Approximately 33 million people were estimated to be living with HIV in 2007, two-thirds of whom live in sub-Saharan Africa . Sexually transmitted infections (STIs) enhance the transmission of HIV, and STI control is recommended for HIV prevention [2,3].
The bacterium Mycoplasma genitalium was first isolated in 1980 from the urethra of two men with nongonococcal urethritis . Mycoplasma genitalium infects both men and women and is most frequently detected in the urogenital tract . The literature conclusively supports the sexual transmissibility of M. genitalium, based on concordance rates among sexual partners and DNA sequence typing of M. genitalium strains in sexual partners [6,7]. M. genitalium infection is common, particularly among HIV-infected individuals in sub-Saharan Africa, where the prevalence is 11–33% [8–13]. There are limited longitudinal data on the natural history of M. genitalium infection, but in one study, which included DNA sequencing, M. genitalium infection persisted for over 21 months of follow-up [14,15]. Similarly, there are little data on incidence of M. genitalium, but the same study, among Kenyan sex workers, found that incidence of M. genitalium (22.7 per 100 person-years) was higher than that for Chlamydia trachomatis or Neisseria gonorrhoeae. The World Health Organization is considering incorporating M. genitalium treatment into the STI syndromic management guidelines for male and female genital discharge algorithms but require further data on the epidemiology of M. genitalium [F. Ndowa, personal communication].
Aetiological diagnosis of M. genitalium is currently limited by the lack of a commercially available diagnostic test. As culture of M. genitalium is difficult due to the fastidious nature and slow growth of the bacteria, in-house nucleic acid amplification tests (NAAT) are the most commonly available diagnostic tools. Due to the low bacterial load in some patients , a very low limit of detection is required, and NAATs are highly sensitive as well as have the capacity to achieve more than 99% specificity [17,18]. Serological antibody tests have also been used for diagnosis and are capable of achieving a similar level of sensitivity as NAATs .
Like other STIs, M. genitalium may play an important role in HIV acquisition and transmission. This theory first emerged when, in 1990, M. genitalium was identified in the blood of an AIDS patient . Since then, a number of studies have provided support for this theory. In one study, urethral inoculation of primates resulted in acute inflammation and shedding [21,22]. Various in-vitro studies [21–23] have shown that the inflammatory response and mucosal disruption (increased expression of cytokine genes, polymorphonuclear leucocyte response) associated with the introduction of M. genitalium may increase susceptibility or transmissibility or both of HIV infection. Mycoplasma genitalium can increase the HIV-associated cytopathic effects [23,24], enhancing HIV viral replication, thereby increasing transmissibility and accelerating disease progression [25,26]. An association has also been shown between HIV viral budding and mycoplasma attachment, suggesting that adherence of mycoplasmas to HIV-infected lymphocytes may trigger viral production or release .
The aim of this article is to systematically review the epidemiological literature on the association of M. genitalium and HIV infection, and to summarize the data with a meta-analysis. Exploring the relationship between these two infections furthers our understanding of modifiable cofactors of HIV. Findings from this analysis, together with emerging information on testing and treatment of M. genitalium, may provide a basis for continued research and policy towards M. genitalium prevention and control as a potential additional HIV prevention strategy.
The objective of our literature review was to identify all published epidemiological studies of HIV infection among adults that included data on M. genitalium. From a total of 70 available databases, we selected those most likely to contain relevant citations. A computerized search of the Medline, Embase, Biosis, Globalhealth, and CAB Abstracts databases was conducted on the Ovid platform for articles published prior to 1 June 2008. The search was restricted to studies in humans, with no restriction on language. Conference abstracts were excluded, as these are often based on preliminary data and may contain insufficient detail for inclusion.
The search strategy was refined after pilot searches to check whether preidentified relevant studies had been correctly identified. The final search strategy was as follows: [‘HIV infections’ or ‘acquired immunodeficiency syndrome’ or ‘AIDS-related opportunistic infections’ or ‘HIV seropositivity’ or (HIV or human immune*deficiency virus or acquired immune*deficiency virus or HIV-infection or HIV-seropositiv* or seroconversion or HIV-positive or HIV-negative or HIV-uninfected) found in title, original title, abstract, name of substance word, or subject heading word] and [(mycoplasma genitalium or (mycoplasma or mycoplasma infection)] found in title, original title, abstract, name of substance word, or subject heading word. The symbol ‘*’ is used for truncation (i.e. permitting any letter, symbol or space).
Further to this, we searched Africa HealthLine (1966 and earlier to December 2007) and AIDSearch (1980 to present) for [‘mycoplasma’ or ‘genitalium’] and [‘HIV’ or ‘AIDS’ or ‘human immunodeficiency virus’ or ‘acquired immune deficiency syndrome’]. To ensure that we were not biasing our study by limiting our search to words found only in the title, abstract, or key words, we performed an advanced search on Google Scholar with all words [‘mycoplasma’ ‘genitalium’ ‘HIV’] located anywhere in the article, from 1990 to 2008 in the following subject areas: Biology, Life Sciences, and Environmental Science; Medicine, Pharmacology, and Veterinary Science; Social Sciences, Arts, and Humanities. We also handsearched the 1993, 17 (vol) supplement 1 of the journal, Clinical Infectious Diseases, pertaining to Mycoplasmas. Finally, the references from all relevant studies were examined for additional relevant citations. Initially, the citations identified were evaluated on the basis of their titles, abstracts, and/or key words. Nonrelevant studies, such as those of mycoplasma infections other than M. genitalium, studies that did not test for both M. genitalium and HIV infection, studies in animals, and studies of nongenital mycoplasma infections such as pulmonary infection, were excluded. The full text of potentially relevant articles was read and data were extracted using a standardized form.
The primary measure of effect estimated was the odds ratio (OR). Random-effects meta-analysis was used to estimate the summary OR and 95% confidence interval (CI). It is plausible that the strength of association between HIV and M. genitalium varies due to the prevalence of HIV cofactors associated with M. genitalium, such as urethritis. The association may also vary according to geographical region, due to the background prevalence of HIV, and/or primary mode of HIV transmission. We thus conducted two a-priori defined sub-group analyses by type of control group (healthy controls without specific infection or condition vs. all other controls); and geographical region (sub-Saharan Africa studies vs. all other studies). As both M. genitalium and HIV infection are sexually transmitted, sexual behaviour is likely to be an important confounding factor, and any adjustment for potential confounding was noted and adjusted data were used in summary analysis wherever available.
Heterogeneity was assessed by calculating the weighted sum of the squared difference between the pooled estimate and the estimate from each individual study. Due to the small number of studies and inherent low power of these tests for heterogeneity, we also conducted a meta-inference analysis to detect which studies were contributing to any between-study heterogeneity. Analysis of publication bias was conducted using the Begg's rank correlation test  and Egger's regression asymmetry test . The random-effects model was implemented using the method of DerSimonian & Laird . Stata Intercooled statistical software (version 10; StataCorp LP, College Station, Texas, USA) was used for all analyses.
After exclusion of duplicate citations from the eight databases, a total of 897 potentially eligible references were identified (Fig. 1). The title and abstracts of each of these were reviewed for relevance. Thirty-five potentially relevant articles were retained for full text review. Reasons for exclusion from among these 35 articles are given in Fig. 1. One study  included stratified analysis among men and women, and the two groups were included as separate studies in the meta-analysis. There was insufficient information on M. genitalium and HIV in six articles, and the authors were contacted, leading to additional data for three of these, which were included in the meta-analysis [30–32]. In one study , there were just five HIV-positive participants, none of whom were M. genitalium-infected. This study was included in the meta-analysis by assigning 0.1 as the number of M. genitalium-positive people in the HIV-positive group in order to estimate an OR and 95% CI.
Eligible studies are summarized in Table 1, grouped by geographical region [8–11,13,19,30–40]. A total of 19 studies were included in our meta-analysis, with a total of 11 110 participants. Studies were from sub-Saharan Africa (n = 10), the United States (n = 6), Europe (n = 2), and South America (n = 1). All studies were cross-sectional or case–control, with the exception of one partner study  and one longitudinal study . Populations included general populations (n = 8), STI clinic attendees (n = 10), injection drug users (n = 5), men who have sex with men (n = 2), sex workers (n = 2), and couples (n = 1). Nine studies included more than one target population. The prevalence of M. genitalium ranged from 3.1% to 47.5%.
Association of Mycoplasma genitalium and HIV infection
The unadjusted OR and 95% CI for each study are shown in Table 1. Four studies [10,14,37] adjusted for confounding factors, including sociodemographic factors (age, marital status), sexual behaviour [herpes simplex virus type 2 (HSV2), other infections, partner HIV status, number of sexual partners], and circumcision status. Adjusted results for these studies are included in Table 1.
Seventeen of the 19 studies found that individuals with M. genitalium infection were more likely to be HIV-infected than M. genitalium-negative individuals, and the association was statistically significant (P < 0.05) in 12 of the studies. The ORs for these 17 studies ranged from 1.40 (95% CI = 1.13–1.72) to 5.96 (95% CI = 0.73–48.62). The three studies for which both crude and adjusted data were available reported a stronger association between M. gentialium and HIV infection in adjusted analysis.
Two studies found highly statistically significant reduced odds of HIV among M. genitalium-infected individuals (OR = 0.16, 95% CI = 0.08–0.30; OR = 0.12, 95% CI = 0.03–0.43) [32,34]. In both studies, the control groups consisted of male STD clinic attendees (restricted to those with clinical symptoms of urethritis in one study ), and the prevalence of M. genitalium was high (9.1% and 14.6%, respectively) compared to HIV-positive individuals in these studies (1.9% and 0%, respectively). STD clinic attendees are arguably a poor comparison group for the association of M. genitalium and HIV, as M. genitalium is associated with urethritis in men and may have a causal role in nongonoccocol urethritis .
Overall, individuals infected with M. genitalium were twice as likely to be HIV-seropositive (summary OR = 2.01, 95% CI = 1.44–2.79; Table 2, Fig. 2a). However, there was strong evidence of between-study heterogeneity (P < 0.001) and the summary OR should be interpreted with caution.
Exploration of heterogeneity and bias
To explore the possible reasons for the heterogeneity, we stratified our analysis by a-priori defined population sub-groups. Forest plots are presented for each of these population sub-groups (Fig. 2b, c). Using random-effects meta-analysis, the strongest association was among the 10 studies in sub-Saharan Africa (summary OR = 2.60, 95% CI = 2.17–3.11) among whom there was little evidence of between-study heterogeneity (P = 0.78), and among the eight studies with healthy control populations in which there was a similar strong association between M. genitalium infection and HIV (summary OR = 2.57, 95% CI = 2.05–3.22; P for heterogeneity = 0.41). By contrast, there was weaker association among the nine non-African studies (summary OR = 1.44, 95% CI = 0.77–2.67; P heterogeneity < 0.001) and among the 11 studies with other control populations (summary OR = 1.96, 95% CI = 1.73–2.21; P for heterogeneity < 0.001).
Using meta-inference analysis, we identified three studies as contributing to the overall heterogeneity [32,34,40]. These included both studies that had urethritis patients as control groups and showed reduced odds of HIV among M. genitalium-infected individuals. When these studies were excluded from analyses, there was no statistical evidence of heterogeneity (P = 0.69), with a summary OR of 2.72 (95% CI = 2.40–3.08).
Funnel graphs of the random-effects meta-analysis of ORs are presented in Fig. 3a–c. The funnel graph for all studies combined appears slightly asymmetrical around the pooled estimate, with smaller studies tending to report a stronger association, suggesting the possibility of publication bias. However, neither the Begg's test nor Egger's test indicated significant bias (Begg's test P = 0. 94, Egger's test P = 0. 86). For the sub-groups of studies with healthy control populations and those in sub-Saharan Africa, there was no visual or statistical indication of publication bias (healthy controls: Begg's test P = 0.54, Egger's test P = 0.24; sub-Saharan Africa: Begg's test P = 0.37, Egger's test P = 0.61).
This first systematic review and meta-analysis of the relationship between M. genitalium and HIV infection provides strong evidence of an association. Overall, we found a statistically significant two-fold increased odds of HIV among M. genitalium-infected populations. However, there was significant heterogeneity between studies. This is likely due to the diversity of study populations, different modes of HIV transmission, testing methods, variation in exposure to HIV, and the prevalence of other STIs. It is also likely that in two of the studies from the USA, the HIV positive and negative groups were not drawn from the same population, though the publications did not present adequate information to confirm this [19,40]. The between-study heterogeneity indicates that the association of M. genitalium and HIV varies between populations. Indeed, when we divided our studies into those in sub-Saharan African populations and those with healthy control populations, we found that the between-study heterogeneity was substantially reduced and the odds of HIV infection associated with M. genitalium actually increased.
The strongest associations were seen among populations in sub-Saharan Africa, and when comparing HIV-positive participants with a healthy control population. In sub-Saharan Africa, HIV is primarily sexually transmitted and hence one might expect a stronger association of M. genitalium with HIV infection than in studies involving populations with other modes of HIV transmission, such as among injection drug users. The weaker effect in nonhealthy control populations may be because some of the control (i.e. HIV-negative) populations included participants with infections associated with M. genitalium, such as urethritis, pelvic inflammatory disease, and salpingitis . This would tend to underestimate the true association of M. genitalium and HIV.
However, in these sub-group analyses, the number of studies was small and there was relatively little power to detect between-study heterogeneity. Due to this acknowledged lack of a robust test, we further explored sources of heterogeneity through meta-inference analysis and identified individual studies that contributed to this heterogeneity. In addition to both studies that had urethritis patients as their control group, one of the studies suspected of having drawn cases and controls from different populations was also identified. When these studies were removed from our analysis, there was no longer any evidence of heterogeneity between studies, and the strong association between HIV and M. genitalium remained.
There were several limitations to this study. Perhaps, most importantly, almost all the studies were cross-sectional, precluding knowledge of which infection preceded the other, and few studies adjusted for confounding. Further work is needed to understand the possible causal association between M. genitalium and HIV. We cannot, therefore, easily distinguish between three possible theories: the association between HIV infection and M. genitalium may be due to confounding; HIV infection may increase the risk of M. genitalium infection; or M. genitalium infection may increase the risk of HIV acquisition or transmission. We will discuss each of these issues in turn.
Confounding of the association between HIV infection and Mycoplasma genitalium
As M. genitalium and HIV are both sexually transmitted, it is likely that HIV-positive participants have higher risk behaviour and are, therefore, more likely to be exposed to M. genitalium. It is therefore striking that, in the studies that did present both univariate and multivariable results, the association became stronger after adjustment for confounding [10,37]. This indicates that confounding by sexual behaviour may not be a major confounder of the association between M. genitalium and HIV infection.
HIV infection as a risk factor for Mycoplasma genitalium
Only one longitudinal study of M. genitalium included HIV testing, and this study found that HIV was a significant risk factor for M. genitalium acquisition in a group of high-risk women (hazard ratio = 2.17, 95% CI = 1.27–3.71) . Further, studies in our review showed that M. genitalium prevalence was higher among AIDS patients (43.7%), compared to 27.3% among asymptomatic HIV-infected persons, and 11.3% among controls. This may indicate that M. genitalium is an opportunistic infection in immunocompromised individuals. However, few studies have collected data on stage of HIV disease, and studies that have included CD4 cell count testing have not found an association between M. genitalium and the stage of HIV disease [38,43]. It may be that the higher rate of M. genitalium detection in AIDS patients is a result of increased antibiotic use in this population, particularly broad-spectrum antibiotics, against which M. genitalium is resistant.
Mycoplasma genitalium as a risk factor for HIV acquisition or transmission
A recent study found that a high M. genitalium organism burden was associated with increased detection of genital HIV DNA , and hence infectivity. Another study  showed that M. genitalium infection was significantly associated with HIV seroconcordance in couples. It is plausible that, like other STIs, the inflammatory response associated with M. genitalium infection may contribute to increased risk of HIV. To our knowledge, there are no published studies of M. genitalium as a risk factor for HIV acquisition. Future cohort and prospective discordant couple studies are needed to provide data on the timing and nature of the two infections.
Systematic reviews can be vulnerable to identification and publication biases due to the difficulty in identifying all eligible articles, and because studies that yield significant results may be more likely to be submitted and published . However, publication bias is unlikely in this review, as most studies did not have the association between M. genitalium and HIV as the primary outcome, but rather reported these results as a secondary analysis. In addition, through personal communication, we obtained data from some of the studies that mentioned testing for both M. genitalium and HIV, even if the results were not presented. While we were not able to acquire results from all such studies, these were small studies with few HIV-positive participants, and contribute relatively little to the summary OR. The decision to exclude conference abstracts likely decreased the possibility of publication bias, as abstracts are more likely to report significant results. Finally, the results of the funnel plot also suggest that publication bias was not a major problem in this meta-analysis.
In the absence of a commercially available test kit for M. genitalium detection, a variety of testing methods were used in the studies included in our review. Thirteen studies used PCR to detect M. genitalium, but nine different variations of PCR detection were used [8–11,13,31–36,38]. Five studies used a method in addition to PCR, four also using culture (three of which failed to isolate M. genitalium) [35–38] and one using the commercial transcription-mediated amplification test, Aptima Combo 2, in development by Gen-Probe Incorporated (San Diego, California, USA) . One study used only the Aptima Combo 2 test . Four studies used serology, including two different antibody tests [19,37,39,40]. Furthermore, a variety of samples were used to test for M. genitalium, including serum, urine, cervical, urethral, and endometrial samples. Due to these varying samples and testing methods, there is likely to be some misclassification of M. genitalium infection, which may have underestimated any association if this was nondifferential with respect to HIV infection. Many of the studies in our analysis used stored samples; thus, there could be loss of sensitivity from freeze-thaw cycles that some samples will have gone through. Again, we would expect any such nondifferential misclassification of infection to underestimate the true association with HIV. Despite these inconsistencies, as well as the lack of a healthy control population in many studies, the consistency of the results suggests that the association between M. genitalium and HIV is a robust and transferable finding.
The observed association with risk of HIV in cross-sectional studies, and the high prevalence of M. genitalium among HIV-infected individuals, highlights the need for further investigation of this potentially important STI, and the development of a commercially available assay for M. genitalium diagnosis is needed. Future epidemiological studies should address the causal association through longitudinal study designs and randomized controlled trials. Careful consideration should also be given to systematic testing and treatment of individuals in high-risk populations, and this may prove to be a potential strategy to prevent HIV transmission.
The search strategy was developed by S.N.M. and H.A.W. Both authors contributed to the study selection process. Statistical analysis was conducted by S.N.M. The preliminary draft of this article was written by S.N.M., and the final version was approved by both authors. The authors would like to thank Jacques Pepin, Gabriela Paz Bailey, and Thomas Quinn for providing additional data for inclusion in this meta-analysis.
H.A.W. is funded by the UK Medical Research Council.
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