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.

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.

Role of genital mycoplasmata and other bacteria in urolithiasis.

Urease-producing bacteria have been shown to affect the formation of infection stones by splitting urea into ammonia, bicarbonate and carbonate. An increase in alkaline pH results in urinary supersaturation of the ions. The increase in ammonia also causes injury to the urothelial glycosaminoglycan layer. Non-urease-producing bacteria have been speculated to form urinary stones. Midstream voided bladder urine and fractured stone nidus samples from 72 patients undergoing surgery for urolithiasis were cultured on specific media for genital mycoplasmata and on conventional media. Urine samples were obtained from a control group of 40 healthy subjects. Genital mycoplasmata and other bacteria were evaluated with regard to the composition of urinary stones. Compared with other origins of stones, the relation between isolation of Ureaplasma urealyticum and infection stone disease was statistically proven. Isolation of genital mycoplasmata was significantly higher in women than in men in the study group. The urinary stones comprised 84.7% calcium stones, 8.3% uric acid stones and 6.9% infection (magnesium ammonium phosphate) stones. Coagulase-negative Staphylococci, Escherichia coli, Corynebacterium spp., Enterobacterium spp. and U. urealyticum were cultured from stone samples. The results suggests that non-urease-producing bacteria, as well as urease-producing bacteria, may influence the formation of urinary stones.

Fatal encephalitis caused by Mycoplasma pneumoniae in a 9-year-old girl.

A case of fatal encephalitis in a 9-year-old girl is described. Serology showed high titre antibodies against Mycoplasma pneumoniae. In addition M. pneumoniae was detected in cerebrospinal fluid by polymerase chain reaction. Direct invasion of the central nervous system as opposed to a secondary immunologic reaction to a M. pneumoniae infection of the respiratory tract in the pathogenesis of encephalitis is discussed.

[Ureaplasma urealyticum--a new problem pathogen in neonatology]. / Ureaplasma urealyticum--ein neuer Problemkeim in der Neonatologie.

Some previous studies showed that Ureaplasma urealyticum is the most common germ that appears in the birthway of pregnant women and which is also frequently found in skin swabs and secretions of newborn and premature babies. The colonization of pregnant women by Ureaplasma urealyticum makes a premature birth more likely. Another factor of risk for a premature infant is a premature rupture of membranes for more than 24 hours which also makes an infection possible. There exists an association between pulmonary infection by Ureaplasma urealyticum and the development of a bronchopulmonary dysplasia especially for premature babies. According to our observations acute exacerbations of severe pneumonia can appear even after month. An attempt of therapy of pulmonary infection should be undertaken with erythromycin, if sensitive serotypes are present. In the case of erythromycin resistance chloramphenicol can be used but only under frequent controls of blood levels. We were able to observe rapid improvements with this effective therapy.

Perinatal mycoplasmal infections.

Maternal infections are known to play a major role in perinatal morbidity and mortality. Ureaplasma urealyticum and Mycoplasma hominis are sexually transmissible microorganisms associated with a number of pathologic conditions in the pregnant woman including chorioamnionitis and postpartum fever. They are rapidly gaining recognition as significant causes of perinatal infection, especially in infants born prematurely. This review summarizes current knowledge regarding the epidemiology, pathology, disease spectrum, and clinical manifestations of mycoplasmal and ureaplasmal perinatal infections and presents guidelines for proper diagnosis and treatment.

[Mycoplasma infections of the human genital tract]. / Mikoplazmennye infektsii genital'nogo trakta cheloveka.

It has been shown that genital Mycoplasma may cause local and generalized diseases particular in parturients and premature neonates. For better laboratory diagnosis of Mycoplasma-induced infections, it is necessary to organize the production of culture media and diagnostic kits.

[Detection of Mycoplasma infection in the postpartum period]. / Vyiavlenie mikoplazmennoi infektsii u zhenshchin v poslerodovom periode.

The comparative results of microbiological and serological testing of parturients with uneventful and complicated course of the postpartum period were presented. The data justified the etiological role of M. hominis and U. urealyticum in the development of postpartum complications.

[Mycoplasma and Ureaplasma infections in chronic genital inflammatory processes, abortion and infertility]. / Mikoplazmoz i ureaplazmoz pri khronicheskikh vospalitel'nykh protsessakh genitalii, nevynashivanii beremennosti i besplodii.

A total of 43 females including 13 with sterility, 23 with habitual miscarriage and 7 pregnant women with a history of chronic inflammatory diseases were examined by using various tools to detect their Mycoplasma and Ureaplasma infections. The females had been studied for bacterial and chlamydial infections. Forty of them turned out to be infected with M. hominis and U. urealyticum. In some cases, sanation led to the improvement of the patients' status, the occurrence of pregnancy, its favourable course and termination in subjects with sterility and habitual miscarriage. The value of the findings is discussed in the paper.

Survey of genitourinary organisms in a population of sexually active adolescent males admitted to a chemical dependency unit.

Sixty-two males consecutively admitted to an adolescent chemical dependency unit for treatment over a one-year period were studied. When interviewed, all denied having urogenital symptoms. Forty-eight reported having had sexual intercourse. Of these 48, 34 were screened (urethral swab) for Neisseria gonorrhoeae, Chylamydia trachomatis, Ureaplasma urealyticum, and Gardnerella vaginalis. Eighteen of the 34 males who had complete screening were found to harbor one or more organisms: 3 had Chlamydia, 13 had Ureaplasma, and 11 had Gardnerella. In addition, 61 of the original 62 subjects were tested for serum antibodies to Treponema pallidum and for pyuria. No subjects were found to have syphilis. Pyuria was present in all subjects with Chlamydia but in only two with Ureaplasma or Gardnerella.

[Ocular findings in infection-linked immune phenomena and secondary diseases (the so-called Reiter's syndrome)]. / Okuläre Befunde bei infektionsassoziierten Immunphänomenen und Nachkrankheiten (sog. "Reiter-Syndrome").

Between 1981 and 1987 the authors saw 12 patients with Reiter's syndrome. Lesions included 6 cases of bilateral conjunctivitis in patients with chlamydial infection, 3 of unilateral serofibrinous iridocyclitis in patients with Yersinia enterocolitica, 1 case of bilateral iridocyclitis in a patient with positive chlamydial complement-binding reaction, 1 case of bilateral follicular conjunctivitis following acute gonococcal urethritis, and one case of unilateral serofibrinous iridocyclitis, in a patient with Ureaplasma urealyticum. Immunohistologic work-up of the conjunctival biopsy from the patient with Urea-plasma urealyticum urethritis revealed IgM deposits in the vascular endothelium of conjunctival vessels, C-3 deposits in the conjunctival stroma, and intercellular IgG in the conjunctival epithelium. Since Reiter's syndrome is interpreted as a sequela of secondary immune diseases following a primary infection that may persist in HLA-B27-positive patients, the patients were treated with both topical and systemic anti-infectious and anti-inflammatory agents.

Comparison of the incidence of Ureaplasma urealyticum in infertile men and in donors of semen.

569 infertile patients and 75 fertile men (donors of semen) were included in our study from 1985 to 1987. We compared the frequency of Ureaplasma urealyticum in semen specimens in these two groups: 40 infertile men (7%) and 4 donors of semen (5.3%) had U. urealyticum in semen cultures. This difference was not statistically significant. We concluded that U. urealyticum was not more frequent in infertile than in fertile men. We also report the results of semen cultures for other bacteria and the parameters of routine semen analysis in these two groups. All infertile patients infected by U. urealyticum were treated with doxycycline: the infection was eradicated in 77.5% of them.

Septic osteomyelitis and polyarthritis with ureaplasma in hypogammaglobulinemia.

We describe a hypogammaglobulinemic woman with a one-year history of destructive septic osteomyelitis and polyarthritis with positive cultures for Ureaplasma urealyticum from joint exudate and blood. The clinical course was complicated by subcutaneous abscesses from which both U. urealyticum and Mycoplasma hominis were grown. Multiple routine cultures had been negative, except for sporadic findings of Staphylococcus epidermidis before specific cultures for mycoplasmas were performed. Therapy with beta-lactam antibiotics, clindamycin, rifampicin, fusidic acid and aminoglycosides had been given without obvious clinical effect. Intravenous doxycycline treatment instituted after microbiological diagnosis had a dramatic effect on the clinical course. The clinical suspicion of mycoplasma and ureaplasma as etiologic agents of orthopaedic infections in hypogammaglobulinemic patients is mandatory in order to perform appropriate cultures.

The genital mycoplasmas.

The smallest free-living, self-replicating organisms known, the mycoplasmas have been the subject of intense research. Of the 12 species that have been found in association with humans, Mycoplasma pneumoniae, M. hominis, and Ureaplasma urealyticum have been clearly shown to have pathogenic properties. The newly described M. genitalium may also have the ability to cause disease. The syndromes with which these organisms have been associated in the genital tract are reviewed, as well as methods of diagnosis and therapy.

Co-existence of N. gonorrhoeae and U. urealyticum in male urethra.

Eight hundred and forty male patients attending sexually transmitted disease (STD) clinic for urethritis were investigated. Out of them, 31.6% had gonococcal urethritis, 16.1% suffered from nongonococcal urethritis due to Ureaplasma urealyticum and in 12.6%, both the organisms were present. Though 14.62% strains of N. Gonorrhoeae were resistant to penicillin, all the strains were sensitive to spectinomycin; while all Ureaplasma strains were sensitive to tetracyclines. As the treatment differs for these two organisms, it is necessary to identify the correct etiological agent.

PIP: The results of a study of 840 men aged 15-55 with urethritis attending a sexually transmitted disease clinic of a general hospital were that 31.6% had cultures isolated for N. gonorrhoeae, 16.1% for U. urealyticum, and 12.6% for both organisms. Bacteria were identified with the standard procedures of Finegold and Martin, and antimicrobial susceptibility testing was described by Cruickshank et al. The presence of microplasmas in patients with N. gonorrhoeae has been found in other investigations. Shepard et al. reported an association in 11.5% of patients compared with the 12.6% reported in this study, although other higher rates have been reported. There is morphological evidence that T strain mycoplasmas attach to cell wall projections on the surface of gonococci, and multiplication and viability are encouraged. Treatment involves penicillin or spectinomycin for gonococcal urethritis and tetracycline for U. urealyticum urethritis, thereby warranting differentiating the strains.