gardnerella vaginalis in high vaginal swabs from adult females in Bahrain: point-prevalence observational study

Shaikha Khaled AL-Enezi (College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain)
Nermin Kamal Saeed (Department of Pathology, Salmaniya Medical Complex, Manama, Bahrain)
Naeema A.A. Mahmood (Department of Obstetrics and Gynaecology, Salmaniya Medical Complex, Manama, Bahrain)
Mohd Shadab (Department of Microbiology, Immunology and Infectious Diseases, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain)
Ali Al Mahmeed (Department of Microbiology, Immunology and Infectious Diseases, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain)
Mohammad Shahid (Department of Microbiology, Immunology and Infectious Diseases, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain)

Arab Gulf Journal of Scientific Research

ISSN: 1985-9899

Article publication date: 6 December 2022

96

Abstract

Purpose

Bacterial vaginosis (BV) is quite common and linked with serious public health issues such as premature delivery and spread of sexually transmitted infections. The study aims to identify different genital mycoplasmas (GM) in high vaginal swabs (HVS) from adult females in Bahrain.

Design/methodology/approach

In total, 401 HVS were collected and cultured on MYCOFAST® RevolutioN 2 test for identification and antibiotic susceptibility. Polymerase chain reaction (PCR) was performed for detection of Mycoplasma genitalium (Mg), Mycoplasma hominis (Mh) and Ureaplasma species. DNA-probe based detection for Gardnerella, Candida and Trichomonas was performed by BD Affirm Assay. Representative PCR amplicons were sequenced by Sanger sequencing.

Findings

In PCR, Ureaplasma sp. was the most common GM, followed by Mg and Mh; the prevalence being 21.2, 5.2 and 1.5%, respectively. On the contrary, 10.7% samples showed positivity for Ureaplasma urealyticum (Uu) and 1.7% for Mh in MYCOFAST® RevolutioN 2. The concordance rates between MYCOFAST® RevolutioN 2 and PCR for Mh and Ureaplasma sp. were 97.7 and 84%, respectively. Considering PCR as gold standard, sensitivity, specificity, positive predictive value, and negative predictive value of MYCOFAST® RevolutioN 2 were 33.3, 98.8, 28.6, 98.9 and 37.7, 96.5, 74.4, 85.2% for Mh and Ureaplasma sp., respectively. The Uu and Mh isolates showed antibiotic-resistance ranging from 53%–58% and 71%–86%, respectively.

Research limitations/implications

The prevalence of Ureaplasma sp. was high. Significant co-occurrence of GM was noticed with BV. MYCOFAST® RevolutioN 2 had lower detection-rate than PCR, so a combination is suggested for wider diagnostic coverage.

Practical implications

The research reflects on status of prevalence of GM in adult females in Bahrain, and their co-occurrence with bacterial vaginosis. Diagnostic approach with combination of tests is suggested for wider coverage. The research has epidemiologic, diagnostic, and therapeutic implications.

Originality/value

This is the first report from the Kingdom of Bahrain reflecting on burden of GM from this geographic location. The diagnostic efficacy of MYCOFAST® RevolutionN 2 test and polymerase chain reaction was evaluated for GM detection.

Keywords

Citation

AL-Enezi, S.K., Saeed, N.K., Mahmood, N.A.A., Shadab, M., Al Mahmeed, A. and Shahid, M. (2022), "Prevalence of genital mycoplasmas and co-occurrence with Gardnerella vaginalis in high vaginal swabs from adult females in Bahrain: point-prevalence observational study", Arab Gulf Journal of Scientific Research, Vol. ahead-of-print No. ahead-of-print. https://doi.org/10.1108/AGJSR-07-2022-0108

Publisher

:

Emerald Publishing Limited

Copyright © 2022, Shaikha Khaled AL-Enezi, Nermin Kamal Saeed, Naeema A.A. Mahmood, Mohd. Shadab, Ali Al Mahmeed and Mohammad Shahid

License

Published in Arab Gulf Journal of Scientific Research. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode


Introduction

The vaginal canal is a home to diverse microbial community that coexists with the host in a symbiotic relationship, known as the vaginal microbiota (VMB) (Andrade Pessoa Morales et al., 2021). VMB is a complex microecosystem that changes with age and menstrual cycle (Andrade Pessoa Morales et al., 2021). Lactobacillus, as the predominant vaginal flora, plays an important role in innate defence by maintaining vaginal pH below 4.5 (Auriemma et al., 2021).

A shift in vaginal flora with diversity of bacterial species, known as bacterial vaginosis (BV), raise the vaginal pH above 4.5 making it vulnerable to infections (Vasundhara, Raju, Hemalatha, Nagpal, & Kumar, 2021). BV may increase predisposition to other sexually transmitted diseases, and may affect 23%–29% women across regions (Foessleitner et al., 2021).

Mycoplasma spp. and Ureaplasma spp. are well-known pathogens affecting human respiratory and urogenital systems, and are responsible for a wide spectrum of inflammatory disorders in neonates, children and adults (Taylor-Robinson & Furr, 1993). Mycoplasmas are eubacteria classed with Mollicutes, and lack cell wall (Taylor-Robinson & Furr, 1993; Yiwen, Yueyue, Lianmei, Cuiming, & Xiaoxing, 2021). M. genitalium, M. hominis, Ureaplasma urealyticum and U. parvum are responsible for urogenital problems in both men and women and are considered as important pathogens for non-gonococcal urethritis (Yiwen et al., 2021). These four species are collectively called as genital mycoplasmas (GM) (Yiwen et al., 2021).

GM cause sexually transmitted infections, which are associated with critical disorders in fertile women, including cervicitis, vaginitis, and if not treated, it can cause infertility and pelvic inflammatory disease (PID) (Kelly, Garland, & Gilbert, 1987).

Ureaplasma urealyticum is a major causative agent for non-chlamydial and non-gonococcal urethritis, cervicitis, chorioamnionitis, preterm delivery and vaginitis (Zheng et al., 2020). Also, in pregnant women, mycoplasma infection can lead to abortion or preterm labour (Zheng et al., 2020).

For diagnosing GM infections, culture method is insufficient (Zhou, Ma, Yang, & Gu, 2018), and serological methods only allow for a retroactive diagnosis (Zhou et al., 2018). Rapid methods have recently become available, including nucleic acid amplification tests (NAAT) such as polymerase chain reaction (PCR) (Coleman & Gaydos, 2018; Adebamowo et al., 2017). Routine screening for reproductive tract infections (RTIs) has been implemented in countries like the USA, Denmark, UK and Sweden (Ma et al., 2021), whereas, in developing countries, routine screening is not performed.

To date, the prevalence of GM, and their co-occurrence with BV, is not clearly reported from the Kingdom of Bahrain, which prompted us to perform this study. Also, we aimed at analysing the utility of MYCOFAST® RevolutioN 2 test, and PCR, in accurately diagnosing these GM.

Materials and methods

Place of study and sample collection

The study was performed in the department of Microbiology, Immunology and Infectious Diseases (DMIID), College of Medicine and Medical Sciences of the Arabian Gulf University, Kingdom of Bahrain. High vaginal swabs (HVS) were collected in Gynaecology Clinic of Salmaniya Medical Complex, Bahrain, from patients presenting with non-specific vaginal discharge. The samples were sent to Microbiology Department at Salmaniya Medical Complex (MDSMC) for testing on BD Affirm assay for Gardnerella, Candida and Trichomonas infections. Further, these HVS samples were sent to DMIID for Mycofast® RevolutioN 2 Test, and molecular testing, for GM {Mycoplasma genitalium (Mg), Mycoplasma hominis (Mh), Ureaplasma sp.(Usp)}.

Through March to August 2021, 401 random HVS samples were collected in AMIES transport media (Deltalab, Spain). The results of the BD Affirm molecular probe assay for Gardnerella, Candida and Trichomonas (only for the positive swabs for GM) were obtained from MDSMC for comparative analyses.

Processing of samples

A new sterile swab was inserted in the transport medium, and 500 μl of mycoplasma preservation and transport solution was added to the transport medium. The tubes were incubated at 37 ˚ C with 5% CO2 for 24 h.

MYCOFAST® RevolutioN 2 test

MYCOFAST® RevolutioN 2 (ELITech – France) test was used for identification and antimicrobial susceptibility of Ureaplasma urealyticum and M. hominis (Redelinghuys, Ehlers, Dreyer, Lombaard, & Kock, 2013). Then, 500 μl of the transport medium was pipetted into 3 ml UMMt-medium vial provided with MYCOFAST® RevolutioN 2 test. After mixing, 100 μl of UMMt solution each was pipetted in 24 wells of the MYCOFAST® tray, followed by adding two drops of mineral oil. Respective trays were sealed with plastic seal and incubated at 37°C for 24 h. Samples with no indication of growth after 24 h were re-incubated for further 24 h.

Growth was inferred by the change of colour of the medium from yellow to red or orange due to the metabolism of urea and arginine, respectively, by Ureaplasma urealyticum and Mycoplasma hominis. The trays had following two divisions:

Black-labelled side (for Ureaplasma urealyticum)

Wells 1 to 3 were for identification of Ureaplasma urealyticum in different concentrations: 10³, 104, 105 CCU/ml. Wells 4 to 13 were for testing antimicrobial susceptibility of Uu. Following antibiotics were used: Levofloxacin (concentrations at 2 and 4 µg/ml), Moxifloxacin (2 and 4 µg/ml), Erythromycin (8 and 16 µg/ml), Tetracycline (1 and 2 µg/ml), Doxycycline (1 and 2 µg/ml).

Red-labelled side (for Mycoplasma hominis)

Well 14th was used for identifying Mycoplasma hominis in a concentration of 104 CCU/ml. Wells 15–24 were for antimicrobial susceptibility testing of Mh. Following antibiotics were used: Levofloxacin (1 and 2 µg/ml), Moxifloxacin (0.25 and 0.5 µg/ml), Clindamycin (0.25 and 0.5 µg/ml), Tetracycline (4 and 8 µg/ml), Doxycycline (4 and 8 µg/ml).

Molecular detection by conventional polymerase chain reaction

PCR was done to detect M. genitalium, M. hominis and Ureaplasma sp. in all samples. Swabs in the transport media were used for molecular detection. DNA was extracted from swabs by using QIAamp DNA mini kit (Qiagen, Crawley, UK). DNA controls procured from vircell MICROBIOLOGISTS were used for each GM species. A 1:10 dilution was prepared from each lyophilised DNA control. The PCR products were analysed by gel electrophoresis on 1.5% agarose.

Polymerase chain reaction cycling and primers

The primers used in this study targeted the urease gene in Ureaplasma, and S16 rRNA in Mycoplasma. Following primers were used: Mycoplasma genitaliumF: 5′-TAC ATGCAA GTC GATCGG AAG TAG C-3′ and R: 5′-AAA CTC CAG CCA TTG CCT GCT AG-3′ (Stellrecht, Woron, Mishrik, & Venezia, 2004); Mycoplasma hominisF: 5′-CAATGGCTAATGCCGGATACGC-3′ and R: 5′-GGTACCGTCAGTCTGCAAT-3′ (Waites, Katz, & Schelonka, 2005; Yoshida, Maeda, Deguchi, Miyazawa, & Ishiko, 2003); Ureaplasma sp. (urealyticum/parvum) – F: 5′-ACGACGTCCATAAGCAACT-3′ and R: 5′-CAATCTGCTCGTGAAGTATTAC-3′ (Timenetsky, Santos, Buzinhani, & Mettifogo, 2006). The expected amplicon sizes were 427, 344 and 429 bp, respectively. Amplification was performed using Applied Biosystems PCR system 9700 thermocycler. PCR reactions for M. genitalium, M. hominis and Ureaplasma sp. were run ranging between 30 and 42 cycles for different species as described by Stellrecht et al., Waites et al., Yoshida et al. and Timenetsky et al., respectively.

DNA sequencing

Representative amplicons of M. genitalium, M. hominis and Ureaplasma sp were commercially sent to MedGenome Labs Pvt Ltd, Bangalore, India, for sanger sequencing.

Statistical analysis

Concordance rate between PCR and MYCOFAST was calculated using formula:

Concordancerate=a+d/a+b+c+d
where, a = samples positive by both PCR and MYCOFAST; b = PCR +ve and MYCOFAST –ve; c = PCR –ve and MYCOFAST +ve; d = both PCR and MYCOFAST negative.

The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated on MedCalc Version 20.027 statistical software package for biomedical research.

Results

Age distribution of the positive cases for genital mycoplasmas

The age of the positive cases ranged from 21–64 years. Most of the cases were between the age of 22 and 39 years; there was a predominance around the age of 34 years.

Prevalence of genital mycoplasmas based on polymerase chain reaction

Out of 401 samples, 102 (25.4%) showed positivity for GM based on molecular testing. From these 102 samples, a total of 112 GM were detected (some samples had positivity for more than one type of GM). Ureaplasma sp. was the most common GM, followed by Mg and Mh. Based on PCR, the prevalence of these species were 21.2% (n = 85), 5.2% (n = 21) and 1.5% (n = 6), respectively (Figure 1a). A total of ten samples were positive for more than one GM species; seven samples showed positive amplicon for M. genitalium + Ureaplasma. sp and three samples were positive for M. hominis + Ureaplasma. sp.

The confirmation of the PCR results was done by repeating the PCR of positive samples twice and using a positive DNA control commercially provided for each species to confirm the targeted amplicons. The representative PCR gels are shown in Figure 2(a)–(c).

MYCOFAST® RevolutioN 2 test

Detection of U. urealyticum and M. hominis

Of the 401 HVS tested, 43 (10.7%) were positive for Ureaplasma urealyticum and seven (1.7%) for M. hominis. The samples positive for M. hominis were concomitantly positive for Ureaplasma sp. The limitation of this test is that it only detects Ureaplasma urealyticum, and M. hominis and does not detect Mycoplasma genitalium and Ureaplasma parvum.

Growth enumeration of MYCOFAST® RevolutioN 2 tests for Ureaplasma urealyticum

The tests showed 38 (88.3%) samples giving growth in 105 CCU/ml enumeration well, whereas only five (11.6%) samples showed growth in 10³/104 CCU/ml enumeration wells.

Antibiotics susceptibility profile

The Ureaplasma urealyticum isolates showed resistance to the tested antibiotics ranging from 53%–58%. The M. hominis isolates were resistant to the tested antibiotics ranging from 71%–86%. The detailed antibiotics susceptibility profiles for Ureaplasma urealyticum and M. hominis are shown in Table 1.

Comparison between polymerase chain reaction and MYCOFAST and concordance rate

A comparative description of the detection of genital mycoplasmas by PCR alone, MYCOFAST alone and combined detection by PCR + MYCOFAST is shown in Figure 1(b). In this study, we got a total of seven positive MYCOFAST® RevolutioN 2 for M. hominis, and two of them were consistent with the PCR results. For U. urealyticum, we got a total of 43 positive samples positive in MYCOFAST® RevolutioN 2 test, and 32 of these were compatible with the PCR results (Figure 1b). The concordance rates between MYCOFAST® RevolutioN 2 test and PCR for M. hominis and Ureaplasma sp. were 97.7 and 84%, respectively.

Sensitivity, specificity, positive predictive value and negative predictive value of MYCOFAST® RevolutioN 2 test

Considering PCR as gold standard, the sensitivity, specificity, PPV and NPV of MYCOFAST® RevolutioN 2 test were 33.3, 98.8, 28.6, 98.9 and 37.7%, 96.5, 74.4, 85.2% for M. hominis and Ureaplasma sp., respectively. The accuracy of the MYCOFAST® RevolutioN 2 test in detecting M. hominis and Ureaplasma sp. was 95.8%–99% (95% CI) and 80%–87% (95% CI), respectively.

Results of high vaginal swabs by BD Affirm molecular assay for vaginosis/vaginitis agents

Out of 102 samples that were positive for GM in PCR, 34 were positive for Gardnerella, 22 were positive for Candida and ten cases showed concomitant infections with Candida and Gardnerella. None of these samples had positivity for Trichomonas. Results are described in Table 2.

Co-occurrence/association of genital mycoplasmas with main etiological agent of vaginosis (Gardnerella vaginalis) and fungal vaginitis (Candida)

On analysing the PCR results with BD Affirm assay, a significant co-occurrence of GM was noticed with Gardnerella vaginalis. As shown in Table 2, 23 (27%; 23/85) Ureaplasma sp., three (50%; 3/6) M. hominis and eight (38%; 8/21) M. genitalium were also positive for Gardnerella vaginalis. Whereas, co-occurrence of Candida was shown in 19 (22.4%) Ureaplasma sp., one (17%) M. hominis and one (5%) M. genitalium positive samples (Table 2).

DNA sequencing

Of the representative amplicons that were sent for commercial sequencing, some failed the QC check, whereas others passed. Blast search analysis of the generated sequences of Ureaplasma sp. confirmed them as Ureaplasma parvum/Ureaplasma urealyticum (majority being Ureaplasma parvum), and the analysis of M. genitalium sequencing results confirmed them as M. genitalium. Unfortunately, the sequencing for M. hominis amplicons failed due to some technical reasons, and we could not send samples again for re-sequencing due to financial constraints. Phylogenetic analyses of the sequences of Ureaplasma sp. and M. genitalium demonstrated genetic variations (Figure 3a and b).

The representative sequences were submitted and published in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with Accession Number ON755316, ON755317, ON755319 for Ureaplasma parvum and ON755318 for Ureaplasma urealyticum.

Discussion

GM are often isolated from female genital tracts. Although their presence (e.g. M. hominis) as a normal flora makes the determination of their pathogenic roles more difficult, several studies have found that vaginal colonisation with these organisms is linked to an increased risk of developing certain pathogenic conditions and linked to pregnancy abnormalities (Sarier et al., 2018). With a growing body of research showing that GM can be clinically significant in some conditions, a reliable and sensitive laboratory approach for their detection is needed (Clegg, Passey, Yoannes, & Michael, 1997). Moreover, the burden of this clinical entity (infection with GM) is not reflected in published literature from our geographic region.

So, from this standpoint, we decided to screen for the prevalence of genital mycoplasmas in adult females in Kingdom of Bahrain who visited hospital with complains of vaginal discharge, firstly, to have a preliminary insight into this health burden of GM occurrence in this geographic region, and secondly, in comparing between a commercial test kit (MYCOFAST) and PCR for detection of these organisms. Moreover, we intended to analyse the co-occurrence of mycoplasmas with Gardnerella vaginalis and Candida, the organisms primarily responsible for BV and fungal vaginitis, respectively.

In our study, a total of 102 (25.4%) samples were positive either for M. hominis, M. genitalium or Ureaplasma sp. by PCR. For all practical purposes, considering PCR as the gold standard, we found the prevalence of M. hominis, M. genitalium and Ureaplasma sp. as 1.5, 5.2 and 21.2%, respectively. We could not find any published study from Bahrain for comparing our results; however, there were fragmentary studies from the nearby Middle Eastern regions. For instance, a study from Makkah reported the prevalence of 14.1% for M. genitalium, and 9.6% for U. urealyticum/parvum (Ashshi et al., 2015). A study from Lebanon reported a prevalence of 35% for Ureaplasma parvum, 4.6% for Ureaplasma urealyticum, 3.6% for Mycoplasma hominis and 0.2% for Mycoplasma genitalium (Hanna et al., 2020). Another study from Iran reported a prevalence of 23.2% for M. genitalium (Mohseni Moghadam, Kheirkhah, Mirshekari, Fasihi Harandi, & Tafsiri, 2014). The results of our study, and the published results from the nearby regions (of the Middle East), reflect a geographical variation in the prevalence of GM, which is obvious.

In MYCOFAST® RevolutioN 2 test, which is a commercial kit based on culture and colorimetric test, of all 401 samples, only 43 (10.7%) samples gave positive results. In comparison to PCR, we got comparatively lower detection rate with this test. Researchers have tried comparing between conventional culture and PCR methods for sensitive and precise identification of GM (de Barbeyrac et al., 1993), and inferred that despite culture being considered gold standard, PCR enabled speedy and accurate identification of GM (Frolund, Bjornelius, Lidbrink, Ahrens, & Jensen, 2014; Sarier et al., 2017). Moreover, culture requires specialised laboratories and has human subjectivity. PCR is therefore extensively employed in laboratories with better experience as opposed to culture (Kusanovic et al., 2020).

It is to be emphasised here that the PCR we used in our study could detect the three types of GM, namely, M. hominis, M. genitalium and Ureaplasma sp. This was in contrast with MYCOFAST® RevolutioN 2, which could not detect M. genitalium. Redelinghuys et al. reported 77 and 80% sensitivity and specificity of MYCOFAST® RevolutioN 2, respectively. They assumed that the MYCOFAST® Revolution 2 assay could be a cost-effective alternative to traditional culture methods, and laboratory employees may be able to offer clinicians with the results in shorter period (Redelinghuys et al., 2013). However, in our study, the sensitivity, specificity, PPV and NPV of MYCOFAST® RevolutioN 2 test were 33.3, 98.8, 28.6, 98.9 and 37.7%, 96.5, 74.4, 85.2% for M. hominis and Ureaplasma sp., respectively. On the other hand, the accuracy of the MYCOFAST® RevolutioN 2 test in detecting M. hominis and Ureaplasma sp. was 95.8%–99% (95% CI) and 80%–87% (95% CI).

Based on MYCOFAST® RevolutioN 2 test, the Ureaplasma sp. isolates showed resistance to the tested antibiotics ranging from 53%–58%, and M. hominis were resistant, ranging from 57%–86%. This was comparable with the global reports on antibiotics susceptibility of GM. The published studies have reported that the antimicrobial resistance of mycoplasmas, especially against Macrolides, Fluoroquinolones and Tetracyclines antibiotics (Bebear et al., 2000; Lendamba, Mbeang Nguema, Onanga, & Mombo, 2022), has increased in previous years. Therefore, minocycline and josamycin are recommended as a new primary choice in anti-mycoplasmas treatment (Lee & Yang, 2020).

In our study, we also compared our PCR results of the positive cases for GM with other vaginitis and vaginosis-related microorganisms like G. vaginalis, Candida and Trichomonas, which were screened for by BD Affirm probe assay. There was a significant correlation between GM and G. vaginalis, i.e. approximately 27% of Ureaplasma sp., 50% of M. hominis and 38% of M. genitalium harbouring cases were also positive for Gardnerella vaginalis. Similar results have been reported in recent past where a strong association between BV and enhanced susceptibility to M. genitalium was suggested (Lokken et al., 2017). In a recent study, Nye, Harris, Pherson and Cartwright (2020) also suggested a strong association between M. genitalium infection and BV in symptomatic females with vaginitis.

For Candida, 22.4% of Ureaplasma sp., 17% of M. hominis and 5% of M. genitalium harbouring cases were also positive for Candida infection. Interestingly, we noticed that the GM were more associated with G. vaginalis than Candida.

The presence of Ureaplasma in the lower genitourinary tract samples equalling 105 CCU/ml is usually considered as the load indicating an infection that should be treated with antibiotics. Ureaplasma colonisation is thought to occur in low quantities in all lower genitourinary tract specimens (urine, vaginal swabs) and is considered as a normal flora (Kokkayil & Dhawan, 2015). Determining load of Ureaplasmas in upper genitourinary tract is not essential as it is always regarded as an infection (Kasprzykowska, Elias, Elias, Maczynska, & Sobieszczanska, 2014). In our study, we got about 38 (88%) Ureaplasma that showed growth of 105 CCU/ml, and only five (12%) samples gave growth only of 10³/104 CCU/ml, which indicates that most of our Ureaplasma species found were true infections.

In nutshell, we found a cumulative prevalence of 25.4% for GM in our study population; the prevalence of Ureaplasma sp. was significantly high as opposed to other GM. Sequencing and BLAST analyses demonstrated predominance of U. parvum in our study. The genetic variation in the derived sequences of Ureaplasma and M. genitalium is worrisome. MYCOFAST® RevolutioN 2 test showed lower sensitivity in detecting Ureaplasma sp. and Mycoplasma hominis, than PCR. Since MYCOFAST® RevolutioN 2 test has limitations in detecting M. genitalium, which can be detected by PCR, and that it has lower sensitivity than PCR, we suggest performing both MYCOFAST® RevolutioN 2 and PCR tests for accurate diagnosis. Moreover, the usage of MYCOFAST® RevolutioN 2 along with PCR is important because MYCOFAST® RevolutioN 2 test will reflect on antibiotics susceptibility of the isolate, which has an added therapeutic advantage. We found a significant co-occurrence of mycoplasmas with BV in our study population. To the best of our knowledge, this is the first report from the Kingdom of Bahrain reflecting on burden of GM from this geographic location.

Acknowledgment

The authors thank Mr Tomy Kaitharath for the technical support provided during the study. The authors are grateful to Dr Parvez A. Khan for helping in statistical analyses. The authors are also grateful to the university REC and Ministry of Health, Kingdom of Bahrain, for approving this study.

Ethical approval and consent to participate: All methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Research and Ethics Committee, CMMS, Arabian Gulf University (E039-PI-4/19) and by the Ministry of Health, Kingdom of Bahrain (AURS/ 403/2020). Written informed consent was obtained from all subjects and/or their legal guardian(s).

Consent for publication: Not applicable.

Availability of data and materials: All data generated or analysed during this study are included in this published article.

Competing interests: The authors declare that they have no competing interests

Funding: Internal grant was provided by the Kuwait Cultural Office in Bahrain (through Arabian Gulf University, Bahrain; Student Project No. E039-PI-4/19) for Shaikhah Al-Enezi towards the study of Master in Laboratory Medicine Programme at the Arabian Gulf University.

Authors’ contributions: MS, NKS, NAAM: conceptualisation; MS, SKA, NKS, NAAM: data analysis; SKA, MohdS (Mohd. Shadab), AAM: methods; SKA, MS: original writing; MS, SKA, NKS, NAAM, MohdS, AAM: review and editing. All authors have reviewed and approved the manuscript.

Figures

(a) Positivity for different types of GM by PCR test. (b) Comparison between the PCR and MYCOFAST® RevolutioN 2 test in detection of GM. *PCR primers were targeting against Ureaplasma sp. (U. urealyticum + U. parvum), whereas MYCOFAST could detect only U. urealyticum

Figure 1

(a) Positivity for different types of GM by PCR test. (b) Comparison between the PCR and MYCOFAST® RevolutioN 2 test in detection of GM. *PCR primers were targeting against Ureaplasma sp. (U. urealyticum + U. parvum), whereas MYCOFAST could detect only U. urealyticum

(a) shows the representative gel having positive amplicons for M. hominis (Lanes1, 2 and 3), (b) representative amplicons of M. genitalium (Lanes1, 2 and 3) and (c) representative amplicons of Ureaplasma sp. (Lanes1, 2 and 3). Lane M is 100 bp ladder

Figure 2

(a) shows the representative gel having positive amplicons for M. hominis (Lanes1, 2 and 3), (b) representative amplicons of M. genitalium (Lanes1, 2 and 3) and (c) representative amplicons of Ureaplasma sp. (Lanes1, 2 and 3). Lane M is 100 bp ladder

(a) Dendrogram showing clonal relation/diversity in our representative sequences of Ureaplasma sp. analysed against U. parvum and U. urealyticum reference sequences (b) Dendrogram showing clonal relation/diversity in our representative Mycoplasma genitalium analysed against M. genitalium ref. sequence

Figure 3

(a) Dendrogram showing clonal relation/diversity in our representative sequences of Ureaplasma sp. analysed against U. parvum and U. urealyticum reference sequences (b) Dendrogram showing clonal relation/diversity in our representative Mycoplasma genitalium analysed against M. genitalium ref. sequence

Antibiotics resistance rates for Ureaplasma urealyticum (n = 43) and M. hominis (n = 7) tested in MYCOFAST® RevolutioN 2 test for respective antibiotics

Antibiotics tested in MYCOFAST® RevolutioN 2 panel% resistance (n) for Ureaplasma urealyticum% resistance (n) for M. hominis
Levofloxacin58% (25)86% (6)
Moxifloxacin56% (24)71% (5)
Erythromycin58% (25)NPP*
Tetracycline53% (23)71% (5)
Doxycycline56% (24)71% (5)
ClindamycinNPP*57% (4)

Note(s): *NPP = Not present in MYCOFAST® RevolutioN 2 panel

The results of BD Affirm molecular assay for Gardnerella, Candida and Trichomonas in 102 PCR positive samples for GM, and co-occurrence of GM with Gardnerella and Candida

BD Affirm molecular assay positivity forNo. of positive samples (%)
Candida22 (21.6%)
Gardnerella34 (33.3%)
Candida + Gardnerella10 (9.8%)
Trichomonas0 (0%)
Co-occurrence of Gardnerella with GM
Gardnerella + Ureaplasma sp.23 (27%)
Gardnerella + M. hominis3 (50%)
Gardnerella + M. genitalium8 (38%)
Co-occurrence of Candida with GM
Candida + Ureaplasma sp.19 (22.4%)
Candida + M. hominis1 (17%)
Candida + M. genitalium1 (5%)

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Corresponding author

Mohammad Shahid can be contacted at: Mohammeds@agu.edu.bh

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