Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Novel PCR for Mycoplasma pneumoniae detection in specimens from patients with various types of respiratory infections(Sri Lanka College of Microbiologists, 2010) Wijesooriya, W.R.P.L.I.; Kok, T.W.; Perera, J.INTRODUCTION: M. pneumoniae is the causative agent of primary atypical pneumonia and causes 20-40% of community acquired pneumonia. Patients mount IgM and IgG antibody responses, which provide useful diagnostic markers. IgM antibodies are not always produced in adults upon reinfection. Specific IgG antibodies increase slowly during the course of illness. Hence, test interpretation needs paired-serum which is not user friendly. Use of molecular diagnostic methods will overcome these. OBJECTIVE: To develop novel PCR primers to detect M. pneumoniae. METHODOLOGY: New forward and reverse primers which exclusively amplify M. pneumoniae-DWk encoding P1 adherent protein were developed. Master mix consisted of distilled water, 25mM-Mgcl2, 10X-PCR-Buffer, 10mM-dNTPS, two primers (10-p.M-Mpn-S (0.50p.M), 10-y.M-Mpn-RS (0.50|iM)) and Taq-Gold (5U/pJ). Purified M. pneumoniae- DNA (M129-B7-ATCC-29342) (20pg/ I) was used to determine PCR sensitivity. Detection limit was expressed as M. pneumoniae-DNA copy number. Each test had positive and negative controls. Specificity of PCR was evaluated using blast search. In addition, specificity was checked in the laboratory by doing the M. pneumoniae PCR with S. pneumoniae, H. influenzae and S. aureus (common respiratory pathogens causing pneumonia) and no positive reactions were observed among them. RESULTS: Limit of detection of M.pneumoniae-PCR was 400 fg of DNA which is equivalent to 10 copies per45pl of reaction mix. Specificity of the designed primer sequences was 100% with GenBank blast search and no cross reactions were observed with other respiratory-pathogens. M.pneumoniae-DNfltwas detected in 52% (13/25) of sero¬logy confirmed (positive IgM +/ IgG seroconversion) cases. CONCLUSION: Novel M. pneumoniae PCR has a sensitivity of 52% when tested with serology confirmed cases and a specificity of 100% when tested against other common respiratory pathogens. Detection limit was 10 copies / 45 pi of reaction mix.Item The use of serology and PCR in the diagnosis of Mycoplasma pneumoniae(Sri Lanka College of Microbiologists, 2010) Wijesooriya, W.R.P.L.I.; Kok, T.W.; Perera, J.; Thilakarathna, Y.; Sunil-Chandra, N.P.INTRODUCTION: M. pneumoniae is the causative agent of primary atypical pneumonia. Patients mount IgM and IgG antibody responses, which are useful diagnostic markers. Tests for specific DNA by polymerase chain reaction (PCR) in respiratory samples offer rapid and highly sensitive detection for M. pneumoniae diagnosis. AIM: To determine the relationship between serology and PCR in the diagnosis of M. pneumoniae inpatients with respiratory tract infections and a control group. METHODOLOGY: Paired sera from 418 adult patients were enrolled (pneumonia - 97, acute bronchitis -183, acute pharyngitis -138). The control group consisted of 87 paired sera from patients without acute respiratory infections. Isotype specific (IgM, IgG) antibodies were tested by M. pneumoniae specific ELISA (IBL-Hamburg-Germany). PCR for M. pneumoniae DNA was done in respiratory samples of serologically positive and age and gender matched serologically negative patients. RESULTS: M. pneumoniae specific IgG was seen in 9.3% (9/97), 5.4% (10/183) and 1.5% (2/138) in patients with pneumonia, acute bronchitis and pharyngitis respectively. IgM was seen in 4.1 % (4/97) and 2.1 % (2/183) and 0% (0/138) respectively. Both IgM and IgG were observed only in patients with pneumonia (2.1% (2/97)). M. pneumoniae DNA was detected in 52% (13/25) of serology confirmed and 15% (4/26) of serology negative cases. CONCLUSION: M. pneumoniae specific DNA was detected in both serologically positive and negative cases. These discordant results showed that with M. pneumoniae infection, both serology and PCR tests should be performed to maximize diagnosis.Item Detection of M. pneumoniae DNA and specific antibodies in relation to duration of illness(Sri Lanka College of Microbiologists, 2009) Wijesooriya, W.R.P.L.I.; Kok, T.W.; Perera, J.; Thilakarathna, Y.; Sunil-Chandra, N.P.INTRODUCTION: M. pneumoniae is the causative agent of primary atypical pneumonia. Patients mount IgM and IgG antibody responses, which provide useful diagnostic markers. Tests for specific antibodies-and DMA amplification by poiymerase chain reaction (PCR) in respiratory samples are now widely used for this infection. The timing of specimen collection is the one most important component to influence test sensitivity, amongst other test parameters. AIM: To determine optimum sampling time for detection of M. pneumoniae specific IgG/IgM antibodies and DNA by PCR. DESIGN, SETTING AND METHOD: A prospective clinical study was carried out involving 418 adult patients in Colombo North Teaching Hospital, Ragama and Chest Hospital, Welisara. (Pneumonia -97, acute bronchitis - 183, pharyngitis - 138). M. pneumoniae specific IgG and IgM were tested in paired sera using ELISA kits (IBL-Hamburg-Germany). PCRfor M. pneumoniae DNA was done for serologically positive and serologically negative patients. Each positive result was analysed in relation to duration of illness. RESULTS: IgM was detected in 37.5% (3/8) of patients on days 1-10 , 37.5% (3/8) on, days 11-20 , 12.5% (1/8) days 21 -30 and 12.5% (1/8) days 31 -40 post onset of illness (poi). IgG was detected in 48% (11/23) of patients on days 11-20, 22% (5/23) days 21-30 poi. M. pneumoniae DNA was detected in 94% (16/17) during the first 15 days of illness. Three seronegative patients (3/4, 75%) were negative for M. pneumoniae DNA >15 days poi. CONCLUSION: IgM response, higher during the first 20 days of illness than IgG which was detected during days 11-20, post onset of illness. M. pneumoniae DNA was detected within the first two weeks of illness.Item Paired sera IgG test detects more Mycoplasma pneumonias infections than the single IgM test(Sri Lanka College of Microbiologists, 2009) Wijesooriya, W.R.P.L.I.; Kok, T.W.; Perera, J.; Thilakarathna, Y.; Sunil-Chandra, N.P.INTRODUCTION: M. pneumoniae is the causative agent of primary atypical pneumonia. Patients mount an IgM and IgG antibody response, which are useful diagnostic markers. The single serum test for IgM specific antibodies may be attractive for rapid laboratory diagnosis, due to delays or non-provision of the convalescent phase serum sample by patients. IgM antibodies are not always produced in adults upon reinfection. AIMS: To evaluate the diagnostic value of paired serum IgG testing compared to single serum IgM for diagnosis of M. pneumoniae infection. DESIGN, SETTING AND METHOD: A prospective clinical study was done involving 418 adult patients in Colombo North Teaching Hospital, Ragama and Chest Hospital, Welisara. {Pneumonia-97, acute bronchitis-183, pharyngitis-138). Control group-87 adults with no acute respiratory infections. M. pneumoniae specific IgG and IgM were tested in paired sera (taken 2-3 weeks apart) using an ELISA kit (IBL-Hamburg-Germany). RESULTS: Patients with >12 U/ml IgM response or IgG sero-conversion were considered positive for this infection. IgM response was detected in 27% (6/22) (4 - pneumonia, 2 - acute bronchitis) of the study population. IgG sero-conversion was detected in 64% (14/22) (9 - pneumonia, 10 - acute bronchitis, 2 - pharyngitis) and 9% (2/22) (2 -pneumonia) by both antibody types. In this study population, IgM specific antibodies were detected in 36% (8/22).There were no IgG responders in the control group but 2% (2/87) showed positive IgM response. CONCLUSION: Specific IgG testing with paired serum samples detect more cases of M. pneumoniae infection than the use of a single serum IgM test.