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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty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 Detection, identification, and antimicrobial susceptibility of Campylobacter spp. and Salmonella spp. fromfree-ranging Nonhuman Primates in Sri Lanka(Wildlife Disease Association, 2019) Tegner, C.; Sunil-Chandra, N.P.; Wijesooriya, W.R.P.L.I.; Perera, B.V.; Hansson, I.; Fahlman, A.ABSTRACT: Infections with Campylobacter spp. and Salmonella spp. are the most frequently reported causes of human bacterial enteritis. Warm-blooded animals, including livestock, pets, and wildlife, can be carriers of the bacteria and may contaminate the environment and food products. The present study investigated the occurrence of Campylobacter spp. and Salmonella spp. in fecal pat samples from free-ranging toque macaques (Macaca sinica) and tufted gray langurs (Semnopithecus priam) collected in March-May 2015 in Sri Lanka. In 58 samples from toque macaques, Campylobacter jejuni was isolated in 10 (17%), Campylobacter coli in four (7%), and Salmonella enterica subsp. enterica serovar Virchow in two (3%). None of the bacteria were isolated in the 40 samples from tufted gray langurs. Pulse-field gel electrophoresis and multilocus sequence typing identified six profiles and four clonal complexes of C. jejuni. The isolated Campylobacter spp. showed varying susceptibility to antimicrobial substances. All Campylobacter spp. isolates were susceptible to chloramphenicol, erythromycin, florfenicol, gentamicin, and streptomycin. Four of the C. jejuni were resistant to at least one of the following: ampicillin, ciprofloxacin, nalidixic acid, and tetracycline, and one of the isolates was multidrug resistant. All four C. coli were resistant to ampicillin, whereas the two Salmonella Virchow strains were susceptible to all antibiotics tested. The presence of Campylobacter spp. and Salmonella spp. in toque macaques may have an impact on the conservation of endangered primates and public health in Sri Lanka. KEYWORDS: Campylobacter spp .; Antimicrobial resistance; PFGE; Salmonella spp; conservation; nonhuman primates.Item Enteric pathogens of zoonotic concern in non-human primates in Sri Lanka(European Wildlife Disease Association (EWDA, 2016) Tegner, C.; Sunil-Chandra, N.P.; Ingrid, H.; Perera, V.; Wijesooriya, W.R.P.L.I.; Fahlman, A.Zoonotic disease is a two-way street where humans and other animals are interchanging pathogens. We investigated the occurrence of the potentially zoonotic Campylobacter spp., Salmonella spp. and group A rotaviruses in faecal samples from free-ranging toque macaques and tufted gray langurs in Sri Lanka. Samples were opportunistically collected from primate troops with close human contact at five sites. Standardized culturing was used to detect the bacteria and an ELISA-based dipstick test was used for detection of group A rotaviruses antigens. Genotyping was performed using pulse field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) and the isolates' sensitivity to selected antibiotics was tested with VetMIC TM (National Veterinary Institute, Uppsala, Sweden) panels Camp EU, CLIN GN and GN-mo (version 4). All 98 samples tested negative for rotavirus. The 40 samples from gray langurs were also all negative for Campylobacter spp. and Salmonella spp. Of the 58 samples collected from toque macaques, C. jejuni was isolated from ten, C. coli from four and Salmonella enterica enterica subsp. Virchow from two of the samples. The fact that neither of the bacteria were isolated from tufted gray langur samples could reflect a true difference between the primate species. However, this should be interpreted in the light of a relatively small sample size. Resistance to ampicillin, ciprofloxacin, nalidixic acid and tetracycline was identified in four C. jejuni isolates, of which three were multidrug resistant. In addition, all C. jejuni showed undetectable MIC-values to colistin, while all C. coli were sensitive to the substance. All C. coli were resistant to ampicillin. The S. Virchow isolates were sensitive to all antibiotics tested for. Six strains of C. jejuni were identified using PFGE and MLST clonal complexes were assigned to all isolates. Sequence types were assigned to seven out of ten C. jejuni. The detection of antibiotic resistant zoonotic bacteria in free-ranging toque macaques with close human contact may have implications for both non-human primate conservation and public health in Sri Lanka and beyondItem 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 Prevalence of antibodies to herpes simplex virus types 1 and 2 (hsv-1 and hsv-2) Amongst non-high risk and High risk populations in Sri Lanka(Sri Lanka College of Microbiologists, 2001) Sunil-Chandra, N.P.; Kumarage, J.; Wijesooriya, W.R.P.L.I.; Jayasinghe, S.M.A.S.INTRODUCTION: HSV-1 and HSV-2 infects both the ora cavity and the genital tract whilst the HSV-i generally causes genital infection. Both c these human herpesviruses cause botl primary and recurrent infections leading ft a lifelong persistence of antibodies. Mol infections of either type are asymptomatic Detection of type specific antibodies hai important implications in the diagnosis of HS' infection in sexually active adults ar> prevention of mother to child transmissiof Recently, problems associated with commo epitopes which elicit cross reactiv antibodies in infected individuals have bee overcome by new HSV type specif! serological assays using the gG1 protein < HSV-1 and gG-2 of HSV-2 as antigens. Th study determine the burden and the epidemiology of type specific HSV infection amongst Sri Lankan populations. OBJECTIVE: To estimate and compare age and gender specific seroprevalences of HSV-1 and HSV-2 amongst non-high risk and high risk populations from Sri Lanka. DESIGN: A prospective study among selected target groups. Setting: Children (aged 1-12 years) and non-high risk adults (aged 13-89 years) and blood donors (aged 15-54 years) reported to Teaching Hospital, Ragama, expectant mothers (aged 14-44 years) of Kelaniya Medical Officer of Health (MOH) division, and the patients attended Central STD Clinic Colombo (aged 4-79 years) during the years 2000 and 2001 were included in this study. METHODS: Single sample of blood was obtained from each of 433 children, 757 ante-natal women, 1374 non-high risk adults, 929 blood donors, and 676 STD clinic attendees. Samples were tested for IgG class antibody responses to HSV infections using FDA approved type specific ELISA assay (MRL) at the Dept . of Microbiology, Faculty of Medicine , University of Kelaniya. RESULTS: Overall seroprevalence of HSV-1 among children, ante-natal women, blood donors, adult patients and STD attendees was 51.3%, 75.2%, 79.3%, 75.9% and 78.7% respectively whilst the seroprevalence of HSV-2 was 4.6%, 8.3%, 10.9%, 19.8% and 39.6% respectively. Age and gender specific differences in seroprevalence were observed within study groups.Item Evaluation of a rapid whole blood assay for testing dengue patients at point of care(Sri Lanka College of Microbiologists, 2004) Sunil-Chandra, N.P.; Karunasekera, E.W.S.; Somasiri, D.A.D.H.; Samarakoon, S.M.R.M.; Jayawardena, K.A.T.M.; Fernando, W.M.D.; Wijesooriya, W.R.P.L.I.; Garcia, M.INTRODUCTION: Dengue is the most significant mosquito borne viral disease affecting nations from Asia to the Americas. Symptoms associated with dengue infection range in severity. . The presentation of disease is impacted by age, prior exposure to the virus and the infecting strain of virus. The more severe form of the disease (haemorrhagic fever) can lead to mortality are generally associated with Secondary infections. Clinically, the measurement of dengue-specific IgM and elevated IgG, allows for the detection and differentiation of Primary and Secondary dengue infection. This discrimination is particularly important in situations such as outbreaks where the allocation of resources needs to be directed to those at greatest risk. In cities and major regional centers worldwide clinicians have access to traditional serological techniques such as ELISA and HAI that measure IgM and IgG levels. Unfortunately, clinicians in rural and remote areas generally do not have the resources available for this technology. Hence there is high clinical utility in a field diagnostic device which has the ability to rapidly and accurately detect and differentiate dengue infections. OBJECTIVES: To evaluate a novel dengue whole blood assay (PanBio) having the capacity for qualitative detection of both dengue-specific IgM and IgG, and differentiate between primary and secondary dengue with regard to sensitivity and specificity. To meet the demand for testing at the point of care or in the near patient environment, the test was required to have the capacity to detect antibodies in whole blood. DESIGN, SETTING AND METHODS: This assay device was used at the bed site of patients to evaluate its performance. The test is simply performed by adding the specimen to the sample well followed by running buffer to the buffer well, wait 15 minutes and visually reading the results. No additional materials required. 231 hospital inpatients in the Gampaha district of Sri Lanka, using a finger prick drop of blood as the analyte were assessed against PanBio Dengue Capture IgM and IgG ELISA for the period of 6 weeks starting from 10Ih November 2003. The capacity to detect and differentiate presumptive primary and secondary dengue was evaluated. RESULTS: The whole blood dengue cassette was able to detect 151 positive and 80 negative samples where as the ELISA could detect 126 positive samples and 105 negative patients. The detection of IgM and IgG positive samples by the cassette gave a relative sensitivity of 94.5%, specificity of 86% and 87.1% agreement between the assays. The cassette was able to identify 71% of positive samples as primary infections (IgM positive) and 96.7% as secondary infections (IgG positive with or without IgM) compared to ELISA. CONCLUSION: These data indicate that the Whole Blood Dengue Cassette has good utility in the detection of primary and secondary dengue with a very high accuracy in discriminating patients at greatest risk and represents a valuable field based assay to support the clinical evaluation of patients presenting with symptoms suggestive of dengue fever. ACKNOWLEDGEMENTS: PanBio Ltd, Australia for the financial assistance and Directors of Teaching hospital Ragama and Base hospitals of Negombo, Gampaha and Wathupitiwala.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 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.