New LIAISON® AutOmAted ImmuNOASSAyS for the detection of MycoplasMa pneuMoniae Igg and Igm antibodies in human serum/plasma specimens

F. Capuano*, B. Grassi*, L. Pallavicini*, Z. Greenberg+, M. Lipson+. *DiaSorin SpA, Saluggia, IT; +Savyon Diagnostics Ltd, Ashdod, IL.

CLINICAL dIAgNOStICS metHOdS ANd ReSuLtS

PAIRed SeRA

SeROLOgy

CONCLuSION

Mycoplasmas are the smallest self-replicating organisms that are capable of cell-free existance (Figure 1).Due to the lack of a cell wall, mycoplasmas do not respond to penicillins and other beta-lactams used for the treatment of bacterial pneumonia and can be considered an elusive pathogen.Infection occurs worldwide, being endemic in most areas and cyclic epidemics of the virus are observed every 3 - 7 years, usually in the early autumn. The infection has an insidious onset with malaise, myalgia, sore throat or hea-dache and increasing chest symptoms by one to five days. Children aged 2 - 12 are infected more often than adults and infection sequelae are also influenced by age.As clinical findings are often insufficient to distinguish between Mycoplasma pneumoniae and pneumonia caused by other pathogens, correct etiologic determination depends on differential laboratory diagnosis.Culture is 100% specific but is time-consuming and relative intensive and complement fixation does not enable differentiation between antibody class. Due to this serology can be very helpful for clinicians to establish clinical status of the patient.

AGREEMENTS: A study was performed on 465 specimens (IgG) and 445 specimens (IgM) belonging to a population with signs and symptoms of atypical pneumonia, collected in different laboratories. The specimens were also tested by a reference ELISA method and consensus with additional serological data was applied to define the expected results for discrepant specimens (Table 3).

In addition, 49 paired samples were tested, collected at least two weeks apart from patients with recent Mycoplasma pneumoniae infection. Specimens were classified using LIAISON® Myco-plasma pneumoniae IgG and a commercially available EIA method for detection of Mycoplasma pneumoniae IgG.Diagnostic concordance on paired samples was 95.9% (47/49) - 95% confidence interval: 86.0-99.5%.

• 14 / 49 paired samples (28.6%) concordantly graded as negative

• 18 / 49 paired samples (36.7%) concordantly graded as current infection (range of LIAISON IgG titer ratios: from 3.3 to 210, MEDIAN: 33)

• 7 / 49 paired samples (14.3%) concordantly graded as past infection

• 2 / 49 paired samples (4.1%) discordantly graded past infection or negative Both assays classified the result as no current infection.

Following initial infection, the normal immune system responds by rapidly producing antibodies that peak after three to six weeks, followed by gradual decline over months to years. As a result of the long incubation period, antibody response is often evident by the time symptoms appear.Increased Mycoplasma pneumoniae-specific IgM levels alone can often be interpreted as evidence of acute infection, since this antibody typically appears within one week of the initial infection and approximately two weeks before IgG antibody.However, the presence of IgM is considered most significant in paediatric populations, where there have been fewer opportunities for repeated exposures. Adults who have been infected repeatedly over a period of years may not respond to mycoplasmal antigens with a brisk IgM response. In these cases, reinfection leads directly to IgG response; therefore, the presence of a negative IgM test does not rule out acute infection. When it does occur, the IgM response may persist for months or years following infection and in these cases a positive IgM test result may not reflect current or recent infection.Determination of IgG antibodies has proved necessary since patients may develop neither an IgM or an IgA response. The IgG antibody response is the last to appear and IgG levels remain elevated for at least one year after infection (Figure 2).

A significant increase of IgG concentration of two consecutive withdrawals allows the diagnosis of current or recent infection.The LIAISON® Mycoplasma pneumoniae IgG & IgM assays are the first fully automated solution for Mycoplasma pneumonia antibody detection, and uses chemiluminescence immunoassay (CLIA) technology for the semi-quantitative (IgG) and qualitative (IgM) determination of specific IgG antibodies to Mycoplasma pneumoniae in human serum or plasma samples.The interpretation of results are based on the combination of IgG and IgM antibody detection (Table 1-2).

The unique practical and technological advantages of the LIAISON® system have been combined to incorporate the best available choices of reagents and antigens.The LIAISON® Mycoplasma pneumoniae IgG uses a recombinant antigen against the 170-kDA p1 adhesion protein of Mycoplasma pneumoniae. (Figure 3).The LIAISON® Mycoplasma pneumoniae IgM, in addition to the P1 antigen, incorporates the whole-cell lysate. (Figure 4).

We have developed the first fully automated solution for Mycoplasma pneumoniae antibody detec-tion with specificity and sensitivity comparable to assays currently available on the market, for a new flexible approach to Mycoplasma pneumoniae diagnosis.

recombinant antigen

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Sample

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Sample Diluent

FIRST INCUBATION

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Conjugate 200μL

SECOND INCUBATION FIRST INCUBATION

Sample

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Stripping

PREDIL.

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recombinant antigen

Whole cell lysate

SECOND INCUBATION

Agreement With reference ELISA test On expected results

For positive 92.3% (96/104) 98.8% (323/327) results (95% confidence interval: 85.4-96.6%) (95% confidence interval: 95.5-98.6%)

For negative 89.5% (323/361) 94.2%(130/138) results (95% confidence interval: 85.8-92.4%) (95% confidence interval: 88.9-97.4%) Overall 90.1% (419/465) (95% confidence interval: 87.0-92.7%) -

For positive 97.0% (96/99) 97.8% (315/322) results (95% confidence interval: 85.4-96.6%) (95% confidence interval: 95.5-99.1%)

For negative 94.3% (313/332) 99.1%(110/111) results (95% confidence interval: 85.8-92.4%) (95% confidence interval: 95.0-100%) Overall 91.9% (409/445) (95% confidence interval: 87.0-92.7%) -

LIAISON®

Mycoplasmapneumoniae

IgG

LIAISON®

Mycoplasmapneumoniae

IgM

IgG IgM Interpretation of results (single bleed)

No evidence of infection. A negative result for IgM antibodies to Mycoplasma pneumoniae generally indicates that the patient has not been infected, but does not exclude the possibility of infection. Adults who have been infected repeatedly over a period of years may not respond to mycoplasmal antigens with a strong IgM response.

Test a second sample, collected two to four weeks later in parallel with the first sample.

Test a second sample, collected two to four weeks later in parallel with the first sample.

Negative Negative

Negative or Positive Positive

Positive Negative

Table 1

Table 3

Table 2

Figure 3: LIAISON® Mycoplasma pneumoniae IgG Figure 4: LIAISON® Mycoplasma pneumoniae IgM

IgG IgM Interpretation of results (paired sera)

A significant increased (three-fold or greater) of Mycoplasma pneumoniae IgG titer between two consecutive bleeds is considered suggestive of acute infection or re-infection even in the absence of IgM.

Stable or decreased Mycoplasma pneumoniae IgG concentration in the absence of IgM or in the presence of low IgM levels may indicate either recent or past infection.

Indication of acute infection.

x 3 + / – Significant increase

= /cc + / – LOW Stable or decreases absence or low levels

Negative +

Figure 1

Figure 2

21st ECCMID / 27th ICC 2011 - Milano, Italy 7-10 May 2011