Thesis (D Phil. (Microbiology))--2010. / Mycoplasma genitalium is the smallest existing self-replicating prokaryote, lacks a cell wall and has a genome consisting of only 580 kilo base pairs. It has characteristic pear/flask-like morphology with a terminal tip organelle used for attachment. Many researchers, mainly in developed countries, have investigated the role the organism plays in the aetiology of male urethritis and the majority of studies show an association between M. genitalium and male urethritis. In this study, the modified Koch’s postulates were applied to answer the question whether M. genitalium is a true pathogen, or merely a passenger, invading already inflamed or damaged cells. A total of 300 urine specimens were collected from adult males with symptoms and/or signs of urethritis and 75 from asymptomatic men. In the first study, three molecular assays; viz, a commercial conventional PCR test, a real-time PCR (q- PCR) test and a transcription mediated amplification (TMA) assay were evaluated for the detection of M. genitalium. The comparison between the assays was based on the extended gold standard concept, where a specimen was deemed positive when any two nucleic acid amplification tests were positive. In the second study, the specimens were tested for four common urethral pathogens (N. gonorrhoeae, C. trachomatis, T. vaginalis and M. genitalium) using TMA assays. Finally, the bacterial loads for M. genitalium were determined using the q-PCR assay. v All three assays tested were highly specific (98-99%) for the detection of M. genitalium. However, where q-PCR and TMA demonstrated high sensitivities (96% and 100%), the sensitivity of the conventional PCR assay was low (78%). One or more pathogens were detected in a total of 129 (43%) men with urethritis. M. genitalium was the most frequently detected pathogen in men with urethritis (129; 43%), and significantly more (p= 0.04) than in asymptomatic men (7; 9.0%). There is a strong association with M. genitalium bacterial load and clinical urethritis. Patients with urethral discharge had significantly higher M. genitalium concentrations than those with only burning on micturition (p<0.001), and the bacterial concentrations in men with symptoms and/or signs of urethritis were significantly higher than that in asymptomatic men (p=0.02). As the number of organisms increased, the severity of the symptoms increased; an indication of the role that the organism plays in disease progression. In conclusion, by applying the modified Koch postulates, it was shown that Mycoplasma genitalium is by no means a passenger, but rather an important cause of adult male urethritis that should be taken into account when making diagnosis and when designing treatment strategies.

Thesis (MSc (Med)(Microbiology))--University of Limpopo (Medunsa Campus), 2010. / Mycoplasma genitalium, a human mycoplasma species has been established as a cause of nongonococcal urethritis (NGU) in men, particularly in Chlamydia trachomatis-negative patients. It was also shown to play a role in cervicitis and pelvic inflammatory disease (PID) in women. Due to difficulty in culturing, and the lack of routine molecular diagnostic tests, many M. genitalium infections are undetected. The purpose of this study was to evaluate three nucleic acid amplification tests (NAATs) i.e. a recently developed Gen-Probe research only transcription mediated amplification (TMA) assay, a conventional polymerase chain reaction (PCR) assay and a real-time PCR (q-PCR) assay for the detection of M. genitalium in urine specimens of men with symptoms of urethritis. To evaluate the three assays, 300 urine specimens were collected between June 2007 and July 2008 from sexually active male patients presenting with discharge (N=94) and/or burning on micturition (N=206) to a private medical practitioner in Silverton, Pretoria. A specimen was considered positive by extension of the gold standard i.e. if any two of the three assays were positive. This was used to calculate the sensitivity and specificity of each method. TMA detected M. genitalium in 62 (21%), PCR in 43 (14%) and q-PCR in 48 (16%) of the 300 patients. The sensitivities of the assays were 100% (TMA), 92% (q-PCR) and 78% (PCR), with specificities of 90% (TMA), 95% (q-PCR) and 97% (PCR). The sensitivity of the TMA assay was higher than that of the q-PCR and PCR assays. The lower sensitivity obtained by the q-PCR assay might have been due to inhibition and limitations in the amount of the DNA template. However, the q-PCR assay was easy to perform as it combines amplification and detection thus eliminating further handling of PCR products. The PCR, although with a higher specificity, was the least desirable in terms of testing time and problems with subjectivity when reading agarose gels. v We concluded that the Gen-Probe TMA assay is a highly sensitive method for detection of M. genitalium in urine specimens of men. The use of Gen-Probe TMA and the q-PCR assay, will increase the detection of M. genitalium in clinical specimens at this catchment area.

Au sein des mycoplasmes pathogènes pour l’homme, il existe des mycoplasmes à tropisme respiratoire, parmi lesquels M. pneumoniae, et d’autres dont le tropisme est la sphère urogénitale, comme M. genitalium. M. genitalium est un agent émergent dont l’épidémiologie est encore mal connue. Il est responsable d’infections sexuellement transmissibles, urétrites chez l’homme et cervicites chez la femme. M. pneumoniae est responsable d’infections respiratoires aiguës chez l’enfant et l’adulte jeune. M. genitalium est une espèce extrêmement fastidieuse dont la culture est exceptionnelle à partir de prélèvements de patients. Dans le but d'enrichir notre collection de prélèvements positifs pour M. genitalium un protocole de recherche clinique (FeminIST) proposant un dépistage systématique par PCR du portage de M. genitalium chez les femmes infectées par le VIH de la cohorte Aquitaine a été mis en place. Les méthodes génotypiques ont été largement appliquées pour le typage moléculaire des Mollicutes, mais peu de méthodes simples, automatisées et discriminantes l’ont été à M. genitalium et M. pneumoniae. La MLVA (« Multi-Locus Variable-Number of Tandem-Repeats Analysis ») est une méthode qui analyse le polymorphisme associé aux répétitions en tandem, présentant de nombreux avantages, comme un pouvoir discriminant élevé et la possibilité d’être réalisée directement à partir des prélèvements. Cette technique a été appliquée à M. genitalium et M. pneumoniae et comparée aux méthodes de typage déjà disponibles. Six et cinq VNTR ont été respectivement identifiés comme discriminants pour M. genitalium et M. pneumoniae. Les PCR ont été multiplexées et les amorces marquées pour faciliter et automatiser l’analyse réalisée par électrophorèse capillaire. La méthode a été réalisée sur notre collection de 265 souches cliniques de M. pneumoniae et directement à partir de 123 prélèvements positifs de M. genitalium. La MLVA a permis de typer 89,4 % des prélèvements positifs pour M. genitalium et toutes les souches de M. pneumoniae. Elle s’est révélée plus discriminante que les autres méthodes pour les deux espèces. Les représentations hiérarchiques des résultats confirment l’hétérogénéité de l’espèce M. genitalium et, en revanche, l’homogénéité de l’espèce M. pneumoniae. En résumé, la MLVA s’avère être un outil de typage moléculaire performant pour M. genitalium et M. pneumoniae donnant des résultats facilement échangeables entre laboratoires. / Human pathogenic mycoplasmas include respiratory tract species, such as M. pneumoniae and urogenital species, such as M. genitalium. M. genitalium is an emerging agent for which epidemiology is unclear. It is involved in sexually transmitted infections, mainly urethritis in men and cervicitis in women. M. pneumoniae is responsible for acute respiratory infections especially in children. M. genitalium is a fastidious species for which culture remains extremely difficult. In order to extend our collection of samples positive for M. genitalium, a clinical research study (FeminIST) was conducted. It consisted in a PCR screening for M. genitalium in the urogenital tract of HIV-infected women of the Aquitaine cohort. Genotyping methods have been widely applied to Mollicutes, but few simple and automatized methods have been developed for M. genitalium and M. pneumoniae. The MLVA (Multi-Locus Variable-Number Tandem-Repeats Analysis) method analyzes the genome polymorphism associated with tandem repeats. Its advantages are a high discriminatory power and the possibility of being used directly from clinical samples. This technique was applied to M. genitalium and M. pneumoniae and compared with other available genotyping methods. Six and five VNTR were selected for M. genitalium and M. pneumoniae, respectively. The use of multiplex PCR and capillary electrophoresis enabled a high-throughput analysis and allowed an easy interpretation of the results. The method was applied to our collection of 265 M. pneumoniae clinical strains and used directly from 123 clinical samples positive for M. genitalium. 89.4% of M. genitalium PCR-positive samples and all the M. pneumoniae isolates were amplified and typed. We showed a higher discriminatory power for our MLVA than for other genotyping methods, without the need of a fastidious sequencing step. The hierarchical representation of results confirms the M. genitalium species heterogeneity and the M. pneumoniae species homogeneity. MLVA appears to be a good tool for molecular typing of these two mycoplasma species, allowing an easy exchange of data between laboratories.

Mycoplasma genitalium (M. genitalium) är en liten bakterie utan cellvägg som kan smitta sexuellt och efter en veckas inkubationstid orsaka genitala infektioner. Bakterien är svårodlad, men med PCR-teknik kan DNA-sekvens, specifik för M. genitalium, detekteras i prover från uretra/vagina. M. genitalium-infektionen ger ofta inga symptom. Symptom som sveda, klåda, blödningsrubbningar och flytningar kan dock ses vid M. genitalium-infektion, men dessa symptom liknar C. trachomatis-infektion, en annan vanlig sexuellt överförbar infektion. M. genitalium-bakterien har påvisats i prov från patienter med uretrit och cervicit men även i uppåtstigande infektioner, som benämns med det engelska namnet Pelvic Inflammatory Disease (PID). Obehandlade i tid, kan dessa uppåtstigande infektioner orsaka infertilitet, ektopisk graviditet och kroniska buksmärtor. Detta litteraturarbete har som syfte att redovisa hur vanligt M. genitalium- infektion är bland unga kvinnor. I några av studierna undersöks prevalensen av infektionen bland olika riskgrupper med eller utan symptom. Andra studier undersöker prevalensen av M. genitalium-infektion bland kvinnor som har diagnostiken cervicit och PID. I en studie påvisades en prevalens av M. genitalium-infektion på 2,1 % bland unga kvinnor som rekryterats inom primärvården. En betydligt högre prevalens på 22 % för samma infektion fann man hos riskgrupper för sexuellt överförbara infektioner (STI). I samma studie var 54 % av kvinnorna med M. genitalium-infektion smittade med även andra sexuellt överförbara patogener.  Arbetet undersöker också om behandlingen med doxycyklin, ett vanligt tetracyklin som används vid behandling av C. trachomatis-infektion, även fungerar för att behandla M. genitalium-infektionen. I två av studierna användes 1 g azitromycin (en makrolid) som singeldos för eradikering av M. genitalium. Detta gav en eradikeringsfrekvens mellan 85 - 91 %, vilket är betydligt högre än för doxycyklin på17-37 %. Azitromycinbehandling med 500 mg dag 1 och 250 mg dag 2 till 5 gav en eradikeringsfrekvens på 96 %. En retrospektiv studie visade att kvinnor diagnostiserade med PID och positiva för M. genitalium – infektion hade fått dåliga eradikerings- och behandlingsresultat med en standardbehandling som bestod av cefoxitin och doxycyklin. Dessa kvinnor drabbades senare i livet av infertilitet, kronisk smärta och återfall av PID. Utifrån de studier som användes i arbetet framkom att M. genitalium-infektion år 2010-2013 har en prevalens på mellan 2 till 4 % bland unga kvinnor mellan 17och 27 år och att det finns behov av screening och kontroll av infektionen. Incidens av M. genitalium-infektion ligger på ungefär 1 % per 100 kvinnoår. Det behövs nya behandlingsstrategier som undviker doxycyklin och cefoxitin, vilka har en dålig eller helt overksam effekt för eradikering av M. genitalium-bakterien.  Screening för både M. genitalium- och C. trachomatis – infektioner behövs för att välja ett antibiotikum som båda är känsliga för.

Mycoplasma (M.) genitalium é um importante agente de doença sexualmente transmissível sendo, responsável por uma série de desordens do trato urogenital humano. A aderência do micoplasma é um dos principais fatores de virulência na sua patogenicidade e conseqüente colonização nas células hospedeiras. Neste estudo, obte-se a curva de crescimento de duas cepas de M. genitalium (G37 e 1019V) utilizando-se da dosagem proteíca (BCA), densidade óptica (OD600) e PCR em tempo real. A cepa referencial G37 é de alta passagem e foi isolada de homens e a 1019V é de baixa passagem, sendo recentemente isolada de amostra clínica de cérvix humana. Utilizando-se da fase logarítimica obtida pela curva, comparou-se a dinâmica de interação destas cepas na célula epitelial de carcinoma de cérvix humana (HeLa) e na célula endometrial humana (EM42), em diferentes intervalos de tempo com auxílio de microscopia confocal. Apesar destas cepas divergirem na seqüência dos genes relacionados a aderência houve poucas variações entre as curvas de crescimento. A aderência e a invasão de M. genitalium nas células não fagocíticas confirmou os dados de literatura. Entretanto, após 30 minutos de contato com as células, detectou-se o antígeno de aderência de ambas as cepas na região intranuclear Este achado, indica uma nova característica desta espécie ainda não conhecida entre os molicutes. / Mycoplasma genitalium (Mg) is an important cause of sexual transmitted disease and has been implicated in a range of genital tract disorders.The adherence of mycoplasmas is a key virulence attribute, pathogenic features and consequences of host-cell colonization. Herein, we characterize growth properties of two Mg strains (G37 and 1019V) using BCA assay, OD600, CCU assay, real-time PCR. Based upon these strategies, we compared the behavior of similarly grown Mg variants coincubated with HeLa cervical epithelioid cells and EM42 endometrial cells over a dynamic time course.using laser scanning confocal microscopy. Mg G37 is a multiply passaged type strain isolated from a male while 1019V was recently isolated from human cervical samples and only minimally passaged. Both strains further diverge by sequence heterogeneities within adherence-related MG191 and MG192 genes. Despite these differences, our results identified only subtle variations in axenic growth for the two strains. Further and consistent with previous studies, a subset of adherent Mg organisms invaded host cells. However, intranuclear localization was observed, which occurred as early as 30 minutes after infection.

Mycoplasma (M.) genitalium é um importante agente de doença sexualmente transmissível sendo, responsável por uma série de desordens do trato urogenital humano. A aderência do micoplasma é um dos principais fatores de virulência na sua patogenicidade e conseqüente colonização nas células hospedeiras. Neste estudo, obte-se a curva de crescimento de duas cepas de M. genitalium (G37 e 1019V) utilizando-se da dosagem proteíca (BCA), densidade óptica (OD600) e PCR em tempo real. A cepa referencial G37 é de alta passagem e foi isolada de homens e a 1019V é de baixa passagem, sendo recentemente isolada de amostra clínica de cérvix humana. Utilizando-se da fase logarítimica obtida pela curva, comparou-se a dinâmica de interação destas cepas na célula epitelial de carcinoma de cérvix humana (HeLa) e na célula endometrial humana (EM42), em diferentes intervalos de tempo com auxílio de microscopia confocal. Apesar destas cepas divergirem na seqüência dos genes relacionados a aderência houve poucas variações entre as curvas de crescimento. A aderência e a invasão de M. genitalium nas células não fagocíticas confirmou os dados de literatura. Entretanto, após 30 minutos de contato com as células, detectou-se o antígeno de aderência de ambas as cepas na região intranuclear Este achado, indica uma nova característica desta espécie ainda não conhecida entre os molicutes. / Mycoplasma genitalium (Mg) is an important cause of sexual transmitted disease and has been implicated in a range of genital tract disorders.The adherence of mycoplasmas is a key virulence attribute, pathogenic features and consequences of host-cell colonization. Herein, we characterize growth properties of two Mg strains (G37 and 1019V) using BCA assay, OD600, CCU assay, real-time PCR. Based upon these strategies, we compared the behavior of similarly grown Mg variants coincubated with HeLa cervical epithelioid cells and EM42 endometrial cells over a dynamic time course.using laser scanning confocal microscopy. Mg G37 is a multiply passaged type strain isolated from a male while 1019V was recently isolated from human cervical samples and only minimally passaged. Both strains further diverge by sequence heterogeneities within adherence-related MG191 and MG192 genes. Despite these differences, our results identified only subtle variations in axenic growth for the two strains. Further and consistent with previous studies, a subset of adherent Mg organisms invaded host cells. However, intranuclear localization was observed, which occurred as early as 30 minutes after infection.

<p>Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by <em>Chlamydia trachomatis</em> or <em>Neisseria gonorrhoeae, </em>bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, <em>Mycoplasma genitalium</em> was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of <em>M. genitalium</em> infection became more feasible.</p><p>The aim in paper <strong>I</strong> was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time <em>Mycoplasma genitalium</em> adhesin protein (MgPa) gene PCR) for detection of <em>M. genitalium</em>. The study also determined the prevalence of <em>M. genitalium</em>. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of <em>M. genitalium</em> and 213 of these specimens were used in the PCR comparative study. The prevalence of <em>M. genitalium</em> infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, <em>M. genitalium </em>DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of <em>M. genitalium</em> in urogenital specimens from men and women.</p><p>The aim in paper <strong>II</strong> was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of <em>Mycoplasma genitalium </em>by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. <em>M. genitalium</em> was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of <em>M. genitalium</em> positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.</p>

Philosophiae Doctor - PhD / Sexually transmitted infections (STIs) are a problem for both developed and developing countries. Sub-Saharan Africa has the highest rates in the 15-49 years old group. The discovery that these infections playa vital role in the transmission of HIV raised their profile and made their control one of the central strategies of stopping the HIV/AIDS epidemic. In response to the challenge of improving the quality of care for people infected with STIs in the public health sector, the South African Ministry of Health adopted the syndromic management approach, recommended by the World Health Organisation as suitable for resource-poor settings, for use in primary health care clinics. In addition to providing guidelines on clinical management of STIs, the syndromic approach requires health providers to counsel and educate patients about STIs, encourage patients to complete treatment even if symptoms abate, promote condom use and the treatment of all sexual partners. While the management guidelines are clear and detailed around the diagnostic and medication issues, the processes of education and counseling are not as clearly outlined. Furthermore, although the syndromic approach is a viable way of providing good quality care to larger sections of the population than could be serviced through dedicated STI clinics, it requires health providers working in primary health care clinics, most of whom are professional nurses, to perform some tasks for which they may not be adequately trained. This study evaluated the quality of care for persons infected with ST!s by examining the extent to which the syndromic approach was being implemented in primary health care clinics. Interviews, using semi-structured interview schedules, were conducted with ST! patients and health providers in twenty-four clinics located in four provinces. In depth qualitative interviews were also conducted with a sub-sample of the patients. For further triangulation the methods of participant observation, through the use of simulated patients, and focus group discussions with various community groups were used. The findings of the study indicate that although primary health care clinics in South Africa are well-resourced, the management of patients with ST!s is inadequate. Adherence to the various aspects of syndromic management was poor. Similar to other studies in South Africa, the attitudes of health providers towards patients with ST!s were found to be problematic, a finding that has implications for health-seeking behaviours. The thesis argues that a large part of the problem is related to the multiple roles that nurses have to play in primary health care settings, as well as the content and methodology of the training of nurses who manage ST! patients. It further argues for the constitution of the basic health team at primary health clinics to be multi-disciplinary, and for a multi-disciplinary input in the training of health providers.

Indiana University-Purdue University Indianapolis (IUPUI) / Inflammation of the urethra—urethritis—is commonly diagnosed in men and women who have sexually transmitted infections (STI). Characteristic signs and symptoms of urethritis include urethral discharge and burning pain during urination (dysuria). However, these findings are non-specific and can be elicited by STI for which optimal treatment approaches differ. We wanted to investigate if immunoglobulins (antibodies) in the urine of men with acute urethritis could determine the etiologies of these cases. Previously, we conducted an observational case-control study of biological males to compare the urethral microbiota of participants with unambiguous, laboratory-confirmed urethritis (cases) and participants without urethral inflammation (controls). This revealed that nearly 2 in 5 men with nongonococcal urethritis tested negative for all common STI. We identified atypical urethral pathogens in approximately 1/3 of these STI-negative individuals using shotgun metagenomic sequencing. However, we did not detect microorganisms suspected to be urethral pathogens in the remaining 2/3 of STI-negative participants. We hypothesized that these men with “pathogen-negative” urethritis had persisting inflammation from a recent STI that already cleared spontaneously by the time of testing. We observed that urine IgA antibodies against Chlamydia trachomatis (Ctr) infectious particles were significantly more prevalent among men with pathogen-negative urethritis compared to controls. In contrast, we found that the prevalence of urine anti-Ctr IgA was similar between controls and urethritis cases with atypical infections. However, our efforts to detect antibodies against another common STI, Mycoplasma genitalium (Mgen), were complicated by low abundance in urine and the unexpected prevalence of Mgen-reactive antibodies among controls. Collectively, our results suggest that signs and symptoms of urethritis can continue after the causative STI(s) have been eliminated. Furthermore, male urine represents a practical, non-invasive source of pathogen-reactive antibodies that could be evaluated using point-of-care diagnostic tests to elucidate urethritis etiologies. Importantly, our results also suggest that sexual partners of men with pathogen-negative, nongonococcal urethritis are an unrecognized chlamydia reservoir. / 2024-05-22

No description available.