Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia

Background & Objectives: Mycoplasma pneumoniae is the most important and common cause of community-acquired pneumonia (CAP). The conventional detection methods (culture and serology) lack sensitivity. PCR offers a better approach for rapid detection but is prone to carry over contamination during manipulation of amplification products. Quantitative real-time PCR (qRT-PCR) method offers an attractive alternative detection method. In the present study, qRT-PCR, PCR and serology methods were used to detect M. pneumoniae infection in cases of pneumonias and findings compared. Methods: A total of 134 samples consisting of blood (for serology) and respiratory secretions (for PCR and qRT-PCR) from 134 patients were collected. The blood samples were tested for IgG, IgM and IgA using commercially available kits. For standardization of PCR of M. pneumoniae P1 gene was cloned in pGEMTEasy vector. Specific primers and reporter sequence were designed and procured for this fragment. The qRT-PCR assay was performed to prepare the standard curve for M. pneumoniae positive control DNA template and detection in patient samples. Results: Of the 134 patients, 26 (19%) were positive for antibodies against M. pneumoniae. IgG was positive in 14.92 per cent (20) cases, IgM in 4.47 per cent (6) and IgA was positive in 5.22 per cent (7) cases. In the qRT-PCR assay 19 per cent (26) samples were positive. Of the 26 qRT-PCR positive samples, nine could be detected by serology. PCR was positive for 25 samples. An extra sample negative by PCR was detected by qRT-PCR. Thus, real-time PCR assay, PCR and serology in combination could detect M. pneumoniae infection in 43 patients. Interpretation & Conclusions: The study shows that 17 patients were detected by serology alone, 17 were detected by qRT-PCR only and nine patients were positive by both serology and real-time PCR. Of the 134 samples tested, 25 were positive by conventional PCR, but qRT-PCR could detect one more sample that was negative by PCR and serology. These results suggest that a combination of two or three methods may be required for reliable identification of CAP due to M. pneumoniae

Identification of an N-terminal 27 kDa fragment of Mycoplasma pneumoniae P116 protein as specific immunogen in M. pneumoniae infections

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma pneumoniae </it>is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of <it>M. pneumoniae </it>to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes.</p> <p>Methods</p> <p>A 609 bp fragment P116_(N-27),corresponding to the N-terminal region of <it>M. pneumoniae </it>P116 gene was cloned and expressed. A C-terminal fragment P1_(C-40),of P1 protein of <it>M. pneumoniae </it>was also expressed. Three IgM ELISA assays based on P116_(N-27),P1_(C-40)and (P116 _(N-27)+ P1_(C-40)) proteins were optimized and a detailed analysis comparing the reactivity of these proteins with a commercial kit was carried out. Comparative statistical analysis of these assays was performed with the SPSS version 15.0.</p> <p>Results</p> <p>The expressed P116_(N-27)protein was well recognized by the patient sera and was immunogenic in rabbit. P1_(C-40)of <it>M. pneumoniae </it>was also immunogenic in rabbit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116_(N-27),P1_(C-40)and (P116_(N-27)+ P1_(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, and there was a good correlation and association between them.</p> <p>Conclusion</p> <p>This study shows that an N-terminal fragment of P116 protein holds a promise for serodiagnosis of <it>M. pneumoniae </it>infection. The IgM ELISA assays based on the recombinant proteins seem to be suitable for the use in serodiagnosis of acute <it>M. pneumoniae </it>infections. The use of short recombinant fragments of P116 and P1 proteins as specific antigens may eliminate the risk of cross-reactions and help to develop a specific and sensitive immunodiagnostic assay for <it>M. pneumoniae </it>detection.</p

Quantitative and qualitative study of intestinal flora in neonates

ackground: In the neonatal period the developing intestinal barrier function provides a sub-optimal mucosal defense against infection. Establishment of the normal commensal micro-flora plays a vital role in this process. Aims: To determine aerobic and anaerobic bacteria by quantitative and qualitative methods from faecal samples of neonates. Settings and Design: A prospective study was carried out in two groups in a tertiary care hospital, Group A-comprised preterm infant and in group B-full term infants. Materials and Methods: Sixty two preterm infants with the weight 0.05). On day 21 there was a significant change in colonization of both bacterial sp (P < 0.05). Potential pathogenic aerobic bacteria dominate the intestinal flora of premature babies nursed in neonatal unit. There is a need to investigate interventions to offset this imbalance in gut micro-ecology of premature babies

Multiplex polymerase chain reaction of genetic markers for detection of potentially pathogenic environmental Legionella pneumophila isolates

Background & objectives: Genomic constitution of the bacterium Legionella pneumophila plays an important role in providing them a pathogenic potential. Here, we report the standardization and application of multiplex polymerase chain reaction (PCR) for the detection of molecular markers of pathogenic potential in L. pneumophila in hospital environment. Methods: Culture of the standard strains of L. pneumophila was performed in buffered charcoal-yeast extract agar with L-cysteine at p H 6.9. Primers were designed for multiplex PCR, and standardization for the detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water samples and 200 swabs were collected from the hospital environment. The bacterium was isolated from the hospital environment by culture and confirmed by 16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L. pneumophila isolates were studied using the standardized multiplex PCR. Results: The PCR was sensitive to detect 0.1 ng/μl DNA and specific for the two standard strains used in the study. Of the 45 hospital isolates tested, 11 isolates had four markers, 12 isolates had three markers, 10 isolates had two markers, nine isolates had one marker and three isolates had none of the markers. None of the isolates had all the five markers. Interpretation & conclusions: The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila