key: cord-0701646-r67p0mtk authors: Busson, Laurent; Van den Wijngaert, Sigi; Dahma, Hafid; Decolvenaer, Marc; Di Cesare, Lina; Martin, Agnes; Vasseur, Liesbet; Vandenberg, Olivier title: Evaluation of 10 serological assays for diagnosing Mycoplasma pneumoniae infection() date: 2013-03-26 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2013.02.027 sha: fbc2586454df58414b6940edfc78688e5dac5775 doc_id: 701646 cord_uid: r67p0mtk In this study, the performance of 10 serological assays for the diagnosis of Mycoplasma pneumoniae infection was evaluated. A total of 145 sera from 120 patients were tested. They were obtained from patients who were serologically positive for M. pneumoniae infection as well as from patients who were infected with micro-organisms that may cause interstitial pneumonia. The following assays were utilized: SeroMP IgM and IgG, SeroMP recombinant IgM, IgA and IgG, Liaison M. pneumoniae IgM and IgG and M. pneumoniae IgM, IgA and IgG ELISA Medac. The SeroMP Recombinant and Liaison assays both showed low IgM specificity, and crossreactivity was mainly observed in groups of patients with acute cytomegalovirus and Epstein-Barr virus infections. For IgA, the Medac assay was less specific than the SeroMP Recombinant assay. Discrepancies between the four tests were observed in IgG analyses, and due to the lack of a gold standard, 22 results were removed prior to determining the sensitivity and specificity. Therefore, the overall performance of IgG assays may be overstated; nevertheless, the SeroMP assay demonstrated a lack of sensitivity. The seroprevalence of IgG appears to be very low, raising concerns regarding whether the serological techniques can detect IgG levels over time. Serology remains a biological tool of choice for diagnosing M. pneumoniae infection, but improvement and standardization of the assays are needed, particularly for the determination of IgG. Mycoplasma pneumoniae is a leading cause of bacterial community-acquired pneumonia (Cillóniz et al., 2012; Strålin et al., 2006) , accounting for 15-20% of cases, and up to 40% in children. It may also be responsible for upper respiratory tract infections and extrapulmonary manifestations. This infection is endemic, with epidemic peaks occurring every 4 to 7 years (Bébéar, 2007) , as was observed in several European countries from 2010 to 2011 (Jacobs, 2012) . Laboratory diagnosis was previously performed with cultures of the organism, but this technique is slow and less sensitive than serological or nucleic acid amplification assays (She et al., 2010) . The nucleic acid amplification assays appear to be the most sensitive methods; however, those techniques cannot distinguish between asymptomatic and acute infections (Dorigo-Zetsma et al., 2001; Foy, 1993; Ieven and Goossens, 1997; Loens et al., 2003) . Serology still has a place of choice in the diagnosis of M. pneumoniae infection, but this must rely on the analysis of two coupled sera taken at 2-3 weeks. Serological diagnosis can confirm a recent infection when there is an apparition of the IgG or when there is a significant increase in IgG levels between the two sera because upon reinfection, IgM may not be present (Waites and Talkington, 2004) . Complement fixation has been replaced with a variety of commercially available techniques, specifically with the enzyme-linked immunosorbent assays (ELISA), which allow for a precise quantification of IgM, IgA or IgG (Bébéar, 2007) . The aim of this work is to evaluate the performance of 10 serological assays for the diagnosis of M. pneumoniae infection. A total of 145 sera from 120 patients (54 women and 66 men) were evaluated. Fifty sera were coupled, and the interval between the two sera collections varied from 5 days to 5 months. The average age of the patients was 23.63 years (median: 15.5 years). Sera were sorted into different groups (Table 1) The results were classified as negative, equivocal or positive, according to the cut-off values that were specified by the manufacturers. As there is no gold standard for M. pneumoniae serology, the sorting of the results into true or false positives or negatives was performed by matching the clinical data with the serological profile. If the interpretation was unclear, a paired sample was analyzed, if available. Despite our careful interpretation, some discrepant results could not be sorted and were not taken into account when calculating the performance of the assays. Of the 145 samples that were evaluated, this occurred for 3 IgM, 5 IgA and 22 IgG results. Table 2 shows the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) results for each assay. The PPV and the NPV are shown for informative purposes, as they depend on the disease prevalence in the population. For IgM, the specificity was recalculated after removing 25 sera with non-specific IgM results, as the Table 1 List of sera. Criteria high proportion of those sera (25/145) could have a negative impact on the calculation. Table 3 shows the 22 IgG results that could not be categorized as true or false positives or negatives. Five sera were in the non-specific IgM group, 2 were in the Chlamydia pneumoniae group, 1 was in the respiratory syncytial virus (RSV) group, 3 were in the influenza group, 4 were in the acute EBV infection group, 4 were in the acute cytomegalovirus (CMV) infection group, 2 were in the varicella zoster virus (VZV) group, and 1 was in the measles group. The group of sera that had a higher number of IgM false positive results from all of the assays (excluding the non-specific IgM group) included sera from patients with acute EBV infection; this crossreactivity has been previously described (Beersma et al., 2005) . Table 4 shows the mean, the standard deviation (SD) and the median values of the false positive results for IgM, omitting the group with nonspecific IgM results. Each assay had a sensitivity of 100% for the detection of IgM, IgA, and IgG, except for the detection of IgG using the SeroMP and SeroMP Recombinant assays, which had a sensitivity of 61.76% and 97.05%, respectively. The SeroMP test was used in our routine practice, and in our experience, IgG disappear sometimes over time, which could explain the low sensitivity of this test. For the SeroMP Recombinant assay, only one sample had a false negative result in a 32-year-old patient with an acute M. pneumoniae infection. The control serum obtained one week later showed a high IgG titer. The three other assays showed a positive IgG result in the first serum sample. The IgM tests had a higher specificity with the SeroMP and Medac assays compared to the two other tests, which can be partially explained by the lower number of false positive results that were obtained in the group of sera taken from patients with acute CMV infection. The Liaison and SeroMP Recombinant assays also produced a few more false positive results, distributed among the other groups of sera. When the group of non-specific M. pneumoniae IgM was taken into account the specificity of the assays is lower. The significance of those false positives IgM results could not be explained, but in 3 cases, these were obtained from patients undergoing polyclonal-based stimulation of the immune system, as confirmed by the numerous serological tests showing IgM perturbations. Three additional false positive samples were obtained from patients with Streptococcus pyogenes infections (positive for anti-streptolysin O or anti-Streptococcus deoxyribonuclease B); however, a clear link could not be drawn from these observations. With IgA, the SeroMP test appears to be more specific than the Medac test. False-positive results were obtained with the Medac test, mainly in the group selected for its non-specific IgM results. The IgG specificity is 100%, except for the SeroMP and Liaison tests, which produced a few false positive results very close to the cut off value. IgM usually appear within 1 week of an initial infection and can persist for months or years following infection. Thus, a positive IgM result does not always implicate an acute infection. Upon reinfection, the IgM response can sometimes be absent (Thacker and Talkington, 2000) . IgG generally appear 2 weeks after IgM. They could be considered as the most important parameter in M. pneumoniae serology because the serological diagnosis is confirmed upon apparition or significant augmentation of IgG between the acute-and convalescent-phase sera taken within 2-3 weeks. This is particularly important in reinfection cases, as the IgM response may be absent. IgA are produced in the early phase of the disease, rise quickly to peak levels and then decrease more rapidly than IgM or IgG. IgA are believed to be infrequently synthesized in children. In this work, 9 children less than 15 years of age were included in the group of serologically confirmed M. pneumoniae infections. Two 3-year-old children produced IgM, but not IgA, whereas the 7 others, including one 1-year-old child, produced IgA and IgM. The advantage of detecting IgA is that these antibodies usually appear in reinfections and thus could already help to suspect this state in an acute-phase serum sample that is positive for IgA and IgG without waiting for the results of the convalescent-phase serum that would show a rise in IgG levels (Table 5) . For financial and practical reasons, the determination of IgM and IgG alone is performed in our routine practice. Therefore, analysis of a convalescent-phase serum is required when an isolated IgG-positive result is obtained from acute-phase serum because this could incorrectly be interpreted as a serological scar whereas the patient is experiencing a reinfection. As mentioned, of the 145 sera that were included in this work, 22 IgG results could not be classified due to the discrepancies between the assays that are reported in Table 3 and due to the lack of a gold standard for M. pneumoniae serology. In all of the discrepant cases, the SeroMP Recombinant assay yielded a negative result. Such discrepancies in IgG were also observed by other authors (Beersma et al., 2005; Talkington et al., 2004) . The seroprevalence of IgG was determined in the groups of sera, excluding M. pneumoniae infection and non-specific IgM cases. With the 88 sera remaining, the seroprevalence was different depending on the assay used, and there was an agreement among all 4 assays for only one sample ( Table 6 ). The very low seroprevalence of IgG in general is very striking, particularly in patients over the age of 65 (9.09% for SeroMP and 0% for the 3 other assays, obtained from 11 samples from patients over the age of 65). These percentages are far from the 40-50% seroprevalence of IgG in adulthood and 60% over the age of 65, as described by Tuuminen et al. (2000) . These observations raise several questions: (1) are the serological assays able to correctly detect M. pneumoniae IgG in acute phases and over time; (2) are the cut-offs of these assays too high; and (3) are the M. pneumoniae IgG lasting over time? In this work, the performance of 10 serological assays for the diagnosis of M. pneumoniae was evaluated. For IgM, the SeroMP Recombinant and the Liaison tests obtained lower specificities, and cross-reactivity was mainly observed in sera from acute EBV and CMV infections. For IgA, the SeroMP Recombinant assay appears to be more specific than the Medac assay. The interest in determining IgA levels may be critical to early detection of reinfections because the IgM response may be absent. For IgG tests, the performances were comparable, except for the SeroMP test, which had several false negative results that led to a low sensitivity. However, 22 sera had to be excluded from the IgG results prior to the sensitivity and specificity calculations due to discrepancies between the assays, and thus, the performances may be overstated. These findings implicate the need for improving and standardizing the serological M. pneumoniae assays, particularly for IgG determination. A recent article reports the attempts to develop a blotting technique that improves the performance of the serological assays (Dumke et al., 2012) . Pathogenesis and laboratory diagnosis of Mycoplasma pneumoniae infections. Revue française d'allergologie et d'immunologie Evaluation of 12 commercial tests and the complement fixation test for Mycoplasma pneumoniaespecific immunoglobulin G (IgG) and IgM antibodies, with PCR used as the "Gold Standard Community-acquired pneumonia in outpatients: etiology and outcomes Molecular detection of Mycoplasma pneumoniae in adults with communityacquired pneumonia requiring hospitalization Optimized serodiagnosis of Mycoplasma pneumoniae infections Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory Mycoplasma pneumoniae: now in the focus of clinicians and epidemiologists Molecular diagnosis of Mycoplasma pneumoniae respiratory tract infections Limited utility of Mycoplasma pneumoniae and Chlamydophila pneumoniae for diagnosis of respiratory tract infections Etiologic diagnosis of adult bacterial pneumonia by culture and PCR applied to respiratory tract samples Analysis of eight commercial enzyme immunoassay tests for detection of antibodies to Mycoplasma pneumoniae in human serum Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum Prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae immunoglobulin G and A antibodies in a healthy Finnish population as analyzed by quantitative enzyme immunoassays Mycoplasma pneumoniae and its role as human pathogen The author acknowledges the serology team from the Porte de Hal Laboratory, and also Anne Dediste and Delphine Martiny for their comments and remarks.