Mycoplasma pneumoniae is not an important cause of lower respiratory tract infections in the European GRACE study: a combination of methods is necessary for early detection of cases

Ieven M.1, Lammens C.1, Loens K.1, Vanderstraeten A.1, Ursi D.1, Dettlaff S.2, Goossens H.1 and the GRACE study team1 University of Antwerp, Antwerp, Belgium; 2 Medac GmbH, Wedel, Germany

Objectives: Diagnosis of atypical bacteria such as M. pneumoniae (MP) is often based on serology, which may require up to 3 weeks for a definite result. PCR based assays offer earlier diagnosis; however often respiratory specimens are not available from patients in a primary care setting. The aim of this study was to evaluate the prevalence of MP infection and compare serology and real-time PCR on both sputum and nasopharyngeal flocked swabs (NPFS) for diagnosis of MP.

Figure 1: The 16 primary care networks involved in this study

This project is supported through Priority 1 (Life Sciences, Genomics and Biotechnology for Health) of European Union's FP6, Contract number: LSHM-CT-2005-518226

•This is the largest etiologic study on LRTI in primary care ever done. Prevalence of MP infection in the first winter period was low. •Detection of MP IgM antibodies in the early phase of LRTI is too insensitive. •A combination of IgM and PCR on sputum is the most sensitive method for early detection of a MP infection. •Bacterial loads are higher in sputum samples; these are therefore superior to NPFS for PCR detection.

Patients: From 10/2007 through 04/2010, a total of 3102 adult patients with LRTI in the community and 2984 controls were enrolled in a 3 year prospective study in 16 primary care networks PCNs in 11 European countries. (Fig. 1). Samples: Nasopharyngeal flocked swabs (NPFS, Copan, Brescia, Italy), sputa, and paired sera were collected and sent to the local laboratory to be frozen until transport to the central lab in Antwerp. Methods: Nucleic acids were extracted by using the NucliSEns EasyMAG (Biomérieux, France). On a subset of 374 patients from whom sputa were available in the first winter period, in-house real-time PCR for MP was performed (1) both on the sputum and NPFS. Sputa were scored according to the number of leucocytes (WBC) and squamous epithelial cells (SEC): specimens with ratios of WBC/epithelial cells ≥1 were defined as good quality sputa, ratios <1 were considered low quality sputa. IgG and IgM serology was performed using Mycoplasma pneumoniae-IgG/IgM-ELISA (Medac GmbH, Wedel, Germany) for detection of IgM or a IgG seroconversion or significant rise in anti MP IgG in sera collected 3-4 weeks apart; paired sera were available from 313 patients.

Correspondence: Greet Ieven, leader WP3, greet.ieven@uza.be Vaccine & Infectious Disease Institute (VAXINFECTIO) University Hospital Antwerp, Wilrijkstraat 10, B-2650 Edegem, Belgium

www.grace-lrti.org

OBJECTIVESOBJECTIVES

MATERIALS & METHODSMATERIALS & METHODS

RESULTSRESULTS CONCLUSIONSCONCLUSIONS

•MP was detected in 2.9% (9/313) of the studied adults with CA-LRTI by a IgG seroconversion or significant rise in IgG. (Table 1)•3/9 (33%) M. pneumoniae IgG positive patients were also found positive for M. pneumoniae IgM. •In 4/374 (1.1%) of the sputa MP was detected by PCR, with a mean Ct value of 19.62 (range 16.17-25.93)•1/374 (0.3%) was also positive in the NPFS with a Ct value of 34.27. •All patients positive by PCR were also positive by IgG seroconversion; 6/9 IgG positive patients were positive in the acute phase by either IgM or PCR.•PCR positivity in sputum was not related to sputum quality. (Table 2)

References1.Ursi, D., et al. 2003. Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control. J. Microbiol. Methods 55, 149-153.

Table 1. M. pneumoniae: IgG, IgM and PCR results

Table 2. M. pneumoniae: positivity in function of sputum quality

*AU/ml were calculated after 1:10 dilution of the serum.

Patient Nr Nr of days between sampling

IgG V1 (AU/ml)

IgG V2 (AU/ml)

IgG interpretation

IgM V1 (AU/ml)

IgM V2 (AU/ml)

PCR sputum (Ct)

1 37 223.6 (+) 683.6 (+) Significant rise 1.54 (+) 1.48 (+) Pos (25.93) 2 97 <9 (-) 24.7 (+) Seroconversion 1.07(±) 1.19 (+) Neg 3 27 <9 (-) 95.4 (+) Seroconversion <0.9 (-) <0.9 (-) Pos (18.88) 4 29 <9 (-) 108.3 (+) Seroconversion <0.9 (-) <0.9 (-) Pos (16.17) 5* 32 142.0 (+) 149.5 (+) High IgG

concentration in both sera

<0.9 (-) <0.9 (-) Neg

6 22 <9 (-) 33.2 (+) Seroconversion <0.9 (-) <0.9 (-) Neg 7 29 <9 (-) 38.8 (+) Seroconversion <0.9 (-) <0.9 (-) Neg 8 56 12.0 (+) 27.4 (+) Significant rise 1.42 (+) 1.39 (+) Neg 9* 29 238.0 (+) 227.6 (+) High IgG

concentration in both sera

2.03 (+) 1.96 (+) Neg

10 NA ND ND NA ND ND Pos (17.52)

Patient Nr PCR sputum (ct)

Sputum Nr WBC/microscopic

field

Sputum Nr SEC/microscopic

field

Sputum Nr RBC/microscopic

field

WBC>SEC

1 Pos (25.93) 0 2 0 No 2 Neg 5 1 0 Yes 3 Pos (18.88) 6 1 0 Yes 4 Pos (16.17) 4 3 0 Yes 5 Neg 4 1 0 Yes 6 Neg 4 0 0 Yes 7 Neg 1 1 1 Equal 8 Neg 2 1 0 Yes 9 Neg 0 1 0 No 10 Pos (17.52) 1 0 4 Yes