An epidemic of Mycoplasma pneumoniae in Manitoba : A serological report

OBJECTIVES
To report an epidemic of Mycoplasma pneumoniae in Manitoba and to discuss the limitations of the serodiagnostic tests used.


DESIGN
A retrospective analysis of the results of a province-wide serological testing for respiratory infections caused by M pneumoniae, using a complement fixation test and an indirect immunofluorescent antibody test for the detection of immunoglobulin (Ig) M antibodies.


MATERIAL
From April 1, 1987, to March 31, 1991, 12,804 sera were tested and a serological diagnosis of recent M pneumoniae infections were established in 509 (3.97%). From April 1 to September 30, 1991, an additional 2088 persons were tested; the 158 (7.5%) recent cases of M pneumoniae were subjected to analysis.


RESULTS
Compared with the previous three years, an increase in the number of recent cases of M pneumoniae was first noticed in July 1990 which persisted until September 1991. Of 856 single sera tested, 59 (6.8%) were recent M pneumoniae infections and 56 (96.1%) of these were positive for IgM antibodies. Of the 616 persons who submitted paired sera, 99 (16%) were recent infections, but only 46 (46.4%) had IgM antibodies. Primary infections (ie, positive for IgM antibodies) were detected in 102 (64.5%) and reinfections (ie, positive complement fixation test only) in the remaining 56 persons with recent M pneumoniae infections. Primary infections were detected more frequently in the 'under 16' than in the 'over 16' year age group (75% versus 55.8% of the recent cases of M pneumoniae in each age group). Reinfections were more common in the older age group. Of the 158 recent cases of M pneumoniae, 30.3% had a pneumonia; of these, 21 (55.2%) were under the age of 16 years.


DISCUSSION
M pneumoniae is an important cause of morbidity. Serological tests are used for the diagnosis despite their limitations. The detection of IgM antibodies in acute serum establishes a diagnosis of primary M pneumoniae; however, their absence does not exclude M pneumoniae. A second (convalescent) blood test is required to diagnose all primary infections. To diagnose all reinfections, paired sera should be tested by complement fixation.


SUMMARY
Manitoba experienced an epidemic of M pneumoniae in 1990-91. Properly selected serological tests can provide a specific and rapid diagnosis.

occur worldwide and range in severity from asymptomatic to mild respiratory infections, atypical pneumonia and extrapulmonary complications.If diagnosed early, M pneumoniae infections respond well to treatment with tetracycline or erythromycin.There is no pathognomonic clinical feature .The specific diagnosis of M pneumoniae requires laboratory testing but there is no 'gold' standard as cultivation rarely is attempted (1).Traditionally, the diagnosis of M pneumoniae has relied on the complement fixation test (2) with theM pneumoniae antigen included in a battery of tests for respiratory viruses.For proper interpretation of results, the complement fixation test requires the simultaneous testing of acute and convalescent sera, thus delaying the diagnosis.Tests for cold agglutinins lack sensitivity and specificity (3) .Recently, a number of tests for the detection of specific immunoglobulin (Ig) M antibodies have been developed, eg, indirect fluorescent antibody (IFA-IgM).The specific IgM response has been studied (4); the detection of IgM antibodies in a single acute serum establishes an etiological diagnosis and thus allows treatment to be started earlier.
The present report on the serodiagnosis of M pneumoniae in Manitoba shows that an epidemic was ex- perienced in 1990-91.The limitations of serological testing are discussed.

METHODOLOGY
In Manitoba, the Cadham Provincial Laboratory is the sole provider of serodiagnostic testing for M pneumoniae and respiratory viruses, using a complement fixation test.In addition, an IFA test for the detection of IgM antibodies has been performed since 1987.The antigen used in the complement fixation test is an ether extract of mycoplasma organisms (Microbix Biosystems Inc) and the test is performed as standardized by the Centers for Disease Control (2).usually on paired sera.For the IFA-IGM test, a whole organism antigen (Behr• ing Diagnostics, Behringwerke AG , Marburg, Germany) is spotted on the slide and the conjugate is a sheep antihuman IgM (Wellcome Diagnostics, Dartford, Kent, United Kingdom).All positive sera are absorbed by Gull sorb (Gull Laboratories, Inc, Utah) to ensure the removal of all IgG antibodies and thus increase test specificity.The test is performed as previously described (5) on single, as well as on paired, sera.Criteria for the diagnosis of a recent M pneumoniae infection include a seroconversion, a fourfold rise in complement fixation antibodies, static high (at least 1: 128) complement fixation antibody levels or the presence of IgM antibodies.
Results of complement fixation and IFA-IgM tests were compared.In persons with serological indication of a recent M pneumoniae infection, the number of sera required to establish the diagnosis, the age of the patient (younger or older than 16 years) and available clinical information (ie, pneumonia or upper respir• atory tract infection) were recorded.

RESULTS
The number of sera tested each year, the number considered recent M pneumoniae infections and the basis for such a classification are recorded in the Cadham Provincial Laboratory annual reports and sum• marized in Table 1 for April 1987 to March 1991.Of the 12,804 persons tested, 509 (3.97%) were considered recent infections; of these, only 288 (56.5%) were posi• tive for IgM antibodies.
More cases were diagnosed in 1990-91 than in the entire previous three years.On average, one to eight recent cases of M pneumoniae were reported every month, reflecting the endemic level of M pneumoniae in Manitoba; the number of recent M pneumoniae infections started to increase in July 1990, peaked through the winter months , dropped slightly from April to June 1991, but increased again to a higher than average level until the end of September 1991 (Figure 1).
Three points were of particular interest: a seroconversion was detected by IFA-IgM in 22 and by complement fixation in 44 persons; static high complement fixation antibodies were found in 53 persons who were negative by IFA-IgM testing; and in 24 persons, IgM antibodies were detected in both blood samples submitted.Of the 158 recent cases of M pneumoniae, only 72 (45.5%) were under the age of 16 years and of these 54 (75%) were positive by IFA-IgM.Of the 86 recent cases of M pneumoniae who were 16 years of age or older.only 30 (34.8%) were IgM-positive.
The clinical information given on the requisition indicated an upper respiratory illness in 60 (38%) of the 158 recent cases; of these, 31 (51.6%) were under the age of 16 years.A pneumonia was specified in an additional 48 persons (ie, 30.3%). of whom 21 (55.2%) were under the age of 16 years.

DISCUSSION
In 1990-91 , Manitoba experienced an increase in the number of recent M pneumoniae infections diagnosed by serological testing.As reviewed by Broughton (6), epidemic outbreaks occur at irregular intervals and can persist for several months, with the incidence of infection three to five times the level seen in other periods.Figure 1 clearly shows that an epidemic occurred in Manitoba in 1990-91 and persisted for over a year.
When it became obvious that Manitoba was experiencing an epidemic of M pneumoniae, questions were raised over the value of testing single versus paired sera, the age group more likely to be infected and the relevance of serological testing to the clinical diagnosis.In an attempt to address these issues, data collected from April 1 to September 30, 1991 , were analyzed.Despite the limitations inherent in any retrospective study.this analysis revealed some useful information.
The presence of IgM antibodies indicates a primary infection while their absence, in a person with seroconversion or a fourfold rise in complement fixation antibody titres, points to a reinfection (7).Reinfections are common (8).In the present study, primary infections  Both the complement fixation and IFA-IgM tests have their limitations.The specificity of the complement fixation text has been challenged; unspecific reactions are due mainly to the glycolipid antigen in current use.Specificity will be improved in the future when the 168 kDa antigen (9) becomes available commercially.Kenny et al (1) reported a sensitivity of 90% for the complement fixation when compared with cultures for M pneumoniae; in their comparison, low static complement fixation antibody was indicative of recent infections.In the present study, low static complement fiXations were not counted to avoid nonspecific reactions; thus the actual number of genuine cases of recent M pneumoniae was probably underestimated.
Static high complement fixation antibodies (ie, 1:128 or less) were included as recent cases because complement fixation antibodies may be high at the time of acute illness and fourfold changes in titre may occur in a short time interval (6).The complement fixation test plays an important role in the serodiagnosis of M pneumoniae despite the need to collect a second blood sample two to three weeks after the first one; without the complement fiXation test.56 (35.4%) of 158 recent M pneumoniae cases would have been missed.Sillis (7).reviewing the limitations of testing for IgM antibodies, reported that 40 of 50 persons (80%) with IgM antibodies detected by IFA were culture-positive and represented genuine primary infections.In primary infections, IgM antibodies appear seven to 14 days after the onset of illness, peak between days 10 and 30 and disappear after 12 to 26 days (4).Using an IgM capture enzyme-linked immunosorbent assay (ELISA).Vikerfors et al (l 0) found that on day 8, 40% of their study cases of M pneumoniae had detectable IgM antibodies while on day 10 post onset of illness, 75% were positive for IgM antibodies.Therefore, while the detection of IgM antibodies in an acute serum established the diagnosis of M pneumoniae, their absence does not exclude M pneumoniae.A second blood test is required to diagnose all primary infections.Both tests (ie, complement fiXation and a test for the detection of IgM antibodies) should be performed on the second blood sample to detect all reinfections as well as primary infections.
In summary, Manitoba has experienced an epidemic of M pneumoniae infections.Acute and convalescent sera must be tested by carefully selected serological tests to ensure the diagnosis of all recent primary infections as well as reinfections.M pneumoniae causes significant morbidity in all age groups.

TABLE 1
Number of cases of recent Mycoplasma pneumoniae infections diagnosed serologically on an annual basis fromApril1, 1987toMarch31, 1991 1) Number of cases ojMycoplasma pneumoniae irifection