Identification of Mycoplasma genitalium from clinical swabs by direct PCR [version 1; peer review: 1 approved, 1 approved with reservations]

Mycoplasma genitalium is one of the smallest self-replicating organisms. It is an obligate parasite found in the human genital tract. In men, the bacteria cause both acute and chronic non-gonococcal urethritis (NGU). In women, it has been associated with pelvic inflammatory disease and cervicitis among other related infections. Treatment of M. genitalium related infections has been effective using antibiotics such as the macrolides (e.g. azithromycin) and fluoroquinolones. However, there have been recorded cases of resistance to these antibiotics in various parts of the world as a result of a mutation in the 23SrRNA gene, although the antibiotic resistance has not been well established. The aim of this study was to detect M. genitalium in 352 swab samples collected from a clinic for sex workers in Nairobi, Kenya. DNA was extracted from the swabs and stored as a crude extract at -31°C. The swab lysates were subjected to direct polymerase chain reaction using primers that specifically target the 16S rRNA gene for M. genitalium. A total of 29 samples tested positive for M. genitalium. The data results showed a M. genitalium prevalence of 8.24% among sex workers in Nairobi, Kenya.


Introduction
Mycoplasma genitalium is an emerging sexually transmitted disease that was first identified and isolated in 1980 1 from men with non-gonococcal urethritis (NGU). Its epidemiology in connection to other STI syndromes been established since nucleic acid amplification assay development in the early 1990s 2 . The bacteria have been detected in substantial amounts from men with urethritis and women with cervicitis 3 . M. genitalium prevalence in the general population has been studied and found to be ranging between 1-3% 4 .
M. genitalium is found in roughly 15% of men with NGU and in 22% of men with non-chlamydial NGU. However, the associated infections do not have unique clinical symptoms, making it difficult to use clinical signs as a mode of identification 5 . Cervicitis has been described as the female version of male urethritis. M. genitalium is found in 10% of women with cervicitis 6 . Chlamydial coinfections in women with cervicitis are also common in some settings 7 . M. genitalium is a very fastidious bacterium and culturing of the bacterium is exhaustive and time consuming.
The introduction of polymerase chain reaction (PCR) assays has provided the necessary data for its clinical prevalence 8 . Many assays have been developed for the detection of M. genitalium in human specimens [8][9][10][11][12][13][14][15][16][17] . Most of these assays are mainly based on the PCR detection technique. Use of these PCR tests have shown that the disease spectrum is similar to those caused by Chlamydia trachomatis and Neisseria gonorrhoeae in both males and females 13 . However, these assays differ in their target DNA sequences, specimen preparation and amplicon detection methods. Many of these detection methods target the 16S rRNA and the MgPa protein genes. Conventional and, more recently, real-time PCR assays have been applied. Most of the detection studies have been conducted in the U.S.A, Europe and Australia, with various strains being discovered. In line with the detection of the bacterial species and its related infections in Africa, more studies need to be conducted on possible strains and their epidemiology. Whether the bacteria have links with other sexually transmitted infections can also be investigated. In this study, it is shown that direct PCR can be applied to the detection of M. genitalium from crude DNA extracts. M. genitalium prevalence and characteristics among female sex workers have been studied in Kenya and Uganda 18-21 . Its prevalence has also been studied among males who underwent circumcision in order to prevent HIV acquisition in Kisumu, Kenya 22 . However, most of these studies have focused on conventional, real-time PCR or transcription-mediated amplification assays for the detection of M. genitalium. This study reports the use of direct PCR for M. genitalium detection from crude DNA extracts using specific primers that target the 16S rRNA gene.

Ethical statement
This study was approved by the Jomo Kenyatta University of Agriculture and Technology Institutional Ethics Review Committee (JKUAT-IERC): reference number JKU/2/4/896B. The swab samples were collected with written informed consent for the performance of further analysis.

Source of samples
The samples used in this study were collected as part of the sex workers outreach program (SWOP) central business district clinic in Nairobi, Kenya. As part of this program, patients who showed STI symptoms and consented to the study were sampled by taking vaginal swabs. The specimens were then put into sterile containers and transported to the Pan Africa Hub Laboratory (NUITM-KEMRI) within an hour and stored at -80°C. Anonymized samples were retrieved for use in this study.

Sample preparation
The 352 swab lysates were prepared using the MightyPrep reagent for DNA (TAKARA BIO INC, Kusatsu, Shiga Prefecture, Japan; Cat No: 9182) using the manufacturer's protocol with a slight modification. Swabs were cut and put into 1.5ml Eppendorf tubes. A total of 200uL of the MightyPrep reagent was added to the tubes and later centrifuged at 15krpm for one minute. The tubes were then transferred to a heated block at 95°C with shaking at 800rpm for 15 minutes. Later, the tubes were cooled down by lowering the heat block temperature to 25°C, followed by hard vortexing of each tube for one minute and, finally, centrifugation at 15krpm for two minutes before storage at -31°C.
After vortexing the master mix for five seconds, 18μl was aliquoted to each of the labeled 96 PCR tubes. 2μl of the swab lysates was added to each tube to make a final reaction volume of 20μl and the tubes were finger tapped for five seconds to mix the contents. A positive control (M. genitalium positive sample) and negative control (PCR water) were used. The PCR tubes were placed in the SimpliAmp ™ Thermal Cycler (Applied Biosystems) and run under the following reaction conditions.
An initial antibody inactivation step was carried at 95°C for 15 minutes, followed by 35 cycles of: denaturation at 94°C for 60 seconds, annealing at 67°C for 60 seconds and extension at 72°C for 60 seconds. A final extension step was carried out at 72°C for 10 minutes, followed by the final hold at 4°C for ∞.

Amplification of the PCR products
The PCR products were subjected to another PCR. Master mix components were as described above. 18μl was aliquoted into the PCR tubes. 1μl of the sample products was added to the tubes to make a 19 μl final volume. The PCR tubes were placed in the SimpliAmp™ Thermal Cycler (Applied Biosystems) and run under the following reaction conditions.
An initial antibody inactivation step was carried out at 95°C for 15 minutes, followed by 30 cycles of: denaturation at 94°C for 60 seconds, annealing at 69°C for 60 seconds and extension at 72°C for 60 seconds. A final extension step was carried out at 72°C for 10 minutes, followed by the final hold at 4°C for ∞.
The products were run on a 2.0% agarose gel at 100V for 40 minutes. A 3000bp ladder (Solis BioDyne, Tartu, Estonia) was used. The gels were stained using 2x GelRed for one hour and viewed under an UltraSlim UV Transilluminator.

Results
A total of 352 lysates were analyzed in this study. The results show evidence for the presence of M. genitalium from swabs taken from the female sex workers who were sampled. 352 lysates were prepared using the MightyPrep reagent.  . Clear Mycoplasma genitalium positive bands can be seen at positions 1, 2, 9, 10 and 17 (Sexually transmitted infection lysates S099, S100, S108, S109 and S116, respectively). The positive control (PC) and negative control (NC) are at positions 24 and 25, respectively.  Clear Mycoplasma genitalium positive bands can be seen at positions 9, 10, 11 and 18 (lysates S137, S138, S139 and S146, respectively). The positive control (PC) is shown at position 24 and the negative control (NC) at position 25.

M. genitalium detection
The PCR products were subjected to another amplification reaction. After the reaction, the products were run on a 13-well agarose gel. A 3000bp ladder was loaded on positions 1 and 13, with the positive and negative controls at positions 11 and 12, respectively, as can be seen in Figure 3 and Figure 4. The study was able to detect 29 M. genitalium positive samples out of the 352 lysates. However, the challenge experienced with this method was non-specific amplification, realized from the multiple fragments produced. A possible solution to this is in the use of more precise target-specific primers to prevent the amplification of genes with closely related sequences. Application of this method can be a remedy to the constant loss of DNA due to long extraction processes, at the same time maintaining its quality for further downstream analysis.
M. genitalium prevalence was shown to be at 8.24%. This shows that one out of every eight patients sampled was positive for M. genitalium related infections. Balkus and colleagues in 2018 were able to detect M. genitalium from 25 out of 221 (11.3%) women from Kenya and the US 25 . Prevalence rates of 12.9% 18 and 16% 19 have also been reported among sex workers in Nairobi, Kenya. The prevalence obtained in this study therefore does not show any significant drop in M. genitalium infections. Despite better and improved access to healthcare, M. genitalium infections seem to continue to be a burden. Possible reasons might be due to having multiple sex partners 26 or antibiotic resistance to drugs of choice such as macrolides and fluoroquinolones 27,28 Overall, the prevalence results suggest that more measures need to be taken to control M. genitalium infections. Awareness campaigns need to be carried out to sensitize people on preventive measures rather than taking potential risks that may lead to exposure to the infection. Studies need to be done to investigate M. genitalium drug resistance. This will be helpful in informing policy and practice. As a result, screening can be done in patients to check for resistance before prescribing medication.

Open Peer Review Introduction
The phrase "Most of the detection studies have been conducted in the U.S.A, Europe and Australia, with various strains being discovered" requires citations from literature to exemplify what is stated.
The phrase "In this study, it is shown that direct PCR can be applied to the detection of M. genitalium from crude DNA extracts." should not be placed in this section. It must be moved to the Discussions section.

Methods
In Sample preparation and Direct PCR sections, the authors declare that they used the manufacturer's protocol "with slight modifications". What are these changes and for what purpose were they made? A positive control (M. genitalium positive sample) was used. Where does it come from? Why two PCR amplifications were made? The authors should explain why they have amplified PCR products.  Figure 1 "The ladder is at positions 1 and 26" and "Clear Mycoplasma genitalium positive bands can be seen at positions 1, 2, 9, 10 and 17". How do you explain that in Figure 4 the band corresponding to sample S163 is at a different level than the others?

Discussion
There is too little discussion about the technique used.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly