Performance of Three Microimmunofluorescence Assays for Detection of Chlamydia pneumoniae Immunoglobulin M, G, and A Antibodies

The microimmunofluorescence (MIF) test is considered the “gold standard” for laboratory diagnosis of acute and chronic Chlamydia pneumoniae infection. The performance of a MIF test based on C. pneumoniae antigen from Washington Research Foundation (WRF) was compared with those of assays from Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute C. pneumoniae or Chlamydia psittaci respiratory tract infection confirmed by PCR or culture; group III, 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from patients with acute infections was acceptable (87 and 88%), and in sera from group III, it was excellent (95 and 97%). The determinations of endpoint titers were reproducible with <1 dilution step difference for all three methods, except that the mean IgM antibody titer found by the LAB assay was almost 2 dilution steps higher than that found by the other two methods. Although the three assays use different C. pneumoniae strains as antigens, the detection rates and IgG and IgA endpoint titers were similar. The difference in endpoint titers of IgM antibodies is of no major concern, as the diagnosis of acute C. pneumoniae infection rests on the presence of IgM antibodies, not on their level.     

Rapid fractionation of serum immunoglobulins by high pressure liquid gel permeation chromatography. Application to routine serologic procedures

High pressure liquid chromatography (HPLC) was applied to separation of IgM from IgG and IgA for the detection of virus-specific antibodies by routine serologic methods. Serum samples of 250 μl were fractionated on a HPLC protein column after filtration for use in rubella haemagglutination inhibition after preabsorption with kaolin. The protein fractions were examined for IgM, IgG and IgA content and for cross contamination.The relative recovery after kaolin absorption was satisfactory: IgM > 60%, IgG > 90%, IgA > 90%. The immunoglobulin M was well separated. IgG/IgA contamination of the fraction was < 0.2%/ < 0.5%. The IgM and IgG fractions were used without further treatment in haemagglutination inhibition. complement fixation and enzyme-linked immunosorbent assay.Highly specific results in the diagnosis of acute viral diseases (Rubella, Herpes zoster, cytomegalovirus) and Mycoplasma pneumoniae were obtained.     

Humoral and Cellular Immunity in Children with Mycoplasma pneumoniae Infection: a 1-Year Prospective Study

To determine whether children have persistent abnormalities in cellular and humoral immunity development after acute Mycoplasma pneumoniae infection, serum immunoglobulin G (IgG), IgA, IgM, and IgE levels and lymphocyte phenotypes were determined. There were no changes in the levels of IgG, IgM, IgA, or CD4⁺ or CD19⁺ lymphocytes that were measured in M. pneumoniae-positive patients after 3 months or after 12 months, but there were increases in these in M. pneumoniae-negative patients. Serum IgE increased in M. pneumoniae-positive patients. We have shown alterations in immunity development after M. pneumoniae infection.     

Diagnosis of Mycoplasma pneumoniae Pneumonia in Children

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of Mycoplasma pneumoniae infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for M. pneumoniae-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of M. pneumoniae DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of M. pneumoniae pneumonia in children of any age.     

Prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae Immunoglobulin G and A Antibodies in a Healthy Finnish Population as Analyzed by Quantitative Enzyme Immunoassays

Chlamydia pneumoniae and Mycoplasma pneumoniae immunoglobulin G (IgG) and IgA antibody seroprevalence rates and antibody levels related to age and gender were studied. The samples (n = 742) were collected during a nonepidemic period and analyzed by quantitative enzyme immunoassays (EIAs). Seroprevalence to C. pneumoniae was found to increase sharply in young children, and in the 15- to 19-year-old group it reached levels as high as 70 and 60% for IgG and IgA, respectively. After adolescence, seroprevalence showed a transient decrease and then continued to increase, although less dramatically than in early childhood. In the elderly the seroprevalence of IgG antibodies reached 75 and 100% in women and men, respectively. The corresponding rates of IgA antibodies were 73 and 100%. When a randomly selected subgroup of samples (n = 66) was analyzed in parallel by a microimmunofluorescence test and an EIA for C. pneumoniae IgA antibodies, similar seroprevalence rates were obtained (36 versus 35%). Seroprevalence to M. pneumoniae was already found to increase very sharply in 2- to 4-year-old children, reaching 16% for IgG and 8% for IgA. Seroprevalence to M. pneumoniae also continued to increase in adolescence, but in contrast to that to C. pneumoniae, the increase leveled off at about 40 to 50% in adulthood. In subjects aged over 65 years, prevalence did not exceed 60% for IgG or 35% for IgA. The seroprevalence patterns as well as the medians and variations of levels of C. pneumoniae and M. pneumoniae IgG antibodies were similar to those of corresponding IgA antibodies. Compared to IgG antibodies, IgA antibodies do not seem to be of additional value in the diagnosis of infections caused by these pathogens when single serum specimens are studied.     

Immunoglobulin levels in pneumonia

Serum immunoglobulin levels were measured by radial immunodiffusion in thirty patients with pneumonia and in thirty healthy controls. In pneumonia the mean IgG level increased during the course of the illness. During convalescence the mean IgM and IgG levels were higher in patients than in controls. The results of immunoglobulin estimations were correlated with the results of aetiological investigations. Patients with M. pneumoniae infections showed a significant rise in IgM concentration which may be specific for respiratory infections associated with this organism.     

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.     

Mycoplasma pneumoniae in children with acute and refractory asthma

BackgroundThe presence of Mycoplasma pneumoniae has been associated with worsening asthma in children. Sensitive assays have been developed to detect M pneumoniae–derived community-acquired respiratory distress syndrome (CARDS) toxin.ObjectivesTo identify the frequency and persistence of M pneumoniae detection in respiratory secretions of children with and without asthma and to evaluate antibody responses to M pneumoniae and the impact of M pneumoniae on biological markers, asthma control, and quality of life.MethodsWe enrolled 143 pediatric patients (53 patients with acute asthma, 26 patients with refractory asthma, and 64 healthy controls; age range, 5-17 years) during a 20-month period with 2 to 5 follow-up visits. We detected M pneumoniae using CARDS toxin antigen capture and polymerase chain reaction and P1 adhesin polymerase chain reaction. Immune responses to M pneumoniae were determined by IgG and IgM levels directed against CARDS toxin and P1 adhesin. pH was measured in exhaled breath condensates, and asthma control and quality of life were assessed using the Asthma Control Test and Pediatric Asthma Quality of Life Questionnaire.ResultsM pneumoniae was detected in 64% of patients with acute asthma, 65% with refractory asthma, and 56% of healthy controls. Children with asthma had lower antibody levels to M pneumoniae compared with healthy controls. Exhaled breath condensate pHs and asthma control and quality of life scores were lower in M pneumoniae–positive patients with asthma.ConclusionThe results suggest that M pneumoniae detection is common in children, M pneumoniae detection is associated with worsening asthma, and children with asthma may have poor humoral immune responses to M pneumoniae.     

Comparison of PCR, Culture, and Serological Tests for Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infection in Children

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.     

Comparison of PCR for sputum samples obtained by induced cough and serological tests for diagnosis of Mycoplasma pneumoniae infection in children

Passive agglutination (PA) and immunoglobulin M (IgM), IgA, and IgG enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Mycoplasma pneumoniae were compared with PCR testing of sputum samples obtained from children with lower respiratory tract infections. The sensitivity and specificity of PA were 80.3% and 92.3% at a titer of 1:80. ELISA was found to be less sensitive than PA.     

CHRONIC CHLAMYDIA PNEUMONIAE INFECTION AND BRONCHIAL ASTHMA: IS THERE A LINK?

Purpose: Besides well-defined environmental causes, accumulating evidence suggests that respiratory tract infections play an important role in the pathogenesis of asthma. Among these Chlamydia pneumoniae infection has been discussed as possibly inducing the development of asthma. Methods: This study was designed to investigate the presence of anti chlamydial IgG, IgA, and IgM antibodies by ELISA in serum samples of 60 adults with a clinical history of asthma and 100 healthy age and sex matched controls. All the samples positive for Chlamydial genus specific IgG antibodies were then subjected to Chlamydia pneumoniae species specific IgG antibody ELISA. Results: The IgG anti chlamydial antibody-positivity rate in the patients with bronchial asthma (80%) was significantly higher in all age groups than that in the healthy age and sex matched controls (59%). No significant association was observed for IgA and IgM anti chlamydial antibodies. C. pneumoniae species specific IgG antibody seroprevalence was also found to be significantly higher in all age groups in comparison to controls (61.66% vs 38%). Conclusions: Serological evidence of chronic infection with C. pneumoniae was more frequent in patients with asthma compared with control subjects. Our results support the correlation of bronchial asthma and chronic infection with C. pneumoniae in Indian population.     

Determination of IgG,IgM, and IgA antibodies toMycoplasma pneumoniae by an indirect staphylococcal radioimmunoassay

An indirect staphylococcal radioimmunoassay (SRIA) has been developed for determination ofM. pneumoniae antibodies. This test allows the detection of antibodies in various immunoglobulin (Ig) classes similar to the previously described radioimmunoprecipitation test (RIP). SRIA has two advantages over RIP: first, it uses 100-fold less anti-Ig reagents than RIP; second, bound can be separated from unbound antigen more easily by the relatively heavy staphylococci. SRIA antibodies, belonging to the IgA class of Ig, could be detected in nasal secretions of volunteers infected intranasally with ts H43 ofM. pneumoniae. In sera of patients withM. pneumoniae pneumonia antibodies to the IgG or the IgM class of Igs could be determined separately. This is especially important for an early diagnosis ofM. pneumoniae disease.     

Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of Mycoplasma pneumoniae in pediatric community–acquired lower respiratory tract infections

PurposeThe study was conducted to evaluate the role of Mycoplasma pneumoniae (M. pneumoniae) in children with community-acquired lower respiratory tract infections (LRTIs).MethodsSeventy five children aged 2 months ?12 years with community-acquired LRTIs were investigated for M. pneumoniae etiology employing paired serum samples to assay M. pneumoniae antibodies. Nasopharyngeal aspirates were obtained for the detection of M. pneumoniae by using polymerase chain reaction(PCR) and nested PCR.ResultsM. pneumoniae infection was positive in 24(85.71%) children aged <5 years and 4 (14.29%) ?≥ ?5–12 years and the difference was statistically insignificant (P ?= ?0.18). Difference in prevalence of M. pneumoniae infection across male and female groups was statistically insignificant (P ?= ?0.69). Clinical and radiological profiles across M. pneumoniae positive and negative cases were comparable except bronchopneumonia which was statistically significant (P ?= ?0.04). Serological evidence of M. pneumoniae infection was observed in 26(33%); PCR was positive in 9 (12%) and nested PCR in 10 (13.33%) children. Together, serology, PCR and nested PCR diagnosed M. pneumoniae infection in 28(37.33%) patients. Sensitivity of serology was 77.78%: specificity 68.18%; positive predictive value 25.00% and negative predictive value at 95.74%.ConclusionsSerological and molecular methods in combination is useful for detection of M. pneumoniae. Our data underline the role of M. pneumoniae in community-acquired LRTIs in children of all ages.     

Prevalence and Persistence of Chlamydia pneumoniae Antibodies in Healthy Laboratory Personnel in Finland

The rates of Chlamydia pneumoniae seroconversions suggesting acute primary infections or reinfections and the prevalences of antibodies were followed up among healthy laboratory workers. Annual serum samples were collected from 47 persons in Helsinki from 1958 to 1990 and from 40 persons in Oulu from 1994 to 1999. C. pneumoniae species-specific immunoglobulin G (IgG), IgA, and IgM antibodies were measured by microimmunofluorescence (MIF) in 407 sera from Helsinki. The 185 sera collected in Oulu were tested both by MIF and by commercial enzyme immunoassay (EIA). During the follow-up periods of 31 years in Helsinki and 6 years in Oulu, seroconversions were demonstrated by MIF in 45% and 15% of the study groups, respectively. In Helsinki 9% of the persons seroconverted twice during the follow-up period. By MIF, the total incidence rate per 100 person-years at risk was 6.9 in Helsinki and 4.9 in Oulu, and annual incidence rates varied from 0 to 15.4. By EIA, annual incidence rates in Oulu varied from 0 to 10.8. The seroconversions by MIF were usually not confirmed by EIA and vice versa. Prevalence and persistence rates, respectively, of IgA antibodies were higher in EIA (62% and 26%) than in MIF (26% and 17%), whereas the figures for IgG were quite similar. The prevalence of IgG and IgA antibodies was higher in older persons than in younger ones. The presence of antibodies did not offer protection from reinfection.     

Comparative Analysis of the Humoral Immune Response to Moraxella catarrhalis and Streptococcus pneumoniae Surface Antigens in Children Suffering from Recurrent Acute Otitis Media and Chronic Otitis Media with Effusion

A prospective clinical cohort study was established to investigate the humoral immune response in middle ear fluids (MEF) and serum against bacterial surface proteins in children suffering from recurrent acute otitis media (rAOM) and chronic otitis media with effusion (COME), using Luminex xMAP technology. The association between the humoral immune response and the presence of Moraxella catarrhalis and Streptococcus pneumoniae in the nasopharynx and middle ear was also studied. The levels of antigen-specific IgG, IgA, and IgM showed extensive interindividual variation. No significant differences in anti-M. catarrhalis and anti-S. pneumoniae serum and MEF median fluorescence intensity (MFI) values (anti-M. catarrhalis and antipneumococcal IgG levels) were observed between the rAOM or COME groups for all antigens tested. No significant differences were observed for M. catarrhalis and S. pneumoniae colonization and serum IgG levels against the Moraxella and pneumococcal antigens. Similar to the antibody response in serum, no significant differences in IgG, IgA, and IgM levels in MEF were observed for all M. catarrhalis and S. pneumoniae antigens between OM M. catarrhalis- or S. pneumoniae-positive and OM M. catarrhalis- or S. pneumonia-negative children suffering from either rAOM or COME. Finally, results indicated a strong correlation between antigen-specific serum and MEF IgG levels. We observed no significant in vivo expressed anti-M. catarrhalis or anti-S. pneumoniae humoral immune responses using a range of putative vaccine candidate proteins. Other factors, such as Eustachian tube dysfunction, viral load, and genetic and environmental factors, may play a more important role in the pathogenesis of OM and in particular in the development of rAOM or COME.     

Performance of three microimmunofluorescence assays for detection of Chlamydia pneumoniae immunoglobulin M,G, and A antibodies

The microimmunofluorescence (MIF) test is considered the "gold standard" for laboratory diagnosis of acute and chronic Chlamydia pneumoniae infection. The performance of a MIF test based on C. pneumoniae antigen from Washington Research Foundation (WRF) was compared with those of assays from Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute C. pneumoniae or Chlamydia psittaci respiratory tract infection confirmed by PCR or culture; group III, 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from patients with acute infections was acceptable (87 and 88%), and in sera from group III, it was excellent (95 and 97%). The determinations of endpoint titers were reproducible with <1 dilution step difference for all three methods, except that the mean IgM antibody titer found by the LAB assay was almost 2 dilution steps higher than that found by the other two methods. Although the three assays use different C. pneumoniae strains as antigens, the detection rates and IgG and IgA endpoint titers were similar. The difference in endpoint titers of IgM antibodies is of no major concern, as the diagnosis of acute C. pneumoniae infection rests on the presence of IgM antibodies, not on their level.     

Importance de la PCR dans la gestion d’une épidémie à Mycoplasma pneumoniae au centre hospitalier de Béthune (Pas-de-Calais)

Subject

Molecular amplification (PCR) provides adequate rapid and specific diagnosis of Mycoplasma pneumoniae infection (first agent responsible for community-wide bacterial pneumonia in children above 5 years of age).

Method

Positive (Chlamylège^®, Argène) PCR in nasopharyngeal aspirate, respiratory samples and nasopharyngeal swab and/or positive serological test (ELISA).

Results

Diagnosis of M. pneumoniae infection in 39 cases: 31 between September and December 2008 (30 children and one adult) and eight since June 2009 (three adults and five children). Children (mean age: 3.6 years) were hospitalized in 88.6% of cases, mean hospitalization duration was 2.9 days for respiratory tract infections, mainly due to lack of response to β-lactamines therapy (65.7%). Four adults (mean age: 29.5 years) presented a pneumonia, with hospitalization for three of them with one in intensive care unit. Twenty-eight PCR have proved positive (87%): without associated serology (13), eight negative serologies, IgG and IgM positive (five), and IgG alone (two). Seven patients had only serological test for diagnosis: IgM ± IgG. For two children, IgM positive only in isolation, with a PCR probably false negative.

Conclusion

The sensitivity of the serology in the diagnosis of mycoplasma infection is limited: IgM, which appear traditionally 1 week after clinical signs are mostly inexistent for adults and IgG rise at a later stage. Early diagnosis of child pneumoniae by PCR helped rapidly characterize this epidemic phenomenon and adapt the treatment.     

Titration of Igs contained in an intravenous IgM-enriched preparation against selected pathogens involved in sepsis

The goal of our work was to titer the IgG, IgM and IgA in Pentaglobin® (a preparation enriched in IgM), targeting specific surface antigens of Gram-positive and Gram-negative bacteria as well as a C. albicans strain. Lipopolysaccharides from Gram-negative bacteria, peptidoglycan and lipoteichoic acid from the other microorganisms were extracted and used in several ELISA assays in order to determine the titration of immunoglobulins in Pentaglobin® directed towards the aforementioned surface antigens. Our results showed an overall immunoglobulin titer of at least 10³ in Pentaglobin® with some exceptions for the IgA titers and for some immunoglobulin titers against E. faecalis, K. pneumoniae and P. aeruginosa. According to these results, Pentaglobin® can be considered as a potential adjuvant for antimicrobial therapy.     

Intrathecal Synthesis of Specific Antibodies in Patients with Invasion of the Central Nervous System by Mycoplasma pneumoniae

 Mycoplasma pneumoniae commonly causes respiratory tract infections in humans, but it may also be associated with central nervous system manifestations. The aim of the present study was to determine whether the cerebrospinal fluid taken from patients with neurologic symptoms due to Mycoplasma pneumoniae infection contains specific antibodies and whether the detection of these antibodies can be used for diagnosis. Mycoplasma pneumoniae was isolated from the cerebrospinal fluid taken from nine patients with central nervous system symptoms on admission to the hospital. In addition, Mycoplasma pneumoniae was detected in cerebrospinal fluid using polymerase chain reaction in four other patients. Antibodies to Mycoplasma pneumoniae were detected using the enzyme immunosorbent assay, indirect immunoperoxidase assay and immunoblotting in cerebrospinal fluid samples from 14 of 19 patients included in the study. The indirect immunoperoxidase assay showed high titers of Mycoplasma pneumoniae immunoglobulin G₁ (IgG₁) and IgM antibodies in cerebrospinal fluid samples of some patients with meningoencephalitis or meningitis. Titers of specific IgA, IgG₂ and IgG₃ antibodies were lower, while specific IgG₄ was not detectable. Cerebrospinal fluid samples with higher antibody titers also contained IgA, IgG₁, IgG₂, IgG₃ and IgM antibodies that recognized the P1 adhesin (170 kDa protein) of Mycoplasma pneumoniae. A comparison of antibody titers of concomitant serum/cerebrospinal fluid samples to Mycoplasma pneumoniae and those to measles virus by enzyme immunosorbent assay suggested the intrathecal synthesis of IgG and IgM antibodies to Mycoplasma pneumoniae in patients with acute meningoencephalitis. Data from this study clearly reinforce previous findings that Mycoplasma pneumoniae is an etiologic agent of central nervous system infections in humans.