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.

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 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.

Problems associated with serotyping strains of Ureaplasma urealyticum.

We sought to identify problems associated with serotyping Ureaplasma urealyticum, a human genital tract mycoplasma. We examined the results of serotyping isolates from cases of nongonococcal urethritis and asymptomatic controls and found no indication of correlation between serotype(s) and pathogenic potential. Reproducibility in serotype determination was generally good, i.e., overall agreement of 83% between primary and secondary plating and 87% on multiple, secondary cultures. We examined the reasons for variation in reproducibility and also the selection of a minor antigenic component in a culture. We provide no evidence of changes in the antigenicity of an isolate. We have indicated the means for standardizing reagents and the interpretation of results and have suggested improvements in methodology to allow more objective serotype determination.

Ureaplasma urealyticum: subcultures invalid for antibiotic susceptibility tests.

We tested the antibiotic susceptibilities of 100 Ureaplasma urealyticum strains from 99 patients using a broth-disk method and two types of inocula: urine sediments and overnight broth subcultures of the sediments. Of the 100 ureaplasma-positive urine sediments tested, nine (9%) of the ureaplasmas were found resistant to all four tetracyclines. When overnight broth cultures were used as the inoculum, 54 (54%) were found to be resistant to all four tetracyclines, an increase in resistance of 45 (45%). Thirty-seven susceptible strains remained susceptible upon subculture. The nine resistant strains remained resistant. Loss of susceptibility was not related to the pH or titer of ureaplasmas in the urine sediment inoculum but was related to the pH and titers when subcultures were used as the inoculum. Results of cultures following treatment, available for 53 patients, showed that treatment successes and treatment failures were significantly related to antibiotic susceptibility tests done with urine sediments but not to those done with broth subcultures as the inocula. Because reliable susceptibility testing is essential for appropriate therapy for U. urealyticum infections, all factors influencing this test need to be recognized and defined.

Wound infections after cesarean section with Mycoplasma hominis and Ureaplasma urealyticum. A report of three cases.

Mycoplasma hominis and Ureaplasma urealyticum were isolated from the surgical wounds of three patients who developed endometritis and a wound infection after cesarean section. In all patients, aspiration of the incision yielded a cloudy serosanguinous exudate. Gram stain of the fluid revealed numerous white blood cells but no bacteria. All patients responded to antibiotic therapy and local wound care.

Attempts to produce gynaecological disease in grivet monkeys with Ureaplasma urealyticum.

Attempts were made to infect grivet monkeys with Ureaplasma urealyticum, including strains freshly isolated from patients with infection of the genito-urinary tact, laboratory reference strains and simian strains. The organisms were inoculated directly into the uterine tubes exposed at laparotomy, or through the cervical canal into the uterine cavity before endometrial curettage; but only colonisation, of up to 5 months' duration, was achieved, without evidence of inflammation in the genital tract or elsewhere, and without an antibody response.

Nongonococcal urethritis.

Nongonococcal urethritis is a frequent genital infection, in most cases caused by Chlamydia trachomatis or Ureaplasma urealyticum. Diagnosis requires demonstration of urethritis and exclusion of Neisseria gonorrhoeae infection. Preferred treatment is seven days of tetracycline hydrochloride or doxycycline, to both the patient and partners. Physical sequelae are infrequent but include epididymitis and Reiter's syndrome.

Urethral syndrome in women.

Dysuria remains one of the most common symptoms experienced by adult women. In the past, women with dysuria were generally classified as having cystitis or the acute urethral syndrome based on results of quantitative urine cultures. Recent studies indicate that this terminology is misleading and that the majority of dysuric women have infections with E. coli and S. saprophyticus detected by cultures of midstream urine. Urethritis caused by C. trachomatis or N. gonorrhoeae and vaginitis can also produce symptoms of urinary infection. Proper management of dysuric women requires evaluation for these illnesses and treatment directed at the specific cause of infection.

Acute pelvic inflammatory disease.

Acute pelvic inflammatory disease is one of the most important consequences of sexually transmitted infection. Of sexually active women in the United States, one million (or 1 per cent) develop the infection. The sequelae include infertility (10 per cent), ectopic pregnancy (5 per cent), chronic pain (15 per cent), and recurrent infection (25 per cent). Organisms that cause the infection include Neisseria gonorrhoeae, Chlamydia trachomatis, genital mycoplasma, and a wide variety of facultative and anaerobic bacteria. Prompt recognition and therapy are necessary to reduce the sequelae.

PIP: This discussion of acute pelvic inflammatory disease (PID) -- usually a spontaneous infection that occurs among sexually active, menstruating, nonpregnant women -- covers: pathophysiology; microbial etiology (gonorrhea, chlamydia, genital mycoplasmas, and aerobic and anerobic bacteria); epidemiology (number of sexual partners, age, IUDs, previous PID, previous gonorrhea, untreated male sexual contacts, and perihepatitis associated with PID); diagnosis (physical examination, laboratory examination, culdocentesis, examination of the male partner, cultures, and ultrasonography); treatment; and sequelae (recurrent PID, infertility, ectopic pregnancy, and pain). The majority of infections are caused by bacteria and a polymicrobial bacterial infection is common. Neisseria gonorrhea, Chlamydia trachomatis, and a wide variety of aerobic and anerobic bacteria are most frequently isolated from women with PID. Primary PID is usually and acute infection in which organisms ascend into the uterus and fallopian tubes from the cervix. Chronic active infections are unusual except in neglected cases and in Actinomyces infection, but sterile chronic inflammatory adhesions are common residuals of acute infection. Except for women who have an IUD in place or the 15% who have had uterine instrumentation, spontaneous PID is almost totally confined to women who are sexually active. There is a much higher PID rate among younger than older women. Women who use an IUD for contraception are at least 2-4 times more likely to develop PID than nonusers. Women who have had PID are twice as likely to develop the infection as those who have never had it. A history of a prior uncomplicated gonococcal infection is more common among women with PID than among women without disease. Untreated males with urethral N. gonorrhea and possibly with C. trachomatis infection are an important source of infection both for the initial and for recurrent episodes of PID. Abdominal pain is the most common symptom although the pain may be mild or even absent in at least 5% of patients with PID verified by laparoscopy. In patients who have overt PID, it is possible to establish the diagnosis with reasonable certainty by a combination of history, physical examination, Gram stain of cervical secretions, culdocentesis, and examination of the male sexual partner. Adequate treatment of salpingitis includes an assessment of the severity of the infection, administration of appropriate antibiotics, employment of other health measures, close patient follow-up, and treatment of the male sexual patner. 25% of women with 1 episode of salpingitis develop a subsequent episode.

Prostatitis and epididymitis.

Ascending spread of urethral pathogens may be the mechanisms of infection of the prostate and epididymis. Sexually transmitted organisms cause most epididymitis in young men. Although evidence is mounting to suggest that sexually transmitted organisms may cause prostatitis, data are insufficient at this time to base a therapeutic approach on this concept. The authors present approaches to the management of these two disorders.

Chronic orbital inflammatory disease: parasitisation of orbital leucocytes by mollicute-like organisms.

Chronic orbital inflammatory disease (COID) is usually considered non-infectious and idiopathic. Treatment is empirical, palliative, and may not prevent disease progression. COID occurs in isolation or in association with various systemic diseases. Exophthalmos may be an important presenting sign. Vasculitis, lymphoid infiltrates, and granulomas are common. Mollicute-like organisms (MLO) parasitising and destroying vitreous leucocytes are often found to cause human chronic uveitis when an appropriate search is made. Inoculation of these MLO into mouse eyelids produced chronic uveitis and exophthalmic orbital inflammatory disease. Mollicutes are cell wall deficient bacteria. Extracellular mollicutes cause human and animal diseases characterised by lymphoid infiltrates, immunosuppression, and autoantibody production. Intracellular morphologically similar bacteria are non-cultivable pathogens termed MLO. Identification is based on direct detection in diseased cells by transmission electron microscopy. MLO are cytopathogenic and detection is aided by the alterations they produce. MLO replace the cytoplasm, destroy the organelles, and alter the nucleus. This results in cell proliferation, destruction, and dysfunction. MLO parasitise lymphocytes, monocytes, and polymorphonuclear leucocytes. This report describes orbital leucocytes parasitised by MLO in three patients with isolated COID. Rifampicin treatment of MLO disease is discussed.

The potential of the ovine udder as a model to determine the pathogenicity of bovine ureaplasmas.

Various methods of inducing mastitis in the ovine mammary gland with two bovine ureaplasma strains were investigated. The most successful method was by inoculation of fresh broth cultures on two successive days, 24 h apart. Eight more bovine strains were inoculated by this means and three successfully infected the glands.

Affinity of microorganisms of the genus ureaplasma to the reproductive organs of cattle.

The purpose of this work was to define more precisely the role of Ureaplasma organisms in the aetiology of granular vulvovaginitis and balanoposthitis (GVVBP) of cattle. To contribute to this question the frequency and degree of infection with Ureaplasmas in two main groups of cattle was taken into account: (a) in cattle with symptoms of the mentioned disease, (b) in cattle without clinical symptoms. The samples of semen from 301 sires with symptoms of GVVBP and from 43 healthy sires as also vaginal mucus swabs from 96 cows with GVVBP and from 40 cows mated by the sire infected with Ureaplasma organisms and from 50 cows inseminated with semen which contained Ureaplasma organisms were taken for bacteriological examinations. The control group in relation to the above mentioned cows constituted of 22 heifers free from symptoms of GVVBP and neither inseminated nor mated naturally. It has been shown that on an average 78.1% of sires with pathological changes in the mucosa of the penis or prepuce and only 25.6% of healthy sires were infected with Ureaplasma organisms. The concentration of Ureaplasma organisms was also significantly higher in material obtained from sires with symptoms of the disease than in that from healthy animals. Ureaplasma organisms were demonstrated more frequently (72.7%) in cows with GVVBP than in cows without these symptoms (13.3%). Similarly, as in the material obtained from sires, in the material taken from cows with symptoms of the disease the concentration of Ureaplasma organisms was significantly higher than that in the material originating from the healthy cows. The obtained findings may indicate that Ureaplasma organisms play a role in the aetiology of GVVBP.