Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Is a Superior Diagnostic Tool for the Identification and Differentiation of Mycoplasmas Isolated from Animals.

In veterinary diagnostic laboratories, identification of mycoplasmas is achieved by demanding, cost-intensive, and time-consuming methods that rely on antigenic or genetic identification. Since matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) seems to represent a promising alternative to the currently practiced cumbersome diagnostics, we assessed its applicability for the identification of almost all mycoplasma species isolated from vertebrate animals so far. For generating main spectrum profiles (MSPs), the type strains of 98 Mycoplasma, 11 Acholeplasma, and 5 Ureaplasma species and, in the case of 69 species, 1 to 7 clinical isolates were used. To complete the database, 3 to 7 representatives of 23 undescribed Mycoplasma species isolated from livestock, companion animals, and wildlife were also analyzed. A large in-house library containing 530 MSPs was generated, and the diversity of spectra within a species was assessed by constructing dendrograms based on a similarity matrix. All strains of a given species formed cohesive clusters clearly distinct from all other species. In addition, phylogenetically closely related species also clustered closely but were separated accurately, indicating that the established database was highly robust, reproducible, and reliable. Further validation of the in-house mycoplasma library using 335 independent clinical isolates of 32 mycoplasma species confirmed the robustness of the established database by achieving reliable species identification with log scores of ≥1.80. In summary, MALDI-TOF MS proved to be an excellent method for the identification and differentiation of animal mycoplasmas, combining convenience, ease, speed, precision, and low running costs. Furthermore, this method is a powerful and supportive tool for the taxonomic resolution of animal mycoplasmas.

Development and clinical application of an InvaderPlus® assay for the detection of genital mycoplasmas.

We developed a PCR-based assay involving Invader® technology for detection of the genital mycoplasmas of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. We compared its performance with that of a PCR-microtiter plate hybridization assay, which we developed previously, in detecting genital mycoplasmas in first-voided urine (FVU) specimens from men with non-gonococcal urethritis. The tests targeting each of the genital mycoplasmas were specific for the respective species and could detect as few as 10 copies of the plasmids containing the target genes of each of the genital mycoplasmas per reaction. The assay using the InvaderPlus® method (InvaderPlus® assay) showed very similar performance to that of the PCR-microtiter plate hybridization assay for detecting the genital mycoplasmas in the FVU specimens. In addition, the PCR and endonuclease reaction in the InvaderPlus® assay were carried out simultaneously in one procedure, thus simplifying the assay, leading to time- and labor-savings and a decrease in the risk of specimen contamination. The InvaderPlus® assay could be useful in diagnosing genitourinary tract infections caused by the genital mycoplasmas.

Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women.

BACKGROUND: Genital mycoplasmas colonise up to 80% of sexually mature women and may invade the amniotic cavity during pregnancy and cause complications. Tetracyclines and fluoroquinolones are contraindicated in pregnancy and erythromycin is often used to treat patients. However, increasing resistance to common antimicrobial agents is widely reported. The purpose of this study was to investigate antimicrobial susceptibility patterns of genital mycoplasmas in pregnant women. METHODS: Self-collected vaginal swabs were obtained from 96 pregnant women attending an antenatal clinic in Gauteng, South Africa. Specimens were screened with the Mycofast Revolution assay for the presence of Ureaplasma species and Mycoplasma hominis. The antimicrobial susceptibility to levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline were determined at various breakpoints. A multiplex polymerase chain reaction assay was used to speciate Ureaplasma positive specimens as either U. parvum or U. urealyticum. RESULTS: Seventy-six percent (73/96) of specimens contained Ureaplasma spp., while 39.7% (29/73) of Ureaplasma positive specimens were also positive for M. hominis. Susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% (26/44) and 98% (43/44) respectively. Mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin and tetracycline (both 97% resistance). Resistance of Ureaplasma spp. to erythromycin was 80% (35/44) and tetracycline resistance was detected in 73% (32/44) of Ureaplasma spp. Speciation indicated that U. parvum was the predominant Ureaplasma spp. conferring antimicrobial resistance. CONCLUSIONS: Treatment options for genital mycoplasma infections are becoming limited. More elaborative studies are needed to elucidate the diverse antimicrobial susceptibility patterns found in this study when compared to similar studies. To prevent complications in pregnant women, the foetus and the neonate, routine screening for the presence of genital mycoplasmas is recommended. In addition, it is recommended that antimicrobial susceptibility patterns are determined.

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections.

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex(®) STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO™) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex(®) STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.

Accuracy of urethral swab and urine analysis for the detection of Mycoplasma hominis and Ureaplasma urealyticum in women with lower urinary tract symptoms.

OBJECTIVES: Our objective was to evaluate and compare the accuracy of urethral swabs and urine specimens in the detection of Mycoplasmas in women with lower urinary tract symptoms (LUTS). METHODS: During a urogynecological work-up, including cystometry, we obtained first-void urine, urethral and vaginal swabs in 207 consecutive women at our urogynecological division. Mycoplasma hominis and Ureaplasma urealyticum as well as other microorganisms were detected by standard culture methods. RESULTS: 131 of 207 women reported LUTS. The other 76 formed the controls. Of 207 women 50 (24.2%) had positive cultures for Mycoplasmas. The prevalence of Mycoplasmas in women with LUTS (30.3%) was statistically significant and higher in the group without LUTS (14.5%) (p = 0.011). The detection of M. hominis was most accurate using urethral swab (Specificity 99.9%, PPV 99.6%) compared to the urine specimen (96%, 75%) and vaginal swab (95.1%, 67%). Similar results could be achieved for U. urealyticum (urethral swab: specificity 98.7%, PPV 96.3%; urine specimen: 86.8%, 72%; vaginal swab: 80.5%, 65.2%). CONCLUSION: In the subgroup of women less than 50 years an (detectable) infection due to Mycoplasma or Ureaplasma leads typically to LUTS with normal filling cystometry, whereas no such findings were relevant for the elderly women.

The role of genital mycoplasma infection in female infertility: A systematic review and meta-analysis.

PROBLEM: Recent studies show that lower genital tract infection with genital mycoplasma may be associated with the pathology of female infertility. However, this association remains controversial due to the variable prevalence, sample sizes, and different methods used to diagnose genital mycoplasma infection. The aim of the present meta-analysis was to gain better understanding of the specific impact of genital mycoplasma on female infertility. METHOD OF STUDY: A systematic review of literature on the association of genital mycoplasma (Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum) infection and female infertility was performed using three electronic databases: PubMed, Scopus, and CINAHL, from January 2000 to January 2020. Pooled odds ratio (OR) and 95% confidence intervals for genital mycoplasma infection and female infertility were derived from a fixed effects model. RESULTS: This meta-analysis included eight studies conducted in six countries. Based on the results, women with infertility had a statistically higher odds of having any genital mycoplasma infection (p < .0001) compared to the control group. The pooled OR of all the included studies was 3.82 (95% CI: 2.55, 5.72). There was an unremarkable heterogeneity in all the studies included in this meta-analysis (I2  = 0%, p = .48). A subgroup analysis also showed that M. genitalium, M. hominis, and U. urealyticum infections are significantly associated with female infertility. CONCLUSION: Our meta-analysis showed a significant association between M. genitalium, M. hominis, and U. urealyticum infections and female infertility. This evidence supports the development of guidelines for the diagnosis and treatment of genital mycoplasma infections to prevent female infertility.

Impact of combined mycoplasmataceae and HPV co-infection on females with cervical intraepithelial neoplasia and carcinoma.

PURPOSE: The concurrent prevalence investigation of human papillomavirus (HPV), Mycoplasma hominis (Mh) and Ureaplasma urealyticum (Uu) in women in order to estimate the association of co-infection with cervical lesions. METHODS: The study cohort comprised 120 women with no cervical lesions (control group) and 62 women with abnormal cytological findings from the cervix (cervical intraepithelial lesion/neoplasia) as study group. A combination of molecular analyses was implemented. RESULTS: The presence of HPV infection was shown in 52/62 (83.9%) of women with abnormal cytology. Women with cervix cytological findings were shown to have 17.6 times higher risk for Mh and Uu co-infection (p=0.001). HPV and Uu co-infection were detected with a higher prevalence among women with CIN 3 and invasive cancer. CONCLUSION: These findings are consistent with the notion that microbial co-infections may play an important role in persistent inflammation and progression of cervical lesions.

Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma.

A spiroplasma recovered from allantoic fluids of chick embryos infected with the tick-derived suckling mouse cataract agent was grown in continuous passage on a new artificial culture medium. The cultured organisms induced typical ocular and other disease symptoms in susceptible animals, and were reisolated from involved host tissues. Although spiroplasmas have been previously recognized as plant and insect pathogens, this is the first spiroplasma shown to multiply at 37 degrees C and to be pathogenic for vertebrates.

Identification of a novel Hemoplasma species from pigs in Zhejiang province, China.

Hemoplasmas belong to Mycoplasmataceae (Mollicutes: Mycoplasmatales) and are able to infect a broad range of mammalian species. We investigated prevalence of hemotropic mycoplasma species in pig farms in the region of Zhejiang by a PCR scheme using universal primers targeting 16S rRNA and RNase P RNA gene (rnpB). Representative positive samples from different farms were selected for sequencing of 16S rRNA and the 219bp rnpB gene fragments for phylogenetic analysis. Sequencing analysis of PCR products from first samples identified a novel hemoplasma species present in several pig farms in the region with highest nucleotide identity of 92% to Candidatus Mycoplasma turicensis. A duplex PCR assay was then designed for differential detection of the novel hemoplasma from Mycoplasma parvum/M. suis in field samples. Of 324 blood samples from clinically healthy pigs, 26.5% was positive for this novel hemoplasma species and 50% positive for M. suis/M. parvum, indicating that the novel hemotropic mycoplasma species were of considerably high prevalence in Zhejiang province, China.

Infant pneumonitis associated with cytomegalovirus, Chlamydia, Pneumocystis, and Ureaplasma: a prospective study.

In a prospective study of 104 infants between 1 and 3 months of age hospitalized with pneumonitis, 65 (63%) had evidence of infection with one or more potential respiratory pathogens. Single infections were noted in 48 (74%) whereas mixed infections occurred in 17 (26%) of 65 infected infants. The four most common infections were Chlamydia trachomatis (15/59, 25%), Ureaplasma urealyticum (8/38, 21%), cytomegalovirus (21/104, 20%), and Pneumocystis carinii (19/104, 18%). In sharp contrast, the incidence of these infections in control infants was 0% (0/25), 4% (2/49), 3% (3/97), and 0% (0/64), respectively. The clinical, radiologic, and laboratory characteristics of the pneumonitis syndrome associated with Chlamydia, cytomegalovirus, and Pneumocystis were indistinguishable from each other. Patients with mixed infections had a more severe pneumonitis as measured by the occurrence of apnea and the need of oxygen therapy an mechanical ventilation. The patients enrolled in this study are being followed-up to determine the longitudinal course of these infections.

Experimental infection of the respiratory tract with Mycoplasma pneumoniae.

M. pneumoniae, a common human respiratory pathogen, has been studied experimentally for years using intranasal inoculation of the golden Syrian hamster. Because of recent evidence outlining the role in pulmonary immune development of particle size and depth of mycoplasma deposition in the hamster lung, we developed an aerosol chamber for the reproducible aerosolization of radiolabeled M. pneumoniae. Organisms were labeled to high specific activity by the incorporation of 3H-oleic acid and aerosolized under airflow and humidity conditions creating a mean particle diameter of 2.0 micrometers. Under these conditions, viable mycoplasmas were reproducibly and evenly distributed to all major lobes of the lung. Examination of radioactive clearance and organism viability within the lung during the first 48 hr after aerosolization have suggested a minimal role for macrophage mycoplasmacidal activity and a more prominent role for ciliary clearance. Data from aerosol infections of hamsters with radio-labeled M. pneumoniae should provide a unique opportunity to examine in a highly controlled manner the effects of air pollutants on the initial stages of infection as well as effects on the development of pulmonary immunity and histologic alterations.

Ureaplasma urealyticum colonization of full term infants: perinatal acquisition and persistence during early infancy.

In a prospective study 225 (35%) of 640 pregnant women who delivered at term had vaginal colonization with Ureaplasma urealyticum at the time of delivery. One hundred ninety-three full term infants born to U. urealyticum-colonized mother were cultured from the throat, eyes and vagina within the first 3 days of life. One hundred seven infants (55%) had at least one culture site positive for U. urealyticum (throat 41%, eyes 20%, vagina 40%). Rupture of membranes for greater than or equal to 12 hours and the mode of delivery did not affect vertical transmission of U. urealyticum. We were able to follow 108 infants during the first 3 months of life. Sixty-eight, 33 and 37% of the infants who were initially colonized with U. urealyticum in the throat, eyes and vagina, respectively, were still colonized when the follow-up cultures were obtained 3 months later. Fourteen of the 108 infants whom we followed developed a lower respiratory tract illness. In the pharyngeally colonized infants there was no increased risk for lower respiratory tract illness during early infancy compared with the pharyngeally noncolonized infants.

Vertical transmission of Ureaplasma urealyticum from mothers to preterm infants.

Ureaplasma urealyticum is a common component of the vaginal flora during pregnancy. Although colonization of low birth weight infants with U. urealyticum occurs frequently, the actual rate of vertical transmission of U. urealyticum in preterm infants has not been determined. Sixty-five preterm infants (less than 37 weeks of gestation) born to mothers colonized with U. urealyticum had eye, throat, vagina and rectum cultured for U. urealyticum at 1, 3 and 7 days of age and weekly thereafter for the first month of life while the infants remained in the hospital. Thirty-eight infants (58%) had at least one culture site positive for U. urealyticum (eye, 8%; throat, 37%, vagina, 54%; and rectum, 18%). Vertical transmission was not affected by method of delivery or duration of rupture of amniotic membranes. The rate of vertical transmission of U. urealyticum was higher among infants with birth weight less than 1,000 g (89%) than among those with birth weight of 1,000 g or greater (54%) (P = 0.07). Chronic lung disease developed in 9 of the 65 (14%) infants; 8 were colonized with U. urealyticum. The high rate of ureaplasmal colonization and chronic lung disease in infants less than 1,000 g makes these infants a suitable target population for a clinical treatment trial to determine whether eradication of U. urealyticum would decrease the incidence of chronic lung disease.

Ureaplasma urealyticum colonization and chronic lung disease in low birth weight infants.

Ureaplasma urealyticum is a common component of the vaginal flora during pregnancy. Transmission of U. urealyticum to the low birth weight infant may contribute to neonatal respiratory disease. We studied prospectively 111 infants with birth weights of 2 kg or less who were consecutively admitted to a neonatal intensive care unit during a 7-month period. The infants had eye, throat, vagina and/or rectum cultured for U. urealyticum on days 1, 3 and 7 and weekly thereafter until the time of discharge. Forty-six infants (41%) had at least one culture site positive for U. urealyticum (eye, 9%; throat, 35%; vagina, 34%; and rectum, 13%). Respiratory distress at birth was not associated with U. urealyticum colonization. However, colonization with U. urealyticum was significantly associated with the development of chronic lung disease. Of the infants colonized with U. urealyticum 30% developed chronic lung disease, whereas 8% of those not colonized developed chronic lung disease (P less than 0.05). Duration of positive pressure ventilation and oxygen therapy could not account for the higher incidence of chronic lung disease in the infants colonized with U. urealyticum. Stepwise logistic regression analysis using the profiles of birth weight, need for intubation and status of colonization with U. urealyticum correctly identified 79% of the infants who developed chronic lung disease. Additional studies serologic techniques are needed to confirm the association of U. urealyticum colonization and chronic lung disease in low birth weight infants.

Role of Ureaplasma urealyticum and other pathogens in the development of chronic lung disease of prematurity.

A prospective cohort study enrolling 107 infants weighing less than 1250 g was conducted between September 1, 1986, and November 15, 1987 in order to determine the role of microorganisms on the development of chronic lung disease (CLD). Ureaplasma urealyticum was isolated significantly more frequently from gastric aspirates and nasopharyngeal or endotracheal aspirates from 43 infants developing CLD than from 56 who did not (51% vs. 16%; P less than 0.005). Infants developing CLD, defined by radiographic and blood gas abnormalities, were significantly younger (26 vs. 29 weeks; P less than 0.0001), weighed significantly less (830 vs. 1050 g; P less than 0.0001) and required more ventilatory support (37 vs. 10 were being ventilated and 42 vs. 26 received oxygen supplementation on Day 7) compared with those who did not develop CLD. Viruses were isolated in association with U. urealyticum in two infants developing CLD and in one infant who did not develop CLD. Mycoplasma hominis was isolated from three infants who were colonized with U. urealyticum and developed CLD. Chlamydia trachomatis was not recovered from any patients. From a discriminant analysis it was found that U. urealyticum contributed to the development of CLD along with the effect of ventilatory support, gestational age and severity of initial respiratory disease. The effect of interventions directed against U. urealyticum on the development of CLD deserves further study.

Vertical transmission of Ureaplasma urealyticum in full term infants.

Ureaplasma urealyticum is a common inhabitant of the urogenital tract of pregnant women. Although colonization of newborn infants with U. urealyticum has been documented previously, the actual rate of vertical transmission has not been determined. Cervical cultures for U. urealyticum were performed on 1315 pregnant women on admission to the labor suite. A positive culture was found in 810 (62%). Eye, nasopharyngeal and/or throat, vaginal and rectal cultures were obtained in the first 5 days of life from 132 full term infants born to mothers colonized with U. urealyticum. Fifty-nine infants (45%) had at least one culture site positive for U. urealyticum (eye, 4%; nasopharynx 24%; throat, 16%; vagina, 53%; and rectum, 9%). None of the infants had evidence of disease caused by U. urealyticum during the nursery stay. Vertical transmission was not affected by the method of delivery. However, among the vaginally delivered infants, rupture of membranes greater than 1 hour correlated with an increased rate of vertical transmission of U. urealyticum (52%) compared with rupture of membranes less than or equal to 1 hour (22%) (P less than 0.05). Because vertical transmission of U. urealyticum occurs frequently, caution must be exercised when attributing disease to U. urealyticum based solely on positive cultures of mucosal surfaces.

Placental infection with Mycoplasma homonis and Ureaplasma urealyticum: clinical correlation.

Placentas from a clinical study group of 446 high-risk pregnancies and 108 normal pregnancies were cultured for Mycoplasma hominis and Ureaplasma urealyticum and examined histologically. Results were compared and correlated with the clinical history. The recovery rate of U urealyticum, but not of M hominis, was significantly higher in the clinical study than in the control group. Isolation of both mycoplasmas was associated with polymorphonuclear leukocyte infiltration of placental membranes, fetal surface, and umbilical cord. Recovery of mycoplasma was significantly higher with prolonged membrane rupture, spontaneous abortion, stillbirth, and early neonatal death. Isolation of U urealyticum, but not of M hominis, was associated with prematurity, lower birth weight, and intrauterine growth retardation.

Etiology of recurrent pregnancy losses and outcome of subsequent pregnancies.

Prospective evaluation of 155 couples with two or more consecutive pregnancy losses disclosed uterine morphologic abnormalities in 27%, chromosomal abnormalities in 21 individuals (7.7%, or 15.4% of the couples), and at least one abnormal diagnostic test suggestive of a cause for recurrent pregnancy losses in 106 (68%). A positive test for antinuclear antibody was found in 7.5% of the women, whereas the expected rate in a population of this age is less than 2%. Cervical cultures for Ureaplasma urealyticum (T-strain mycoplasma) were positive in 48% of the women, and 28% of these women had a genetic or uterine abnormality to explain their pregnancy losses. Thyroid function profiles and cervical cultures for Mycoplasma hominis provided no significant information in the evaluation in these couples. With the exception of women with a positive antinuclear antibody, the overall prognosis for later pregnancies was quite good whether the diagnostic evaluation of the couple was normal (77% subsequent live births) or abnormal (71% subsequent live births). The significance of the positive antinuclear antibody in these women is unclear, but further studies and long-term evaluation are necessary to determine the relationship between recurrent pregnancy losses and later development of collagen-vascular diseases.

Effect on birth weight of erythromycin treatment of pregnant women.

To test the hypothesis that treatment with antibiotics prevents low birth weight, pregnant women whose vaginal cultures contained Ureaplasma urealyticum or Mycoplasma hominis (or both) and who gave written informed consent were treated with one of the following: identical looking capsules containing 250 mg of either erythromycin estolate or stearate (active against U urealyticum), or 150 mg of clindamycin hydrochloride (active against M hominis), or placebo, four times daily for six weeks in a randomized double-blind study. Treatment with clindamycin had no effect. Treatment with erythromycin initiated during the second trimester had no effect on mean birth weight or on the frequency of low-birth-weight infants. In contrast, women whose treatment with erythromycin was initiated in the third trimester gave birth to infants with a heavier mean birth weight (3331 g) than infants born to placebo-treated women (3187 g) (P = .042). Similarly, in women whose erythromycin was begun during the third trimester, the birth rate of infants weighing 2500 g or less was 3%, whereas in women treated with placebo, the birth rate for low-birth-weight infants was 12% (P = .047). These data suggest that treatment with erythromycin during the third trimester prevents low birth weight in mycoplasma-colonized pregnant women. Whether the effect is due solely to the action of erythromycin on U urealyticum is uncertain.

Comparative evaluation of clindamycin versus clindamycin plus tobramycin in the treatment of acute pelvic inflammatory disease.

Fifty-one hospitalized women were treated with either clindamycin phosphate alone (N = 23) or a combination of clindamycin phosphate plus tobramycin (N = 28) for community-acquired pelvic inflammatory disease. The overall regimen clinical success rates were 100 and 91% for the clindamycin/tobramycin and clindamycin-alone groups, respectively. Efficacy for patients with positive pre-treatment Neisseria gonorrhoeae cultures was eight of eight (100%) for the clindamycin/tobramycin group and seven of eight (88%) in the clindamycin-alone group. Of the total population studied, 15 of 51 (29%) had positive pre-treatment cultures for Chlamydia trachomatis, and a microbiologic cure was attained in 15 of 15 (100%). However, in two of 15 (14.5%), the patient did not return for the 6-week culture, although the culture at discharge from the hospital was negative. No serious adverse reactions were observed, although oral follow-up treatment with clindamycin had to be discontinued in one patient because of diarrhea, which resolved without additional therapy. The results suggest that intravenous clindamycin alone is a viable alternative to the use of clindamycin/tobramycin for women with acute pelvic inflammatory disease requiring hospitalization.