Unusual Mycoplasma detection in PBMCs of patients with underfined fever.

BACKGROUND AND AIM Infections by Mycoplasma and Chlamydia represent a challenge for the clinician, because of the changing clinical expression and the potential pathological sequelae. The routine microbiological investigations do not always provide elements for definitive diagnosis. We retrospectively evaluated Mycoplasma and Chlamydia spp. in sera and in blood specimens from patients with fever as main symptom in the absence of other clinical or laboratory signs indicating the presence of direct or indirect causal organism. METHODS Serum and PBMC specimens were collected from 34 immunocompetent patients between the ages of 17 and 39 years (mean age, 28•1 years), who attended our outpatient clinic (years 2004-09) because of persistent fever, but pulmonary function test and radiography negative or not significant. Specific serum anti-Mycoplasma pneumoniae and Chlamydia Spp antibodies searched with commercial kits (M. pneumoniae IFA IgM/IgG, Delta Biologicals, Italy; Chlamydophila pneumoniae - Cp Quant IgA/G, Eurospital, Trieste, Italy), were based on M. pneumoniae FH strain (ATCC 15531) cultured in McCoy cells and on purified EB, for C. pneumoniae. For molecular techniques, PBMCs were preliminary screened by PCR for DNA Mycoplasma genus (MGSO/GPO1) and positive results were then analyzed with species-specific primers for human Mycoplasmas. C. pneumoniae and C. trachomatis, were investigated by n-PCR using primers able to amplify fragment 16s rRNA and MOMP genes. The specific products obtained, were confirmed by sequencing with ABI PRISM 3130 DNA Sequencer (Applied Biosystem,The Netherlands). RESULTS Mycoplasma or C. pneumoniae positive serology and or PCR were found in 4 (11.7%) and 9 (26.4%) patients respectively (Fig. 1). Among the patients with Mycoplasma infection, the most prominent symptom was low grade and persistent (for over 3 months) fever associated with fatigue (three cases) or fever with cough or sore throat (one case). These patients had a positive (IgM) serology (four, 11.7%) or PCR (three, 9%) with a positive concordance between both methods in three. M. pneumoniae DNA was found in two (5.8%), while M. hominis in one (2.9%). This last finding was also confirmed in follow-up specimens by two different species-specific primer copies. The sequence analysis also confirmed that the amplified products belonged to M. pneumoniae and M. hominis (Fig. 2). Of the 9 patients with C. pneumoniae positive finding, 6 displayed a positive IgG and IgA serology, while 7, DNA. Their symptoms were: persistent fever with chills and fatigue (two cases) and cough (one), fever alone (three) or associated with CMV infection (one), fever associated with myalgia and pneumonia (1 case, respectively). CONCLUSIONS In general, the distribution of Cp serology and Cp PCR was more abundant than Mp serology or Mp PCR results. These last however, were predominant in patients with fever and fatigue. This suggests that a low percentage of patients with unexplained fever and fatigue without respiratory symptoms may have systemic mycoplasmal infections. Fever is often problematical in absence of clinical or microbiological criteria. The finding of M. hominis DNA in sequential PBMCs should not be underestimated and the use of macrolide therapy may be appropriate.