Detection of Mycoplasma pneumoniae by Two Polymerase Chain Reactions and Role of M. pneumoniae in Acute Respiratory Tract Infections in Pediatric Patients

Abstract

Mycoplasma pneumoniae and viruses in acute respiratory tract infections in children were studied during the winter of 1992-1993 in Antwerp, Belgium. M. pneumoniae was diagnosed in nasopharyngeal aspirates by culture and polymerase chain reaction (PCR). For this, amplification of a fragment of the PI adhesin gene in samples prepared by two methods was compared in two laboratories, and in one laboratory, a fragment of the 16S rRNA gene was amplified. The sensitivity of culture versus PCR was 61.5%. Provided a specific internal control is used, sample preparation by freeze-boiling combined with PCR for the PI gene and amplicon detection by visual inspection of the electrophoresis gel can be recommended, although maximal results are obtained after hybridization. M. pneumoniae was present in 0.5% of patients <2 years old and 6.9% of patients >2. M. pneumoniae was second to respiratory syncytial virus or detected equally in lower respiratory infections.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

“…Serological methods are insufficiently sensitive and require paired serum samples from the acute and convalescent phases of the disease, thus only allowing a retrospective diagnosis (Dorigo-Zetsma et al, 1999;Chia et al, 1988;Sillis, 1990;Fedorko et al, 1995). More rapid, higher sensitivity methods were therefore developed, one of them being the PCR for fragments of the P1 gene or the 16S rRNA gene (Dorigo-Zetsma et al, 1999;Tjhie et al, 1994;Ieven et al, 1996). In the last few years, real-time PCR methods for the diagnosis of M. pneumoniae have been described (Hardegger et al, 2000;Ursi et al, 2003;Templeton et al, 2003).…”

Portación faríngea de Mycoplasma pneumoniae en niños chilenos

“…Para el diagnóstico de M. pneumoniae mediante RPC se amplificaron 209 pb del gen de la adhesina P1, empleando los partidores descritos por Ieven y cols 11 . Para la extracción del ADN se centrifugó una alícuota de 0,4 ml de la muestra clínica a 13.000 r.p.m.…”

“…Por otra parte, la especificidad de la detección de IgM mediante ELISA varía ampliamente entre 25 y 90%, dependiendo del kit comercial utilizado 3,4 . La amplificación del ADN mediante RPC ha sido señalada en algunos estudios como una alternativa sensible y específica para la detección de M. pneumoniae [8][9][10][11][12][13][14] . No obstante, se ha informado portación y persistencia de este microorganismo en la faringe luego de finalizado un cuadro respiratorio, lo que podría limitar la utilidad de esta técnica [1][2][8][9][10]15 .…”

Genotypic Methods for Diagnosis of Mycoplasmal Infections in Humans, Animals, Plants and Cell Cultures

“…PCR primers designed to amplify M. pneumoniae 16S rRNA such these described by van Kuppeveld et al (1992) may cross-react with M. genitalium when present at high level (>104 cfu/ml). A reliable PCR assay for the detection and differentiation of these mycoplasmas would combine the amplification of rRNA and of species-specific target such as the gene encoding the adhesin P1 (Ieven et al, 1996) from M. pneumoniae and MgPa from M. genitaliutn. Although genetic heterogeneity among M. hotninis strains is well documented, 16S rRNA gene has been successfully used to target specific amplification allowing the detection and the identification of this species (Christiansen etal., 1987;Grau et al, 1994).…”

“…One of the most common strategies consists of the use of DNAs from human or non-human mycoplasmas, from human DNA and DNAs from a variety of bacteria as negative controls. The identity of the product is routinely confirmed directly by three-primer PCR (Kai etal., 1993) or seminested (Fink et al, 1995) and nested PCR (Zigangirova et al, 1993) or secondarily by hybridization with a specific probe (Jensen et al, 1991;Palmer et al, 1991;Buck et al, 1992;Wang et al, 1992;Blanchard et al, 1993a,b,c;de Barbeyrac et al, 1993;Grau et al, 1993;Vekris et al, 1995;Berg et al, 1996;Ieven et al, 1996;In Chingbingyong and Hughes, 1996) and/or restriction fragment analysis (Jensen etal., 1991;Palmer et al, 1991;de Barbeyrac et al, 1993;Williamson et al, 1994). Experiments conducted in the authors' laboratory lead them to recommend the hybridization assay since nested PCR may yield false positive signal and three-primer PCR is dependent upon amplification conditions which may vary from one laboratory to another.…”