Synergistic activity between Triphala and selected antibiotics against drug resistant clinical isolates.

BACKGROUND: Triphala is an indigenous medical product used for a variety of diseases. This study was conducted to determine the effect of Triphala on antibiotic properties of gentamicin and oxacillin against multi-drug resistant organisms. METHODS: The checkerboard method was used to determine the synergy of Triphala with gentamicin and oxacillin against multi-drug resistant (MDR) Gram negative bacilli and methicillin-resistant Staphylococcus aureus (MRSA) using 2,3,5-triphenyltetrazolium chloride (TTC) assay. Fractional inhibitory concentration (FIC) index was calculated. RESULTS: When tested alone, the minimum inhibitory concentration (MIC) values of gentamicin for Gram negative isolates ranged from 8 to > 64 µg/ml. The MIC values of gentamicin for the Gram negative isolates ranged from 1 to 32 µg/ml when tested with Triphala. The FIC index was < 1 indicating a synergistic interaction in 10 of the 11 isolates and it was 1 indicating an additive effect in one isolate. The MIC values of oxacillin for MRSA isolates ranged from 4 to > 16 µg/ml with all MICs being equal to or higher than the resistance cut-off level. The MIC level with the addition of Triphala ranged from 0.25 to 4 µg/ml. FIC index was < 1 for all tested isolates indicating a synergistic interaction. CONCLUSIONS: Triphala has synergistic activity with gentamicin against the selected MDR Gram negative bacilli and with oxacillin against MRSA isolates warranting further studies on the possibility of clinical use.

Insight into genetic diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka reveals predominance of the Euro-American lineage.

OBJECTIVE: Sri Lanka is a country where the molecular epidemiology of Mycobacterium tuberculosis (MTB) is poorly explored. Therefore, this study was performed to identify circulating lineages/sub-lineages of MTB and their transmission patterns. METHODS: DNA was extracted from 89 isolates of MTB collected during 2012 and 2013 from new pulmonary tuberculosis patients in Kandy, Sri Lanka and analyzed by spoligotyping, large sequence polymorphism (LSP), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, and drug resistance-associated gene sequencing. RESULTS: The predominant lineage was lineage 4 (Euro-American, 45.9%), followed by lineage 1 (Indo-Oceanic, 29.4%), lineage 2 (East-Asian, 23.5%), and lineage 3 (Central-Asian, 1.2%). Among 26 spoligotype patterns, eight were undesignated or new types and seven of these belonged to lineage 4. Undesignated lineage 4/SIT124 (n=2/8) and SIT3234 (n=8/8) clustered together based on 24-locus MIRU-VNTR typing. The dominant sub-lineage was Beijing/SIT1 (n=19), with the isoniazid resistance katG G944C mutation (Ser315Thr) detected in two of them. CONCLUSIONS: The population structure of MTB in Kandy, Sri Lanka was different from that in the South Asian region. The clonal expansion of locally evolved lineage 4/SIT3234 and detection of the pre-multidrug resistant Beijing isolates from new tuberculosis patients is alarming and will require continuous monitoring.

Evaluation of the 15 and 24-loci MIRU-VNTR genotyping tools with spoligotyping in the identification of Mycobacterium tuberculosis strains and their genetic diversity in molecular epidemiology studies.

BACKGROUND: The transmission dynamics of Mycobacterium tuberculosis (Mtb) using various genotyping tools has been studied globally and a particular tool for genotyping Mtb is the mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR). Tuberculosis (TB) remains an important public health problem worldwide and Sri Lanka being a country of tourist destination; because of major development projects undergoing, it has a high proportion of tourists and immigrants from Asia and Europe that are characterized with highest TB incidences and drug-resistant clinical isolates. Hence, in order to address the question of Mtb genetic diversity, we investigated the discriminatory power of both MIRU-VNTR typing of 15 and 24 loci with spoligotyping to differentiate Mtb isolates. METHOD: Acid-fast bacilli positive sputum samples (n = 150) from first visit patients were collected. Decontamination of sputum and extraction of genomic DNA were carried out using standard techniques. The isolates were characterized by MIRU-VNTR for both the 15 and 24 loci and spoligotyping. RESULTS: In our study population, MIRU-VNTR 15 and 24 loci did not show a significant difference among the identified M. tuberculosis strains. However, MIRU 24 loci yielded an additional strain LAM, which is of T1 origin. 15 loci strain grouping had more clusters of strains grouped together while 24 loci differentiated the same cluster of strains into distinct strain types. CONCLUSION: We conclude that the use of 15-locus MIRU-VNTR typing is sufficient for a first-line epidemiological study to genotype M. tuberculosis, but the additional discriminatory power of 24 loci MIRU-VNTR has been able to differentiate samples within highly homologous groups.

Terminalia bellirica fruit extracts: in-vitro antibacterial activity against selected multidrug-resistant bacteria, radical scavenging activity and cytotoxicity study on BHK-21 cells.

BACKGROUND: Identification of novel sources for developing new antibiotics is imperative with the emergence of antibiotic resistant bacteria. The fruits of Terminalia bellirica (Gaertn) Roxb., widely used in traditional medicine, were evaluated for antibacterial activity against multidrug-resistant (MDR) bacteria, antioxidant activity and cytotoxicity. METHODS: Twelve solvent extracts of T. bellirica fruits were prepared by direct aqueous extraction and sequential extraction with dichloromethane, methanol and water using Soxhlet, bottle-shaker and ultrasound sonicator methods. Antibacterial activity of the extracts was tested against 16 strains MDR bacteria-methicillin-resistant Staphylococcus aureus (MRSA), extended spectrum ß-lactamase (ESBL) producing Escherichia coli and MDR Acinetobacter spp., Klebsiella pneumoniae and Pseudomonas aeruginosa-and 4 control organisms, using the cut-well diffusion method. The minimum inhibitory concentration (MIC) was determined using an agar dilution method. The radical scavenging activity of six antibacterial extracts was screened against 2,2'-diphenyl-2-picrylhydrazyl (DPPH) and correlation was established between EC50 (50% effective concentration) values and the total phenolic content (TPC). Cytotoxicity was determined for the most potent antibacterial extract on baby hamster kidney (BHK-21) cells by Tryphan Blue exclusion method. Statistical analysis was carried out by one-way analysis of variance at significant level p < 0.05 using "SigmaPlot 10" and "R 3.2.0" software. RESULTS: All aqueous and methanol extracts displayed antibacterial activity (MIC 0.25-4 mg/mL) against all strains of MRSA, MDR Acinetobacter spp. and MDR P. aeruginosa. The sequential aqueous extracts (MIC, 4 mg/mL) inhibited ESBL producing-E. coli. None of the extracts exhibited activity against MDR K. pneumoniae (MIC > 5 mg/mL). The sequential methanol extract (Soxhlet) recorded high antibacterial activity and the highest DPPH radical scavenging activity (EC50, 6.99 ± 0.15 ppm) and TPC content (188.71 ± 2.12 GAE mg/g). The IC50 (50% inhibition concentration) values of the most potent antibacterial extract-the direct aqueous extract from reflux method-on BHK-21 cells were 2.62 ± 0.06 and 1.45 ± 0.08 mg/ml with 24 and 48 h exposure, respectively. CONCLUSIONS: Results indicate that T. bellirica fruit is a potential source for developing broad-spectrum antibacterial drugs against MDR bacteria, which are non-toxic to mammalian cells and impart health benefits by high antioxidant activity.

Melioidosis in Sri Lanka.

Until recently, Sri Lanka was not considered a country with endemic melioidosis. However, an increasing number of cases is being reported. National surveillance for melioidosis was instituted after 2008. A total of 250 culture-positive cases was recorded between 2006 and May 2017. Males predominated (71.6%). The age range was wide (2⁻92 years) reflecting a ubiquity of exposure. The majority (201/250, 80%) lived in rural areas. All provinces were affected. Case load increased during the two monsoonal periods (67%). There was representation of every population group including farmers (n = 44), housewives (n = 24), school children (n = 10), professionals (n = 5), businesspersons (n = 6), white-collar workers (n = 10) and blue-collar workers (n = 8). Diabetes was the predominant risk factor (n = 163, 65.2%). Clinical presentations included community-acquired sepsis and pneumonia, superficial and deep abscesses, and septic arthritis. Mortality was 20.4% (51/250). A majority (n = 212) of isolates belonged to the YLF (Yersinia-like fimbrial) clade but 38 were BTFC (B. thailandensis-like flagellum and chemotaxis). A total of 108 isolates was genotyped and 46 sequence types (STs) were identified, 40 being novel. It is clear that melioidosis is endemic in Sri Lanka with a wide geographic and demographic distribution. There is an urgent need to extend surveillance of melioidosis to under-resourced parts of the country and to populations at high risk.

Sri Lankan National Melioidosis Surveillance Program Uncovers a Nationwide Distribution of Invasive Melioidosis.

The epidemiologic status of melioidosis in Sri Lanka was unclear from the few previous case reports. We established laboratory support for a case definition and started a nationwide case-finding study. Suspected Burkholderia pseudomallei isolates were collated, identified by polymerase chain reaction assay, referred for Matrix Assisted Laser Desorption Ionization-Time of Flight analysis and multilocus sequence typing (MLST), and named according to the international MLST database. Between 2006 and early 2014, there were 32 patients with culture-confirmed melioidosis with an increasing annual total and a falling fatality rate. Patients were predominantly from rural communities, diabetic, and male. The major clinical presentations were sepsis, pneumonia, soft tissue and joint infections, and other focal infection. Burkholderia pseudomallei isolates came from all parts of Sri Lanka except the Sabaragamuwa Province, the south central hill country, and parts of northern Sri Lanka. Bacterial isolates belonged to 18 multilocus sequence types, one of which (ST 1137) was associated with septicemia and a single-organ focus (Fisher's exact, P = 0.004). Melioidosis is an established endemic infection throughout Sri Lanka, and is caused by multiple genotypes of B. pseudomallei, which form a distinct geographic group based upon related sequence types (BURST) cluster at the junction of the southeast Asian and Australasian clades.

Acute meningoencephalitis associated with echovirus 9 infection in Sri Lanka, 2009.

The aetiology of acute meningoencephalitis in Sri Lankan children and adults is poorly understood. This study was carried out to determine pathogens responsible for meningoencephalitis in Sri Lanka. A hospital-based cross-sectional study was performed using cerebrospinal fluid samples (22 adult and 17 pediatric) collected from August to December 2009 from patients clinically diagnosed with acute meningoencephalitis at two tertiary care hospitals in Sri Lanka. Routine microbiology for bacterial pathogens together with in-house RT-PCR and PCR assays for the detection of dengue viruses, Japanese encephalitis virus, West Nile virus, chikungunya virus, enteroviruses, mumps virus, measles virus, herpes simplex viruses types 1 and 2, and varicella zoster virus were performed. Bacterial pathogens were not isolated from any patient specimens. However, from nine of the paediatric patients aged 1 month to 10 years (mean age 5.2 years) echovirus 9 (E-9; family Picornaviridae, genus Enterovirus,species Enterovirus B ) was detected by RT-PCR. All nine patients presented with fever, six had headache, and seven had vomiting. Neck stiffness indicating meningitis was present in six of the patients. Phylogenetic analysis of partial VP1 and VP4-VP2 genes showed these E-9 strains to be most closely related to E-9 strains detected in CSF from Korea and France in 2005 and 2006. The remaining patients were negative for all other viruses tested. E-9 was the most common cause of acute meningoencephalitis in the tested paediatric population from Sri Lanka in 2009, which likely reflects circulation of this E-9 strain between Europe and Asia over several years.

Characteristics of rural leptospirosis patients admitted to referral hospitals during the 2008 leptospirosis outbreak in Sri Lanka: implications for developing public health control measures.

To determine the exposure risk factors of highly endemic rural leptospirosis in tropical setting, we conducted a prospective, hospital-based case control study in Sri Lanka. A conceptual hierarchy of variables was used to analyze the data. Case patients included 38 (34%) females and 73 (66%) males with a mean age of 36 yr (SD 12.7 yr). Using piped, chlorinated water for drinking/general purposes (odds ratio [OR] 0.33, 95% confidence interval [CI] 0.16-0.67), paddy fields in the vicinity of home (OR 1.77, 95% CI 1.06-2.97), sighting dogs at home yard/dog ownership (OR 1.79, 95% CI 1.11-2.91), sighting cattle at home yard/cattle ownership (OR 1.69, 95% CI 1.00-2.84), and work in a paddy field (OR 3.02, 95% CI 1.68, 5.41) were the main predictors of leptospirosis among febrile patients. In high endemic tropical settings with rural leptospirosis, risk factors in residential environments, rather than individual exposures, seemed to play a major role in leptospirosis disease transmission.

Antibacterial activity of xanthones from Garcinia mangostana (L.) and their structure-activity relationship studies.

Antibacterial activities of prenylated xanthones from Garcinia mangostana and their synthetic analogues were investigated, and their structure-activity relationships have been studied. γ-Mangostin has shown antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA), vancomycin-resistant Enterococcus (VRE) and vancomycin-sensitive Enterococcus (VSE) strains at MICs 3.13, 6.25, 6.25 and 6.25 µg mL(-1), respectively. In these experiments, gentamicin was used as the positive control. Further, some analogues of γ-mangostin and α-mangostin were synthesised and their activity was tested against MRSA and VRE strains. The analysis of the bioassay results above indicated that, the combination of C-6 and C-3 hydroxyl groups along with the prenyl side chain at C-2 in the 1,3,6,7-tetraoxygenated xanthones from G. mangostana is essential to have a high antibacterial activity.

Scrub typhus among pediatric patients in Dambadeniya: a base hospital in Sri Lanka.

Data on pediatric scrub typhus is uncommon in Sri Lanka and other countries. The objective of this study was to identify the clinical features of patients with scrub typhus at a Base Hospital in Sri Lanka. Sixty patients presenting with suspected scrub typhus were included in the study. Their blood samples were tested for the presence of antibodies against rickettsioses using the reference method. Twenty patients had confirmed scrub typhus and 24 had possible scrub typhus. Their clinical features are discussed in this work.

Detection of rickettsioses and Q fever in Sri Lanka.

Current serological evidence suggests the presence of scrub typhus and spotted fever group (SFG) rickettsiosis in Sri Lanka. Our objective was to identify rickettsial agents/Q fever as aetiological causes for patients who were presumed having rickettsioses by the presence of an eschar or a rash. Sera from patients with unknown origin fever from Matara were tested by immunofluorescence for SFG rickettsial antigens, typhus group rickettsiae, Orientia tsutsugamushi, and Coxiella burnetii antigens. Thirteen (7.3%) of the patients presented with a rash, 11 (6.1%) had an inoculation eschar, and 16 patients recalled a tick or flea bite. We found that 25 (14%) patients had scrub typhus, 6 (3%) SFG rickettsioses, 3 (1.6%) acute Q fever, 3 (1.6%) murine typhus, and 3 (1.6%) were infected by Rickettsia felis. In addition to already described scrub and murine typhus, we found that R. felis and C. burnetii infections should be considered in Sri Lanka.

Seroepidemiology of rickettsioses in Sri Lanka: a patient based study.

BACKGROUND: Rickettsioses are emerging infections in Sri Lanka as shown by the increase in the number of clinically diagnosed rickettsial patients being reported to the Epidemiology Unit, Sri Lanka. However, mapping the disease for the whole island with laboratory confirmed cases has not been previously carried out. METHODS: 615 samples received from 23 hospital representing 8 provinces were tested using ELISA or IFA methods and clinical data was collected using a validated questionnaire. RESULTS: Rash was found among more spotted fever seropositive patients than scrub typhus seropositive patients while the opposite was true for the presence of eschar. Spotted fever and scrub typhus was found in a geographically restricted manner. Consistent temporal patterns were seen for the presentation of patients with rickettsioses in Kandy and Kurunegala districts for 2009 and 2010. CONCLUSIONS: This study expanded knowledge on the distribution of rickettsioses in Sri Lanka and their clinical profiles which in turn helps in the clinical diagnosis of these infections.

Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease.

Global leptospirosis disease burden estimates are hampered by the lack of scientifically sound data from countries with probable high endemicity and limited diagnostic capacities. We describe the seroepidemiologic and clinical characteristics of the leptospirosis outbreak in 2008 in Sri Lanka. Definitive/presumptive case definitions proposed by the World Health Organization Leptospirosis Epidemiology Reference Group were used for case confirmation. Of the 404 possible cases, 155 were confirmed to have leptospirosis. Highest titers of patient seum samples reacted with serovars Pyrogenes (28.7%), Hardjo (18.8%), Javanica (11.5%), and Hebdomadis (11.5%). Sequencing of the 16S ribosomal DNA gene identified six infections: five with Leptospira interrogans and one with L. weilli. In this patient population, acute renal failure was the main complication (14.8%), followed by myocarditis (7.1%) and heart failure (3.9%). The case-fatality rate was 1.3%. This report strengthens the urgent need for increasing laboratory diagnostic capabilities to determine the causes of epidemic and endemic infectious diseases in Sri Lanka, a finding relevant to other tropical regions.

Determinants of leptospirosis in Sri Lanka: study protocol.

BACKGROUND: Leptospirosis is becoming a major public health threat in Sri Lanka as well as in other countries. We designed a case control study to determine the factors associated with local transmission of leptospirosis in Sri Lanka, in order to identify major modifiable determinants of leptospirosis. The purpose of this paper is to describe the study protocol in detail prior to the publishing of the study results, so that the readership will be able to understand and interpret the study results effectively. METHODS: A hospital based partially matched case control design is proposed. The study will be conducted in three selected leptospirosis endemic districts in central Sri Lanka. Case selection will include screening all acute fever patients admitted to selected wards to select probable cases of leptospirosis and case confirmation using an array of standard laboratory criteria. Age and sex matched group of acute fever patients with other confirmed diagnosis will be used as controls. Case to control ratio will be 1:2. A minimum sample of 144 cases is required to detect 20% exposure with 95% two sided confidence level and 80% power. A pre tested interviewer administered structured questionnaire will be used to collect data from participants. Variables included in the proposed study will be evaluated using conceptual hierarch of variables in three levels; Exposure variables as proximal; reservoir and environmental variables as intermediate; socio-demographic variables as distal. This conceptual hierarch hypothesised that the distal and intermediate variables are mediated through the proximal variables but not directly. A logistic regression model will be used to analyse the probable determinants of leptospirosis. This model will evaluate the effect of same level and upper level variables on the outcome leptospirosis, using three blocks. DISCUSSION: The present national control programme of leptospirosis is hampered by lack of baseline data on leptospirosis disease transmission. The present study will be able to provide these essential information for formulation of better control strategies.

Incidence and effects of Varicella Zoster Virus infection on academic activities of medical undergraduates--a five-year follow-up study from Sri Lanka.

BACKGROUND: The adult population in Sri Lanka is having high level of susceptibility for Varicella Zoster Virus (VZV) infection. Among medical undergraduates, 47% are VZV seronegative. The purpose of the present study was to determine the incidence of VZV infection in medical undergraduates in Sri Lanka, and to describe the effects of VZV infection on their academic activities. METHODS: A retrospective cohort of medical undergraduates' susceptible for VZV infection was selected from the University of Peradeniya, Sri Lanka. Data on the incidence of VZV infection (Chickenpox) during their undergraduate period was collected using a self-administered structured questionnaire. A second questionnaire was administered to collect data on the details of VZV infection and the impact of it on their academic activities. VZV incidence rate was calculated as the number of infections per 1,000 person years of exposure. Descriptive statistics were used to describe the impact of VZV infection on academic activities. RESULTS: Out of the 172 susceptible cohort, 153 medical undergraduates were followed up. 47 students reported VZV infection during the follow up period and 43 of them participated in the study. The cumulative incidence of VZV infection during the period of five and half years of medical training was 30.7%. Incidence density of VZV infection among medical undergraduates in this cohort was 65.1 per 1,000 person years of follow-up. A total of 377 working days were lost by 43 students due to the VZV infection, averaging 8.8 days per undergraduate. Total academic losses for the study cohort were; 205 lectures, 17 practicals, 13 dissection sessions, 11 tutorials, 124 days of clinical training and 107 days of professorial clinical appointments. According to their perception they lost 1,927 study hours due to the illness (Median 50 hours per undergraduate). CONCLUSIONS: The incidence of VZV infection among Sri Lankan medical undergraduates is very high and the impact of this infection on academic activities causes severe disruption of their undergraduate life. VZV immunization for susceptible new entrant medical undergraduates is recommended.

Deployable laboratory response to emergence of melioidosis in central Sri Lanka.

A portable molecular diagnostic laboratory was used to provide molecular confirmation of suspected melioidosis cases seen at Peradeniya Hospital, central Sri Lanka. Soil supernatants from rice field and rubber plantation samples also produced PCR-positive results. These procedures could be used for melioidosis field work in other remote locations.

Possible prevalence and transmission of acute respiratory tract infections caused by Streptococcus pneumoniae and Haemophilus influenzae among the internally displaced persons in tsunami disaster evacuation camps of Sri Lanka.

OBJECTIVE: The objective of this prospective study was to investigate the status of acute respiratory tract infections caused by Haemophilus influenzae and Streptococcus pneumoniae in tsunami disaster evacuation camps. METHODS: Nasopharyngeal swabs (NP) of 324 internally displaced persons (IDP) in 3 different tsunami disaster evacuation camps of Sri Lanka were collected between March 18th and 20th, 2005, and analyzed for MIC, beta-lactamase production, serotypes, PCR and pulsed-field gel electrophoresis (PFGE). RESULTS: Many IDP had respiratory symptoms and the prevalence of cough and/or sputum was 84%, 70.5% and 64.7% in the three camps. Twenty-one H. influenzae from 20 IDP and 25 S. pneumoniae from 22 IDP were isolated from the NP. All H. influenzae isolates were nontypeable, and 5 were beta-lactamase producing. Seventeen pneumococci were susceptible, 5 showed intermediate resistance and 3 were fully resistant to penicillin G. Molecular analysis showed the 21 H. influenzae strains had 13 PFGE patterns and 25 pneumococci had 16 PFGE patterns. All 4 different PFGE patterns of H. influenzae strains were detected in a few IDP in camps 1 and 3, and 5 different PFGE patterns of serotype 3, 22A, 9A, 10A and 11A pneumococci were detected in a few IDP in camps 1 and 3. CONCLUSION: Our data indicate acute respiratory tract infections caused by various types of H. influenzae and S. pneumoniae appear to have been prevalent, some of which were potentially transmitted from person to person in tsunami disaster evacuation camps.