Evaluation of four commercial immunoglobulin G (IgG)- and IgM- specific enzyme immunoassays for diagnosis of Mycoplasma pneumoniae infections

The four following commercially available enzyme immunoassays (EIAs) were assessed and compared for their performance in detecting Mycoplasma pneumoniae specific IgG and IgM antibodies: EIA-Platelia, EIA-Bmd, EIA-Sorin and EIA-Biotest. Three groups of patients were investigated: 39 patients (27 children and 12 adults) with respiratory infections and a M. pneumoniae PCR-positive in respiratory specimens (group I; 52 sera), 61 healthy children and adults (group II; 61 sera) and 20 patients with rheumatoid factor, antinuclear antibodies or positive antiviral IgM (group III; 20 sera). In group III, the IgM specificity for the EIA-Platelia, EIA-Bmd, EIA-Biotest and EIA-Sorin was 100%, 90%, 65% and 25%, respectively. In the children from group I, the four EIAs had similar IgM sensitivity (89 to 92%) but a striking difference in IgM sensitivity was observed in adult patients: 16% EIA-Platelia and EIA-Bmd, 50% EIA-Biotest, 58% EIA-Sorin. The sensitivity for IgG was greater with EIA-Bmd and EIA-Biotest, especially in detection of IgG in acute-phase serum : 61% EIA-Bmd and EIA-Biotest, 15% EIA-Platelia and 31% EIA-Sorin. Discrepant and unexpected results were observed in IgM detection from control healthy patients using EIA-Sorin and EIA-Biotest, confirming the lack of specificity of these two EIA-tests and making them inaccurate for routine diagnosis. A high IgG seroprevalence were found in healthy adults by the four EIAs (43-70%). In healthy children, EIA-Bmd and EIA-Biotest gave a higher IgG seroprevalence than EIA-Sorin and EIA-Platelia (45% each for the former as compared to 17% and 20%, respectively, for the latter).These results confirm that the IgM EIA serology test is a valuable tool for the early diagnosis of M. pneumoniae infections in children, as long as the EIA test used is specific. In adults, the difficult interpretation of EIA tests suggests that paired sera, combined with PCR detection on respiratory tract specimens collected on admission of patient, should be required for accurate diagnosis.     

Enzyme immunoassay for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae.

An enzyme immunoassay (EIA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this EIA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection.     

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.     

Importance of methodology in determination of Chlamydia pneumoniae seropositivity in healthy subjects and in patients with coronary atherosclerosis

Enzyme immunoassays (EIAs) for the detection of Chlamydia pneumoniae antibodies were compared to the microimmunofluorescence (MIF) test, the reference method. Furthermore, we assessed the hypothesis that a possible relationship between Chlamydia pneumoniae immunoglobulin G (IgG) antibodies and coronary artery disease is dependent on the type of EIA. Sera from 112 healthy men (mean age, 50.1 years) were tested for antibodies against Chlamydia pneumoniae by five commercial test kits: Focus Chlamydia MIF IgG test, Labsystems Chlamydia pneumoniae IgG EIA (LS EIA), R-Biopharm Elegance Chlamydia pneumoniae IgG EIA (RB EIA), Medac Chlamydia pneumoniae IgG sandwich enzyme-linked immunosorbent assay ELISA (MCp sELISA) and Medac Chlamydia IgG recombinant enzyme-linked immunosorbent assay ELISA (MC rELISA). Sera from 106 consecutive male patients (mean age, 63.6 years) undergoing diagnostic coronary angiography were also examined using the Focus MIF, LS EIA, MCp sELISA, and MC rELISA techniques. The agreement between LS EIA (65 to 83% [controls-patients]) or MC rELISA (49 to 61%) and Focus MIF (78 to 83%) was average to fair (kappa = 0.597 and 0.234, respectively). MCp sELISA and RB EIA showed good agreement with MIF (kappa = 0.686 and 0.665, respectively), with 80 to 89 and 79% of individuals reacting positively. A significant difference in seroprevalence between patients and healthy subjects was observed with the LS EIA, while seropositivities in the two study groups appeared equal when the Focus MIF assay was applied. The MC rELISA and MCp sELISA gave statistically significant differences in antibody seroprevalence in patients with two-vessel disease or when the patient group combined individuals with a two- or a three-vessel disease, respectively. The concordance between MIF and other commonly used serological assays for C. pneumoniae IgG antibody detection is good to fair. The choice of serological assay has important implications for C. pneumoniae antibody seroprevalence, as well as for the relationship between C. pneumoniae seropositivity and coronary artery disease.     

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.     

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.     

Evaluation of four commercial IgG- and IgM-specific enzyme immunoassays for detecting Mycoplasma pneumoniae antibody: comparison with particle agglutination assay

Diagnosis of Mycoplasma pneumoniae infection is important due to its variable clinical manifestations and absence of response to beta-lactams. Introduction of enzyme immunoassays (EIAs) for serologic diagnosis of M. pneumoniae has made it possible to separate the analyses of specific IgG and IgM antibodies. We compared four different commercial EIAs, ImmunoWELL IgG, IgM (GenBio), Medac IgG, IgA, IgM (Medac), Platelia IgG, IgM (Sanofi Pasteur), and Ridascreen IgG, IgA, IgM (r-Biopharm) with indirect particle agglutination assay (PA), Serodia-MycoII (Fujirebio). We tested 91 specimens from 73 pediatric patients (2-17 yr) hospitalized at a tertiary-care hospital between December 2005 and January 2006. The measurements of IgM EIAs were correlated with PA titers (Spearman's correlation coefficient, from 0.89 to 0.92) with high concordance rates, ranging from 82.4% to 92.3%. However, some negative IgM-EIA results in PA-positive specimens indicated that serial samplings with convalescent sera would be necessary to confirm M. pneumoniae infection.     

Comparative detection of measles and rubella IgM and IgG derived from filter paper blood and serum samples.

We compared the use of serum and filter paper blood spots as specimen sources for the detection of measles- and rubella-specific IgM and IgG. We collected capillary blood into microtainer tubes and onto filter paper spots from 60 children and 60 healthy adults. The blood was collected from 12-15-month-old children approximately 3 weeks after primary vaccination with measles, mumps, rubella vaccine, and the sample-pairs were tested for measles-specific IgM and IgG antibodies by using a capture antibody EIA and an indirect EIA, respectively. We tested sample-pairs from a subset of participants for rubella- specific IgM and IgG antibodies by using commercially available capture IgM (Captia) and indirect IgG (Wampole) assays. The concordance of results from serum and filter paper blood spots was high for all assays: 98% for measles IgM, 93% for measles IgG, 94% for rubella IgM, and 93% for rubella IgG, and increased to between 96-100% for all four assays when indeterminate samples were excluded. The correlation coefficients for EIA signals were 0.99 and 0.77 for measles IgM and IgG, respectively, and 0.92 and 0.94 for rubella IgM and IgG, respectively. The cut-off values used for filter paper samples were the same as those used for serum samples for all tests except for the rubella IgM assay. The use of filter paper blood spots is a promising future option for the detection of measles- and rubella-specific antibodies.     

Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The sensitivity of the EIA for the detection of recent rubella virus infection was evaluated by using 115 rubella-IgM-antibody-positive serum specimens, which were confirmed as positive by Rubazyme M (Abbott Diagnostics). In addition, 12 individuals, 2 of whom were exposed to rubella through vaccination and 10 of whom were exposed through natural infection, were studied, and the results were compared with those obtained by indirect EIA (Rubelisa M; Electro-Nucleonics, Inc.) and immunoblotting. The sensitivity of the newly developed EIA with sera from these individuals was 100%. Serum specimens from two patients indicated that the IgM antibodies were detected by the newly developed EIA at the same time as IgM antibodies were detected by immunoblotting and before positive reactions were detected by an indirect EIA. The reference population consisted of 564 healthy blood donors and hospitalized patients (150 serum specimens). In addition, 145 serum specimens commonly giving false-positive reactions in conventional rubella IgM EIAs were studied. With these specimens, no false-positive reactions were observed. Positive IgM responses, which could not be confirmed by immunoblotting, were observed in two samples from the reference population. However, these two samples were rubella IgG positive. The overall specificity of the EIA was 99.8%.     

Evaluation of 12 commercial tests and the complement fixation test for Mycoplasma pneumoniae-specific immunoglobulin G (IgG) and IgM antibodies, with PCR used as the "gold standard"

Serology and nucleic acid amplification are the main diagnostic tools for the diagnosis of Mycoplasma pneumoniae infection. Since no reference standard is generally accepted, serologic assays for M. pneumoniae have not been evaluated on a broad scale. In this study, 12 commercially available serologic assays (for immunoglobulin G [IgG] and IgM) and the complement fixation test (CFT) were evaluated by using M. pneumoniae DNA detection by real-time PCR as the "gold standard." The assays tested were Platelia EIA (Bio-Rad), SeroMP EIA (Savyon), Serion classic EIA (Virion/Serion), Biotest EIA (Biotest), Ridascreen EIA (r-Biopharm), AniLabsystems EIA (Labsystems), Novum EIA (Novum Diagnostica), Diagnosys EIA (MP products), Genzyme/Virotech EIA, ImmunoWell EIA (Genbio), ImmunoCard EIA (Meridian), and SerodiaMycoII microparticle agglutination (Fujirebio). Serum samples (n = 46) from 27 PCR-positive patients with a known first day of disease and sera (n = 33) from PCR-negative controls were obtained from prospective studies of acute lower respiratory tract infections. Additionally, control sera (n = 63) from patients with acute viral or bacterial respiratory infections other than those caused by M. pneumoniae were tested. The results showed low specificities for both the Novum and the ImmunoCard IgM assays. The IgM assays with the best performances in terms of sensitivity and specificity were AniLabsystems (77% and 92%, respectively), SeroMP (71% and 88%, respectively), and CFT (65% and 97%, respectively). Good receiver operating characteristic areas under the curve were found for CFT (0.94), the Platelia assay (0.87), and the AniLabsystems assay (0.85). We conclude that there are few commercial serologic assays for the detection of M. pneumoniae infections with appropriate performances in terms of sensitivity and specificity and that PCR has become increasingly important for the diagnosis of M. pneumoniae infections in defined groups of patients.     

Comparative evaluation of tests for detection of parvovirus B19 IgG and IgM

The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lübeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V‐IgM and ‐IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V‐specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non‐infected, and 29 from other viral recent infections (Epstein‐Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well‐established Biotrin EIAs.     

Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum

The Meridian ImmunoCard (IC), GenBio ImmunoWELL-IgM, and Remel EIA commercial antibody tests are qualitative enzyme immunoassays that detect antibodies to Mycoplasma pneumoniae in serum. These tests were compared to an M. pneumoniae complement fixation (CF) assay, which uses a commercially available antigen component. The Meridian IC and the ImmunoWELL-IgM detect immunoglobulin M (IgM) only; the Remel EIA and the CF test detect both IgM and IgG antibodies. Detection of specific IgM antibody, which appears early in infection, can be, but is not always, indicative of a recent or current infection. Paired serum samples from 64 adult patients with probable M. pneumoniae infection were examined with each of the four tests. Thirty (47%) of the 64 acute-phase sera were IgM positive by Meridian IC, 26 (41%) were positive by Remel EIA, 24 (38%) were positive by CF, and 15 (23%) were positive by ImmunoWELL-IgM. When both the acute- and convalescent-phase serum samples from each patient were examined, 61 (95%) of the 64 patients were positive by CF, 60 patients (94%) were positive by Remel EIA, 52 patients (81%) were IgM positive by the Meridian IC, and 29 patients (45%) were IgM positive by the ImmunoWELL-IgM assay. The Meridian IC was more sensitive than the other tests for early detection of IgM antibodies. However, after examining paired serum samples, we concluded that the detection of IgM alone may not be useful for all cases of mycoplasma infection, especially in an adult population.     

Human cytomegalovirus infection: diagnostic potential of recombinant antigens for cytomegalovirus antibody detection.

Recombinant antigen-based enzyme immunoassays (EIAs) for the detection of human cytomegalovirus (HCMV) specific antibody are believed to yield a higher sensitivity and specificity than virus lysate EIAs. The aim of the present study was to evaluate the accuracy of newly established HCMV assays (Copalis CMV Multiplex, Sorin; Cobas Core CMV IgG and IgM EIAs, Roche Diagnostics; Anti-HCMV recombinant IgG, gB-IgG, IgM and IgA, Biotest; and ETI-CYTOK-G PLUS and M reverse PLUS, Sorin) based on recombinant antigens and/or virus lysate for laboratory diagnosis of HCMV infection. For the assessment of sensitivity, follow-up samples from patients suffering from active HCMV infection were tested. Testing a large number of potentially interfering samples challenged the specificity of the assays. There was no statistically significant difference in the performance of HCMV IgG assays. The results were more heterogeneous for the detection of serological markers of active infection (HCMV IgM, HCMV IgA and anti-CM2). The sensitivities of the different assays ranged between 40.5 and 71.4%. A variable number (17.8-1.7%) of false-positive results were obtained among potentially interfering serum samples. Two of the recombinant antigen based assays showed a high degree of interference with EBV VCA-IgM-positive sera. The best performance was achieved with ETI-CYTOK-M reverse PLUS since it combined the highest sensitivity with specificity. Commercially available assays based on recombinant antigens showed, overall, a poorer performance than the virus lysate EIA.     

Evaluation of two commercially available immunoassays for the detection of hantavirus antibodies in serum samples.

BACKGROUND: hantaviruses are members of the family Bunyaviridae and the spectrum of clinical symptoms in humans may vary from sub-clinical to severe haemorrhagic fever with renal syndrome (HFRS) or pulmonary syndrome (HPS). Several serotypes have been described from which at least five are pathogenic to humans. Each serotype has a different animal reservoir and geographical distribution. In the acute phase of the disease the clinical diagnosis may be confirmed by serology or by polymerase-chain reaction (PCR). OBJECTIVE: to evaluate two commercially available immunoassays using sera from hantavirus suspected and non-hantavirus patients: an enzyme immunoassay (EIA) developed by MRL Diagnostics, for the detection of immunoglobulins M (IgM) and G (IgG) against several hantavirus serotypes and an indirect immunofluorescence assay (IFA) from Progen, based on slides coated with Hantaan virus (HNTV) and Puumala virus (PUUV), infected cells. STUDY DESIGN: a total of 145 serum samples were used for this study. The serum panel included serum samples from patients suspected of mild (n=91), severe (n=10) HFRS and patients with other viral infections (n=44). RESULTS: the agreement between the MRL EIA and the Progen IFA for the detection of IgM and IgG serum antibodies ranged from 87 to 91%, respectively. In the non-hantavirus group one out of 44 samples was positive by the Progen HNTV IgM IFA, none in the Progen PUUV IFA and two samples in the MRL IgM EIA, resulting in specificities of 98, 100 and 95%, respectively. The sensitivities and specificities of the MRL EIAs compared to the Progen overall PUUV and HNTV IFAs were 90 and 91% for IgM, respectively, and 96% for IgG in both immunoassays. CONCLUSIONS: the MRL EIA proved to be relatively sensitive and specific assay for the serological diagnosis of mild and severe HFRS.     

Measurement of sputum antibodies in the diagnosis of acute and chronic respiratory infections associated with Chlamydia pneumoniae.

The aim of this study was to develop methods for the measurement of sputum antibodies in the laboratory diagnosis of acute and chronic lower respiratory tract infections caused by Chlamydia pneumoniae. Paired serum specimens, sputum specimens, and pharyngeal or nasopharyngeal swabs were obtained from 97 patients; 51 of them had community-acquired pneumonia, and 46 had chronic obstructive pulmonary disease (COPD). C. pneumoniae-specific serum immunoglobulin G (IgG), IgA, and IgM antibodies were measured by the microimmunofluorescence (micro-IF) test. For sputa, specific IgA and IgG antibodies were measured by the micro-IF test and secretory IgA (sIgA) was measured by enzyme immune assay (EIA) with C. pneumoniae elementary bodies as the antigen. Sputum IgA and sIgA antibodies to C. pneumoniae were found, respectively, in 52 and 51% of the COPD patients. Elevated levels of stable serum IgG and IgA antibodies (IgG titer of > or = 128 and IgA titer of > or = 40), suggesting chronic infection, were found in 54% of the COPD patients. The sensitivity for the sputum IgA micro-IF test compared with elevated serum antibody levels was 87.5%, and that for the sputum sIgA EIA was 88%; the respective specificities were 90 and 95%. Acute C. pneumoniae infection was diagnosed in seven pneumonia patients, and two (29%) of these patients were positive by sputum EIA antibody measurements. Two pneumonia patients without acute infection had stable elevated IgG and IgA levels in their sera, and both of them were sputum antibody positive. We conclude that the measurement of IgA antibodies to C. pneumonia in sputum is a useful additional diagnostic tool for chronic C. pneumoniae infections.     

Mycoplasma pneumoniae infections: retrospective study in Basse-Normandie, 1997-2005. Epidemiology--diagnostic utility of serology and PCR for a rapid diagnostic

OBJECTIVES: The objective of this study was to evaluate the epidemiology of Mycoplasma pneumoniae (Mpn) infections in Basse-Normandie by a retrospective analysis of serological and PCR data, and to confirm the diagnostic utility of PCR and serology. METHODS: From 1997 to August 2005, 6156 serum samples and 6123 respiratory tract samples were collected from hospitalised patients and evaluated for the diagnosis of Mpn infection by PCR, serological assays, or by the two tests. During the epidemic period (2004-2005), the results of 1489 patients were analysed. RESULTS: Over the 9-y period, the seroprevalence was 40,4% and we reported on 525 cases with serologically or/and PCR proven Mpn infection, according a cyclic pattern spaced out 7 years. During the epidemic period, the seroprevalence increased to 50,2% and the rate of infections was 8.3%. The analysis of the 124 cases of Mpn infection showed typical epidemiological characteristics: a peak of incidence among the children and young adults, a summer-winter pattern and some coinfections with viral strains. For diagnosis of Mpn infection, the comparison of PCR and serological assays among 36 patients showed a concordance of only 41.7%. CONCLUSION: Mpn infections were endemic and outbreaks were observed according cyclic pattern with a high incidence specially in the children. Sensitive and specific tests were now available for early and reliable diagnosis. In children, the combination of the PCR on nasopharyngeal samples and the IgM EIA serology test were recommended. In adults, the PCR was privilegiated.     

Evaluation of a pan-reactive hantavirus enzyme immunoassay and of a hantavirus immunoblot for the diagnosis of nephropathia epidemica.

BACKGROUND: Nephropathia epidemica (NE) caused by the hantavirus serotype Puumala (PUUV) is endemic in large parts of Europe. The prognosis of this disease is usually good. However, a rapid serological diagnosis is important to differentiate NE from potentially more severe renal conditions. OBJECTIVE: To evaluate the diagnostic usefulness of a novel pan-reactive hantavirus enzyme immunoassay (EIA) and of a novel hantavirus immunoblot (IB). STUDY DESIGN: Three groups of serum samples were tested with both assays: 79 samples from 43 patients with acute NE, 27 samples from healthy adults, and 29 tricky samples from patients with autoantibodies, with acute Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infections, and from pregnant women. RESULTS: With the EIA, all but two of the early samples of the NE patients and all of the follow-up samples were positive for hantavirus IgG. All control samples were negative. The IgM EIA was positive in 42 of the 43 primary NE samples. Weak IgM EIA reactions were observed for some of the serum samples from patients with acute EBV and CMV infections. Optimal sensitivity and specificity values for the EIA were achieved when both the IgG and the IgM results were considered for the diagnosis of acute NE. All of the early NE samples reacted with the hantavirus nucleocapsid proteins in the IgG IB and all but one of these samples in the IgM IB. Cross reactions between the PUUV and the Hantaan antigens were very common. Several of the control samples did show borderline or positive bands, but these were mostly bands against only one hantavirus antigen in either the IgG or the IgM IB. The presence of at least three hantavirus bands (PUUV or HTNV) in the IgG and IgM assays was highly predictive of acute NE. CONCLUSION: Both assays were highly sensitive for the diagnosis of acute NE. However, the specificity of the IB IgM was only 76%. The specificity of both the IB and the EIA can be increased by modifications of the result interpretation.     

Evaluation of eight commercial tests for Mycoplasma pneumoniae antibodies in the absence of acute infection

Eight commercially available tests for Mycoplasma pneumoniae (Serodia-Myco II, Labsystems IgM and IgG EIA, IgM and IgG LISA tests, ImmunoWell IgG test and SeroMP IgM and IgG) were compared using 204 single sera from healthy individuals. IgM peaked in late childhood and then declined, while IgG rose progressively into adulthood. Inter-assay agreement was poor. Positivity in Serodia-Myco II and LISA IgG was associated with blood group or Coombs positivity, suggesting non-specific reactions. The study confirmed that single serum serology is unsuitable for the diagnosis of M. pneumoniae infection, and that commercially available tests need further improvement.     

Diagnosis of Oropouche Virus Infection Using a Recombinant Nucleocapsid Protein-Based Enzyme Immunoassay

Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.