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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Mycoplasma pneumoniae DNA detection and specific antibody class response in patients from two tertiary care hospitals in tropical Sri Lanka(Microbiology Society, 2018) Wijesooriya, L.I.; Kok, T.; Perera, J.; Tilakarathne, Y.; Sunil-Chandra, N.P.PURPOSE: Respiratory tract infections are a major cause of global morbidity and mortality. Pneumonia is the ninth leading cause of mortality in Sri Lanka. Atypical pathogens cause about one-fifth of community-acquired pneumonia, while Mycoplasma pneumoniae accounts for about 50 %. This study aimed to determine the seroprevalence of M. pneumoniae respiratory tract infections in Sri Lanka while attempting to understand the relationships between the serology and PCR. METHODOLOGY: Paired sera from 418 adult patients (pneumonia, n=97; bronchitis, n=183; pharyngitis, n=138) and 87 healthy controls were studied. IgM, IgG and IgA antibodies were tested by M. pneumoniae enzyme-linked immunosorbent assay (ELISA). Positive IgM and or IgG seroconversion was considered to be seropositive. M. pneumoniae DNA were tested by PCR in age and gender-matched seropositives and seronegatives. RESULTS: M. pneumoniae IgG was in 14.4 % (14/97), 6.0 % (11/183) and 1.5 % (2/138) of pneumonia, bronchitis and pharyngitis patients, respectively, whilst IgM was in 6.2 % (6/97), 1.1 % (2/183) and 0 % (0/138), respectively. Amongst the pneumonia seropositives, 64.7 % (11/17) showed IgG alone, 17.5 % (3/17) showed IgM alone and 17.5 % (3/17) showed IgM and IgG. Amongst the bronchitis seropositives, 84.6 % (11/13) had IgG alone and 15.4 % (2/13) had IgM alone. In the pharyngitis seropositives, only IgG was detected 100 % (2/2). M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. In pneumonia or bronchitis patients, specific DNA was in 77.8 % (7/10) and 50 % (6/12) of patients, respectively. M. pneumoniae DNA was not found in pharyngitis patients. Of the seropositive PCR-negative pneumonia patients, 66.7 % (2/3) showed IgG alone and 33.3 % (1/3)showed IgM alone. In bronchitis patients, 83.3 % (5/6) showed IgG alone and 16.7 % (1/6) showed IgM alone. Of the seronegative PCR-positive patients, 16.7 % (2/12) had pneumonia and 18.2 % (2/11) had bronchitis. CONCLUSION: The serological evidence for M. pneumoniae infection in Sri Lanka comprised the following prevalences: 17.5 % (17/97), 7.1 % (13/183) and 1.4 % (2/138) in adults with pneumonia, bronchitis or pharyngitis, respectively. M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. IgG was predominant in PCR positives and negatives.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.Item Analysis of data of urine culture isolates of 2013 sent from four laboratories of National Laboratory Based Surveillance of Sri Lanka College of Microbiologists(Sri Lanka College of Microbiologists, 2014) Jayatilleke, S.K.; Karunaratne, G.K.D.; Perera, J.; Perera, R.R.D.P.; Wijesooriya, W.R.P.L.I.; Sunil-Chandra, N.P.OBJECTVES: To determine the aetioiogical agents of midstream urine cultures with a colony count of > 10 5CFU/ml. To analyse the antimicrobial susceptibility of those isolates. METHOD: The National Laboratory Based Surveillance on Antimicrobial Resistance is a collaborative project of the Ministry of Health and the Sri Lanka College of Microbiologists. At the initial phase decided to analyse midstream urine cultures with a colony count of >105 CFU/ml. The specimens were processed according to the standard protocol specified in the laboratory manual in microbiology. Antibiotic susceptibility tests were performed according to the method established in the centre which is either by CLSI method or by Stake's comparative disk diffusion method. Data of 2013 sent by the participating laboratories were analysed using WHONET software. RESULTS: The data was received from four centres. They were Sri Jayewardenapura General Hospital, Lady Ridgeway those isolates. ATotal of 1175 significant isolates were analysed. The majority were Gram negative enteric organisms, com¬monly known as coiforms, with 922 (78.5%) isolates. The others were Enterococcus species 83 (7%), Candida species 60 (5.1%), Pseudomonas species 38 (3.2%), Acinetobacter species 21 (1.8%), Group B beta-haemolytic Streptococcus 20 (1.7%), coagulase negative Staphylococcus species 10 (0.85%), Streptococcus species 9 (0.8%), Staphylococcus aureus 7 (0.6%), and Staphylococcus saprophyticus 5 (0.4%). The susceptibility of coliforms were 11.6% (92/795) to ampicillin, 71.1% (621/873) to nitrofurantoin, 25.9% (223/ 862) to cephalexin, 46% (392/853) to cefuroxime, 29.4% (255/866) to nalidixic acid, 47.8% (422/883) to cefo-taxime, 92.6% (665/718) to meropenem, 70.3% (601/ 855) to gentamicin, 41.6% (341/819) to amoxicillin-clavulanic acid and 38.4% (318/829) to ciprofloxacin. None of the 13 isolates of Acinetobacter species tested were sensitive to meropenem while only 55% (16/29) of Pseudomonas sp. were sensitive to meropenem. 74% (60/81) of Enterococcus species were sensitive to ampicillin. CONCLUSION: Coliforms constitute the commonest organism causing urinary tract infections (UTI). A high resistance rate was noted in coliforms for broad spectrum antibiotics like cefotaxime and ciprofloxacin. Acinetobacter sp. shows a very high resistance rate even for carbapenems. Ampicillin can be recommended as empirical therapy to treat UTI due to enterococcus species.Item Bordete//a pertussis specific Immunoglobulin G antibody levels among asymptomatic individuals aged 4-24 years admitted to two selected hospitals in Sri Lanka(Sri Lanka College of Microbiologists, 2015) Sigera, L.S.M.; Perera, J.; Samaranayake, D.; Ediriweera, E.P.D.S.INTRODUCTION: Pertussis continues to circulate in the community and cases among adolescents and adults have been increasing. Waning of pertussis-specific immunity following natural infection or immunisation may contribute to the persistent circulation. Even though it is not included in the extended programme of immunization in Sri Lanka, the booster doses including the adolescent booster dose of dTap, (acellular pertussis) are included into the list of recommended immunizations in several countries. Even though the protective titre yet not established, information on immunity to pertussis in this age group is needed before any vaccination policy can be considered. OBJECTIVES: To determine the antibody levels against pertussis toxin to determine the need and the optimal age for booster immunization. METHODS: The quantitative determination of specific IgG antibodies to Bordetella pertussis toxin was done by the ELISA using sera of 385 asymptomatic individuals aged 4-24 years admitted to surgical units of Lady Ridgeway Hospital, Colombo and Colombo South Teaching Hospital, Kalubowila. Mann-Whitney U test and Kruskal-Wallis test were used in analysis and p<0.05 was taken as significant. RESULTS: Median age was 12 years (IQR 8-19) with 212 (55.1 %) females. The median (IQR) anti PT antibody level was 3.31 lU/ml (0.73-15.12) and 352 (91%) had anti PT level <55 ID/ml. Median {IQR) anti PT levels were 3.18 ILJ/ml (0.591 -8.00) for 4-7 years, 1.43 Ill/ml (0.336-6.27) for 8-11 years, 4.28 lU/ml (0.978-13.39) for 12-15 years, 6.14 lU/ml (1.44-63.25) for 16-19 years and 4.89 lU/ml {1.11 -16.78) for 20-24 years and all of these difference were statistically significant (Spearman Correlation Coefficient P=0.0121). Females (p<0.003) and those having a sibling above 12 years (p=0.017) had significantly higher anti PT lev els. CONCLUSION: The majority of the study population, especially 8 to 11 years age group had very low anti PT IgG levels. The infection may occur in early adolescents, A booster dose of acellular pertussis vaccine could be considered.Item Analysis of data of urine culture isolates of 2014 sent from seven laboratories of National Laboratory Based Surveillance of Sri Lanka College of Microbiologists(Sri Lanka College of Microbiologists, 2015) Jayatilleke, S.K.; Patabendige, G.; Karunaratne, G.K.D.; Perera, J.; Perera, R.R.D.P.; Wijesooriya, W.R.P.L.I.; Sunil-Chandra, N.P.; Kottahachchi, J.; Athukorala, D.; Dissanayake, P.; Dasanayake, M.OBJECTIVES: To determine the aetiological agents of midstream urine cultures with a colony count of >105 CFU/ml. To analyse the antimicrobial susceptibility patterns of urine culture isolates of 2014. METHOD: The National Laboratory Based surveillance on antimicrobial resistance is a collaborative project of the Ministry of Health and the Sri Lanka College of Microbiologists. In this project midstream urine cultures with a colony count of >105 CFU/ml were analysed. The specimens were processed according to the standard protocol specified in the laboratory manual in microbiology. Antibiotic susceptibility tests were performed according to the method established in the centre which is either by CLSI method or by Stake's comparative disk diffusion method. Data of 2014 sent by the participating laboratories were analysed using WHONET 5.6 software. RESULTS: The data was received from seven centres. They were The National Hospital of Sri Lanka, Sri Jayewardenapura General Hospital, Lady Ridgeway Childrens' Hospital, Faculty of Medicine, Colombo, Faculty of Medicine, Ragama, Faculty of Medicine, Sri Jayewardenapura and North Colombo Teaching Hospital, Ragama. A total of 4441 significant isolates were analysed. The majority were Gram negative enteric organisms, commonly known as conforms, with 3975/4979 (79.8%) isolates. The others were Candida species 408, Enterococcus species 254, Pseudomonas species 194, coagulase negative Staphylococcus species 59, Staphylococcus aureus 36, Acinetobacter species 35 and Group B beta-haemolytic Streptococcus 18. The coliforms from adults who were attending outpatient clinics had 55.2% (112/203) susceptibility to cephalexin andcephradine, 54% (161/298) to amoxycillin/clavulanic acid, 65.1% (278/427) to nitrofurantoin, 48.3% (144/298) to norfloxacin, 63.4% (189/298) to cefotaxime, 97.4% (113/116) to imipenem and 100% (90/90) to meropenem. The adult inward patients had 39.5% (519/1313) susceptibility to cefotaxime, 87.9% (445/506) to meropenem, 62.6% (812/1298) togentamicin and 31.9% (405/1281) to ciprofloxacin. The coliforms from paediatric outpatients had 58.5% (69/118) susceptibility to cephalexin and cephradine, 58.5% (76/130) to amoxycillin/clavulanic acid, 80% (16/20) to nitrofurantoin, 85% (17/20) to cefotaxime and 89.7% (26/29) to meropenem. The paediatric inward patients had 64.6% (53/82) susceptibility to cefotaxime, 90.5% (19/ 21) to meropenem and 80.2% (65/81)togentamicin. CONCLUSION: Coliforms, the commonest organism causing urinary tract infections (UTI), had high resistance rate in in-wardpatients but the resistance was less in outpatients, especially in the paediatric age group.Item Bordetella pertussis serological profile among asymptomatic individuals aged 4-24 years.(Sri Lanka Medical Association, 2014) Sigera, L.S.M.; Perera, J.; Samaranayake, D.; Ediriweera, E.P.D.S.INTRODUCTION AND OBJECTIVES: To determine the antibody levels against pertussis toxin to determine the need and the optimal age for booster immunization. METHODS: The quantitative determination of specific IgG antibodies to Bordetellajiertussistox'm was done by the ELISA using sera of 385 asymptomatic individuals aged 4 -24 years admitted to surgical units of Lady Ridgeway Hospital, Colombo and Colombo South Teaching Hospital, Kalubowila. Mann-Whitney U test and Kruskal-Waliis test were used in analysis and p<0.05 was taken as significant. RESULTS: Median age was 12 years {IQR 8-19) with 212 (55.1%) females. The median (1QR) anti PT antibody level was 3.31 lu/ml (0.73-15.12) and 352 (91%) had anti PT level <55 lU/ml. Median {IQR) anti PT levels were 3.18 ILJ/ml (0.591-8.00) for 4-7 years, 1.43 lU/ml (0.336-6.27) for 8-11 years, 4.28 lU/ml (0.978-13.39) for 12-15 years,6.14 lU/ml (1.44-63.25) for 16-19 years and 4.89 lU/ml (1.11-16.78) for 20-24 years and this difference was statistically significant (p=0.000). Females (p<0.003) and those having a sibling above 12 years (p=0.017) had significantly higher anti PT levels. CONCLUSIONS: The majority of the study population, especially 8 to 11 years age group had very low anti PT IgG levels. The infection may occur in early adolescents. A booster dose of acellular pertussis vaccine could be considered.Item Behaviour of Cytokines IL10, IL6 and IFy during late febrile and immediate defervercent phases of Dengue(Sri Lanka Medical Association, 2014) Weerasinghe, O.M.S.; Premaratna, R.; Gomes, L.; Perera, J.; Silva, S.; Abeyratna, C.; Kasturiratne, A.; Malavige, N.; de Silva, H.J.INTRODUCTION AND OBJECTIVES: Cytokines have been implicated in dengue (DF) pathogenesis. Behaviour of cytokines during the late febrile phase (LFP) and immediate defeversence have not been studied, but may be useful to understand the pathophysiology of disease progression and effect of interventions. METHODS: A preliminary prospective study was performed to investigate 1L-10, IL6 and IFy (pg/ml) responses during the late febrile phase (around fifth day) and immediate defervestence in confirmed (NSlAg positive} dengue patients. Demographic, clinical and laboratory data were collected. Two samples of 1 m! serum were obtained during the above stages of the illness and stored at -80°C to assess cytokine levels. Cytokine levels were compared between phases (LFP and afebrile) and stages (DF, precritical and critical dengue shock syndrome (DSS)). LFP cytokine levels were compared for disease stages using one-way Anova test. RESULTS: 18 patients (11 males, mean age 26 years (SD 10.6)) were studied. There were 3 DF, 9 precritical and 6 criticaj DSS based on national guidelines. Mean temperature during LFP and defervescent phases were 102.07°F (SD 0.98) and 98.53°F (SD 0.26). Median (interquartile range) of IL10, IL6 and IFy in LFP were 164.3 (90.8 - 259.5), 26 (12.7-54.6), 246.5 (117.5-511.8) and during defervescent phase were 17.4 (6A-112.2), 11.9 (4.9-28.2), 2.58 (0.0-58.4) respectively. LFP IL10 significantly correlated with disease stages (F-3.99, P-0.041), IL6 and IFy had no correlation. CONCLUSIONS: All three cytokines rapidly declined with defervecence. IL10 in febrile phase showed significant correlation with disease severity.