Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract.

Mycoplasma genitalium, an organism first isolated from the urethras of two men with nongonococcal urethritis, has been found in throat specimens from military recruits participating in an inactivated Mycoplasma pneumoniae vaccine field trial in 1974-1975. Four of 16 preserved throat isolates, previously identified as strains of M. pneumoniae, have now been shown to be mixtures of M. pneumoniae and M. genitalium. Purification of these mixed mycoplasmas by selection of single colonies confirmed the presence of M. genitalium. Identification of M. genitalium was based upon the occurrence of a species-specific 140-kilodalton protein adhesin in these isolates and their serologic reactivity to an M. genitalium antiserum. The frequent occurrence of both M. pneumoniae and M. genitalium in a number of these throat specimens, in combination with their shared antigenic cross-reactivities, suggests the likelihood that M. genitalium strains are easily missed in the usual laboratory identification procedures. What role M. genitalium may play in human respiratory disease remains to be determined.     

Analysis of eight commercial enzyme immunoassay tests for detection of antibodies to Mycoplasma pneumoniae in human serum

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.     

Immunoblotting patterns with Mycoplasma pneumoniae of serum specimens from infected and non-infected subjects.

Two hundred and ninety-four serum specimens from 248 subjects, whose complement fixation (CF) titres to Mycoplasma pneumoniae were known, were further investigated by IgG immunoblotting. After analysis of M. pneumoniae proteins by SDS-PAGE, nine polypeptides (p) with mol. wts of 180-43 Kda were selected for immunoblotting studies. Antibodies to M. pneumoniae measured by immunoblotting appeared progressively with age; most subjects more than 19 years old gave positive results. For most of the polypeptides, there was an increase in the frequency of band detection when the CF titres were higher. Furthermore, paired serum specimens from 10 patients with M. pneumoniae infection, as demonstrated by a rise in CF antibody titre, were tested for IgG blotting patterns. Generally, p180 (the P1 adhesin of M. pneumoniae), p172 and p84 were shown to be the dominant targets of the immune response to this organism and may have diagnostic value.     

Identification and characterization of immunogenic proteins of mycoplasma genitalium

Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.     

Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene

In order to develop a species-specific PCR for the detection of Mycoplasma genitalium, the sequence of 1,490 bases of the 16S rRNA gene was determined for M. genitalium G37 (type strain) and four Danish isolates of M. genitalium. The sequences of the four Danish strains, mutually different with respect to their MgPa gene, were 100% homologous, although they carried a single common base substitution compared to the type strain. Among members of the Mycoplasma pneumoniae phylogenetic cluster, M. genitalium showed the most-prominent homology to the 16S rRNA sequence of M. pneumoniae (98% homology). From regions showing the least homology to the M. pneumoniae 16S rRNA gene sequence, primers were chosen to amplify DNA from M. genitalium only. Two sets of primers were selected for their ability to detect <10 to 50 M. genitalium genome copies without cross-reactions with M. pneumoniae. The performance of these primers was compared to the performance of two pairs of primers amplifying parts of the MgPa adhesin gene; 1,030 randomly selected specimens submitted for Chlamydia trachomatis culture were screened with one of the 16S rRNA gene primer sets. A total of 41 specimens were found to be positive for this gene; 40 of these could be confirmed by one of the MgPa primer sets, whereas the other MgPa primer set detected only 21 positive specimens out of 40. These results indicate that estimates of the prevalence of M. genitalium in various populations using MgPa PCR primers could be incorrectly low if the PCR primers are located in variable regions of the MgPa gene.     

Antigenic determinants of the attachment protein of Mycoplasma pneumoniae shared by other pathogenic Mycoplasma species.

In previous studies with hyperimmune rabbit antisera, we found evidence of serologic cross-reactivity among Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. Because of certain biologic and morphologic similarities of these species, attempts were made to determine if this cross-reactivity related to the attachment protein (P1) of M. pneumoniae. Monoclonal and monospecific antibodies against P1 were used to probe proteins of the other species by immunoblotting. One of the P1 monoclonal antibodies was reactive with a smaller protein of M. genitalium; rabbit antiserum raised by immunization with P1 excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was found to react with a similar-sized protein of M. gallisepticum. These preliminary findings suggest antigenic sharing among the species examined; however, limitations of the methods used are discussed.     

Cross-hybridization between the cytadhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium and genomic DNA of Mycoplasma gallisepticum

Immunological cross-reactivity was observed between the cytadhesin proteins of Mycoplasma pneumoniae and Mycoplasma genitalium and a 155 kDa protein of Mycoplasma gallisepticum. Furthermore, the cytadhesin genes of M. pneumoniae and M. genitalium were used to demonstrate homology with M. gallisepticum genomic DNA under low stringency conditions suggesting that a family of adhesin-related genes exists among these pathogenic mycoplasmas.     

Absence of Mycoplasma pneumoniae cytadsorption protein P1 in Mycoplasma genitalium and Mycoplasma gallisepticum

Polyclonal and monoclonal antibodies to Mycoplasma pneumoniae protein P1 were nonreactive with whole-cell or soluble preparations of M. genitalium and M. gallisepticum. However, radioimmunoprecipitation performed with hyperimmune rabbit sera raised against each mycoplasma species indicated antigenic cross-reactivity between M. pneumoniae and M. genitalium.     

The use of enzyme-linked immunosorbent assay for detection of Mycoplasma hominis antibodies in infertile women serum samples

BACKGROUND: Besides Chlamydiae trachomatis and Mycoplasma genitalium, Mycoplasma hominis may also cause infertility due to damage of the Fallopian tubes. Therefore serum samples from infertile women were analyzed for antibodies to M. hominis. METHODS: Sera from 304 infertile women were investigated for seropositivity to M. hominis by immunoblotting and a developed ELISA. Women were classified into groups based on the type of infertility: infertile due to lack of passage in Fallopian tubes (TFI, tubal factor infertility), an infertile male partner (MFI, male factor infertility) and unexplained infertility (UFI, unexplained factor infertility). Three M. hominis isolates were used in the immunoblotting analysis and clear differences in patient immunoprofiles were observed between two isolates. For the ELISA we used a mixture of Triton X-114 extracted membrane proteins from those two M. hominis isolates as antigen. RESULTS: Ninety-seven sera (32%) were seropositive to M. hominis when tested by the ELISA. There was a significant correlation between TFI and seropositivity to M. hominis (P = 0.0015, OR = 2.21, CI = 1.35-3.61). We compared the seropositivity of 304 patients to M. hominis with the presence of antibodies against two other bacteria Chlamydiae trachomatis and Mycoplasma genitalium and there was no statistical correlation between those bacteria and M. hominis. CONCLUSION: Our results indicate that M. hominis may be an independent predictor of TFI.     

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.     

Analysis of Eight Commercial Enzyme Immunoassay Tests for Detection of Antibodies to Mycoplasma pneumoniae in Human Serum

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.     

Homologous regions shared by adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium

A lambda gt11 library of Mycoplasma genitalium genomic DNA was generated, and clones were identified using a pool of monoclonal antibodies directed against different epitopes of the 140 kDa adhesin protein. Because the 140 kDa protein of M. genitalium and the 170 kDa P1 adhesin of M. pneumoniae share biological properties such as a tip-associated location, cytadherence function and immunologic crossreactivity, we performed Southern blot analysis using these cloned partial 140 kDa gene fragments and 14 subclones that span the P1 structural gene of M. pneumoniae. Homologous regions of the two genes were identified.     

Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.     

Serum antibody responses in children with rotavirus diarrhea can serve as proxy for protection

We examined sera from 42 patients 1 to 30 months of age for rotavirus immunoglobulin M (IgM), IgA, IgG, and IgG subclasses and sought to determine if serum antibody could serve as a reliable marker for prediction of disease severity. Infants in the first few months of life usually had high maternal IgG titers and, when they were infected with rotavirus, had low IgM titers or no IgM in acute-phase sera and poor seroconversions 3 weeks later, suggesting that maternal antibodies had inhibited viral replication and antibody responses. All patients > or =6 months of age had IgM in acute-phase sera, indicating that IgM is a good marker for acute rotavirus infection. IgG was the best overall predictor of an infection, as the convalescent-phase sera of 81% of the patients had a fourfold rise in the IgG titer. IgA titers in convalescent-phase sera and conversion rates were higher among patients > or =12 months of age than among children younger than 12 months. IgG1 was the predominant subclass detected in the acute-phase sera of some children and in all 28 convalescent-phase serum samples examined. Patients with preexisting acute-phase IgG titers of > or =100 or > or =200 had diarrhea that was less severe or of a shorter duration. These results indicate that serum IgG is the most reliable marker for seroconversion and is a consistent proxy for protection against severe disease.     

Species-specific monoclonal antibody to a 43,000-molecular-weight membrane protein of Mycoplasma pneumoniae.

A murine immunoglobulin G1 monoclonal antibody was produced that binds to a protease-sensitive, periodate-insensitive epitope on a 43,000-molecular-weight Mycoplasma pneumoniae membrane polypeptide. The 43,000-molecular-weight polypeptide appeared to be a major antigenic component of M. pneumoniae, as determined by immunoblot analysis. This monoclonal antibody reacted with 33 different clinical isolates of M. pneumoniae, but not with normal-flora Mycoplasma species or 18 other microorganisms potentially inhabiting the normal or diseased human respiratory tract. This apparent species-specific monoclonal antibody may have application for the detection of M. pneumoniae antigen in clinical specimens.     

Isolation of Mycoplasma genitalium from first-void urine specimens by coculture with Vero cells

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe an improvement of the Vero cell coculture method in which the growth of M. genitalium was monitored by quantitative real-time PCR. Four new M. genitalium strains were isolated from six first-void urine specimens of male Japanese patients with urethritis. In two of them, only M. genitalium was detected: one also contained Ureaplasma urealyticum, and one contained Chlamydia trachomatis, Neisseria gonorrhoeae, U. urealyticum, and Ureaplasma parvum. In the specimens yielding isolates of M. genitalium, growth was documented by quantitative PCR after two to five passages in Vero cells. The complete isolation procedure from the initial inoculation to completion of single-colony cloning took about 1 year. Isolation of M. genitalium from urine specimens proved to be more difficult than from swab specimens. Due to the cytotoxic effect of urine, a procedure involving washing of the urinary sediment was introduced. Furthermore, prolonged storage of the urine specimens before culture was shown to be detrimental to the success of isolation, as shown by the lack of success in attempts to isolate M. genitalium from mailed urine specimens as well as by simulation experiments. High concentrations of penicillin G and amphotericin B were surprisingly inhibitory to the growth of wild-type M. genitalium strains, but penicillin G at 200 IU/ml and polymyxin B at 500 microg/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.     

Isolation of Mycoplasma genitalium strains from the male urethra.

Mycoplasma genitalium is a human mycoplasma species which, on the basis of detection by PCR, has been incriminated as a cause of nongonococcal urethritis. Previously, only two strains from the urogenital tract and five strains from extragenital sites have been isolated. We have developed a method for the isolation of this fastidious microbe. M. genitalium from PCR-positive urethral specimens was initially propagated in Vero cell cultures grown in serum-free medium supplemented with Ultroser HY serum substitute. Growth was monitored by PCR. The M. genitalium strains grown in cell cultures could subsequently be subcultured in modified Friis's FF broth medium. Several passages in broth medium were required before growth on agar medium was attained. A total of 11 urethral specimens positive for M. genitalium by PCR from male patients with urethritis were investigated. Six strains were adapted to growth in broth medium, and four of these strains were cloned. Three specimens were overgrown by other mycoplasmas during propagation in the cell cultures. In only two PCR-positive specimens was propagation of M. genitalium unsuccessful. The use of cell culture combined with PCR monitoring of mycoplasmal growth may prove to be more widely applicable for the isolation of other fastidious mollicutes.     

Naturally Acquired HMW1- and HMW2-Specific Serum Antibodies in Adults and Children Mediate Opsonophagocytic Killing of Nontypeable Haemophilus influenzae

The HMW1 and HMW2 proteins are highly immunogenic adhesins expressed by approximately 75% of nontypeable Haemophilus influenzae (NTHi) strains, and HMW1- and HMW2-specific antibodies can mediate opsonophagocytic killing of NTHi. In this study, we assessed the ability of HMW1- and HMW2-specific antibodies in sera from healthy adults and convalescent-phase sera from children with NTHi otitis media to mediate killing of homologous and heterologous NTHi. The serum samples were examined pre- and postadsorption on HMW1 and HMW2 affinity columns, and affinity-purified antibodies were assessed for ability to mediate killing of homologous and heterologous strains. Adult serum samples mediated the killing of six prototype NTHi strains at titers of <1:10 to 1:1,280. HMW1- and HMW2-adsorbed sera demonstrated unchanged to 8-fold decreased opsonophagocytic titers against the homologous strains. Each affinity-purified antibody preparation mediated the killing of the respective homologous strain at titers of <1:10 to 1:320 and of the five heterologous strains at titers of <1:10 to 1:320, with most preparations killing most heterologous strains to some degree. None of the acute-phase serum samples from children mediated killing, but each convalescent-phase serum sample mediated killing of the infecting strain at titers of 1:40 to 1:640. HMW1- and HMW2-adsorbed convalescent-phase serum samples demonstrated ≥4-fold decreases in titer. Three of four affinity-purified antibody preparations mediated killing of the infecting strain at titers of 1:20 to 1:320, but no killing of representative heterologous strains was observed. HMW1- and HMW2-specific antibodies capable of mediating opsonophagocytic killing are present in the serum from normal adults and develop in convalescent-phase sera of children with NTHi otitis media. Continued investigation of the HMW1 and HMW2 proteins as potential vaccine candidates for the prevention of NTHi disease is warranted.     

Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae and examined the conditions influencing the ability of an indirect immunofluorescence test to detect the specific antigen in respiratory exudates. The antibody did not cross-react with normal human serum or with respiratory exudates from 10 healthy persons. Cross-reactivity of the antibody with species of mycoplasmas other than M. genitalium was fully diminished when absorbed with horse serum and yeast extract, components of the culture medium. Though the absorbed antibody cross-reacted with M. genitalium, the titer was significantly lower than when tested against M. pneumoniae. Two types of antigen-specific fluorescence were observed in clinical specimens: one is large or small fluorescent granular aggregates found in mucus, and the other is fine fluorescent particles diffused on the entire surface of small epithelial cells. Throat smears from 49 patients with serologically confirmed M. pneumoniae infections were examined by our indirect immunofluorescence method. Positive results were obtained in 42 cases, many of which were positive before a rise in serum antibody titer could be demonstrated, indicating that the method is useful for a preliminary diagnosis at an early stage of the infection.     

Norwalk virus antigen and antibody response in an adult volunteer study.

To better define the optimum timing of specimen collection and identify alternate ways to diagnose Norwalk virus outbreaks, we looked at the timing of the antibody response and virus excretion in a human volunteer study. The Norwalk virus antibody titers and antigen in stool specimens were examined by biotin-avidin immunoassay. Our data suggest that in epidemic situations, convalescent-phase sera could be collected as soon as 13 days after the onset of illness and acute-phase sera could be collected as late as 5 days after onset. Our data also suggest that if sufficient serum samples are collected, convalescent-phase case and control serum samples can be used to identify Norwalk virus outbreaks. Antigen detection was much less sensitive than seroconversion for detecting infection.