Suneth Agampodi

Utility of Quantitative Polymerase Chain Reaction in Leptospirosis Diagnosis: Association of Level of Leptospiremia and Clinical Manifestations in Sri Lanka

BACKGROUND Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestations in relation to quantitative leptospiremia. METHODS A new qPCR assay using pathogenic Leptospira-specific 16S ribosomal RNA (rRNA) gene Taqman primers and an optimized temperature stepdown protocol was used to analyze patient blood samples. Serum was compared with whole blood as sample source. Quantitative leptospiremia was compared with clinical manifestations of leptospirosis and outcome. RESULTS The diagnostic sensitivity of qPCR of whole blood and serum was 18.4% (95% confidence interval [CI]: 9.97%-31.4%) and 51.0% (95% CI: 37.5%-64.4%) respectively. The qPCR on suspected cases confirmed infection in 58 of 381 cases (15.2%). Of these, 6 cases confirmed by nested polymerase chain reaction (PCR) and sequencing were serologically negative using a standard but not regionally optimized microscopic agglutination test panel. The bacterial load in serum/blood ranged from 10(2) to 10(6) Leptospira/mL. Median leptospiral load for uncomplicated, renal failure, myocarditis, and multi-organ failure patients were 8616, 11007, 36100, and 15882 Leptospira/mL respectively. The qPCR window of positivity ranged from day 2 to day 15; sensitivity of qPCR was not affected by the length of the interval between the onset of symptoms and sample collection (P = .328). CONCLUSIONS Quantitative PCR shows potential as a valid diagnostic test with a wider window of positivity than previously thought. Quantitative leptospiremia in serum/whole blood samples did not directly correlate with clinical manifestations of outcome in this patient population.

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.

Breastfeeding practices in a public health field practice area in Sri Lanka: a survival analysis

BackgroundExclusive breastfeeding up to the completion of the sixth month of age is the national infant feeding recommendation for Sri Lanka. The objective of the present study was to collect data on exclusive breastfeeding up to six months and to describe the association between exclusive breastfeeding and selected socio-demographic factors.MethodsA clinic based cross-sectional study was conducted in the Medical Officer of Health area, Beruwala, Sri Lanka in June 2006. Mothers with infants aged 4 to 12 months, attending the 19 child welfare clinics in the area were included in the study. Infants with specific feeding problems (cleft lip and palate and primary lactose intolerance) were excluded. Cluster sampling technique was used and consecutive infants fulfilling the inclusion criteria were enrolled. A total of 219 mothers participated in the study. The statistical tests used were survival analysis (Kaplan-Meier survival curves and Cox proportional Hazard model).ResultsAll 219 mothers had initiated breastfeeding. The median duration of exclusive breastfeeding was four months (95% CI 3.75, 4.25). The rates of exclusive breastfeeding at 4 and 6 months were 61.6% (135/219) and 15.5% (24/155) respectively. Bivariate analysis showed that the Muslim ethnicity (p = 0.004), lower levels of parental education (p < 0.001) and being an unemployed mother (p = 0.021) were important associations of early cessation of exclusive breastfeeding. At the time of the study, 62% (135/219) of infants were receiving feeds via a bottle and 23% (51/219) were receiving infant formula. Muslim ethnicity was significantly associated with bottle and formula feeding (p < 0.001). Bottle feeding was also significantly higher among mothers with a low level of education and among employed mothers.ConclusionThe rate of breastfeeding initiation and exclusive breastfeeding up to the fourth month is very high in Medical Officer of Health area, Beruwala, Sri Lanka. However exclusive breastfeeding up to six months is still low and the prevalence of inappropriate feeding practices is high.

Drinking well water and occupational exposure to Herbicides is associated with chronic kidney disease, in Padavi-Sripura, Sri Lanka

BackgroundThe chronic kidney disease of unknown etiology (CKDu) among paddy farmers in was first reported in 1994 and has now become most important public health issue in dry zone of Sri Lanka. The objective was to identify risk factors associated with the epidemic in an area with high prevalence.MethodsA case control study was carried out in Padavi-Sripura hospital in Trincomalee district. CKDu patients were defined using health ministry criteria. All confirmed cases (N = 125) fulfilling the entry criteria were recruited to the study. Control selection (N = 180) was done from people visiting the hospital for CKDu screening. Socio-demographic and data related to usage of applying pesticides and fertilizers were studied. Drinking water was also analyzed using ICP-MS and ELISA to determine the levels of metals and glyphosate.ResultsMajority of patients were farmers (N = 107, 85.6%) and were educated up to ‘Ordinary Level’ (N = 92, 73.6%). We specifically analyzed for the effect modification of, farming by sex, which showed a significantly higher risk for male farmers with OR 4.69 (95% CI 1.06-20.69) in comparison to their female counterparts. In the multivariable analysis the highest risk for CKDu was observed among participants who drank well water (OR 2.52, 95% CI 1.12-5.70) and had history of drinking water from an abandoned well (OR 5.43, 95% CI 2.88-10.26) and spray glyphosate (OR 5.12, 95% CI 2.33-11.26) as a pesticide. Water analysis showed significantly higher amount of hardness, electrical conductivity and glyphosate levels in abandoned wells. In addition Ca, Mg, Ba, Sr, Fe, Ti, V and Sr were high in abandoned wells. Surface water from reservoirs in the endemic area also showed contamination with glyphosate but at a much lower level. Glyphosate was not seen in water samples in the Colombo district.ConclusionThe current study strongly favors the hypothesis that CKDu epidemic among farmers in dry zone of Sri Lanka is associated with, history of drinking water from a well that was abandoned. In addition, it is associated with spraying glyphosate and other pesticides in paddy fields. Farmers do not use personnel protective equipments and wears scanty clothing due to heat when spraying pesticides.

Regional Differences of Leptospirosis in Sri Lanka: Observations from a Flood-Associated Outbreak in 2011

Leptospirosis is known to be an important cause of weather disaster-related infectious disease epidemics. In 2011, an outbreak of leptospirosis occurred in the relatively dry district of Anuradhapura, Sri Lanka where diagnosis was resisted by local practitioners because leptospirosis was not known in the area and the clinical presentation was considered atypical. To identify the causative Leptospira associated with this outbreak, we carried out a cross-sectional study. Consecutive clinically suspected cases in this district were studied during a two-and-a-half-month period. Of 96 clinically suspected cases, 32 (33.3%) were confirmed by qPCR, of which the etiological cause in 26 cases was identified using 16S rDNA sequencing to the species level. Median bacterial load was 4.1×102/mL (inter-quartile range 3.1–6.1×102/mL). In contrast to a 2008 Sri Lankan leptospirosis outbreak in the districts of Kegalle, Kandy, and Matale, in which a predominance of Leptospira interrogans serovars Lai and Geyaweera was found, most cases in the 2011 outbreak were caused by Leptospira kirschneri. Seven (21.9%) confirmed cases had acute renal failure; five (15.6%) had myocarditis; severe thrombocytopenia (<20,000/uL) was seen in five (15.6%) cases. This outbreak of leptospirosis in the relatively dry zone of Sri Lanka due primarily to L. kirschneri was characterized by markedly different clinical presentations and low leptospiremia. These observations and data demonstrate the public health relevance of molecular diagnostics in such settings, possibly related to the microgeographic variations of different Leptospira species, but of particular value to public health intervention in what appears to have been a regionally neglected tropical disease.

Exclusive breastfeeding in Sri Lanka: problems of interpretation of reported rates

Accurate interpretation of reported breastfeeding rates is essential in understanding the true picture of a countrys breastfeeding status. In Sri Lanka, where the reported exclusive breastfeeding (EBF) rate among infants aged from 0 to 5 months is 75%, accurate understanding of this rate is of the utmost importance. The danger of misinterpreting the data and assuming that Sri Lanka has achieved a high EBF rate is that health workers begin to believe that no further effort should be made in this area. This is very dangerous as the potential to further improve rates of EBF will not be addressed. We discuss the interpretation of survey data and various definitions used in the relevant literature. We strongly recommend that interpretation of EBF rates should be done only after careful evaluation of the definitions and survey methods used.

Globalization of leptospirosis through travel and migration

Leptospirosis remains the most widespread zoonotic disease in the world, commonly found in tropical or temperate climates. While previous studies have offered insight into intra-national and intra-regional transmission, few have analyzed transmission across international borders. Our review aimed at examining the impact of human travel and migration on the re-emergence of Leptospirosis. Results suggest that alongside regional environmental and occupational exposure, international travel now constitute a major independent risk factor for disease acquisition. Contribution of travel associated leptospirosis to total caseload is as high as 41.7% in some countries. In countries where longitudinal data is available, a clear increase of proportion of travel-associated leptospirosis over the time is noted. Reporting patterns is clearly showing a gross underestimation of this disease due to lack of diagnostic facilities. The rise in global travel and eco-tourism has led to dramatic changes in the epidemiology of Leptospirosis. We explore the obstacles to prevention, screening and diagnosis of Leptopirosis in health systems of endemic countries and of the returning migrant or traveler. We highlight the need for developing guidelines and preventive strategies of Leptospirosis related to travel and migration, including enhancing awareness of the disease among health professionals in high-income countries.

Utility and Limitations of Direct Multi-Locus Sequence Typing on qPCR-Positive Blood to Determine Infecting Leptospira Strain

Culture-independent molecular characterization of infecting Leptospira human blood specimens from a 2008 outbreak of human leptospirosis in central Sri Lanka was carried out. Of 58 quantitative real-time polymerase chain reaction-positive samples analyzed for seven multi-locus sequence typing (MLST) housekeeping genes (mreA, pfkB, pntA, sucA, tpiA, fadD, and glmU), interpretable data was obtained from 12 samples. Mean bacterial load was 2.2 × 10(5) among specimens with complete MLST profiles compared with 1.3 × 10(4) among specimens without complete MLST profiles; all specimens with complete profiles had at least 4.9 × 10(4) Leptospira/mL (t = 5, P < 0.001). Most (11/12) identified sequence types were ST1 (L. interrogans serovar Lai) and ST44 (L. interrogans serovar Geyaweera). MLST can be used to directly identify infecting Leptospira strains in blood samples obtained during acute illness without the need for culture isolation, but it shows important limitations related to bacterial load.

Inadequacy of the risk factor based approach to detect gestational diabetes mellitus

OBJECTIVE To evaluate the present risk factor based approach in diagnosis of gestational diabetes mellitus (GDM) in Sri Lanka in comparison with new guidelines proposed by the International Association of Diabetes and Pregnancy Study Groups (IADPSG). METHODS A community based cross sectional descriptive study was conducted among pregnant women with gestational age of 24-28 weeks and residing in Anuradhapura district. All eligible pregnant women from selected Medical Officers of Health areas were invited to participate. The 75 g oral glucose tolerance test was carried out among all participants. RESULTS According to IADPSG criteria, 36 (8.9%) of pregnant women had GDM, compared to 29 (7.2%) according to WHO criteria. Prevalence of GDM in the study population (positive by one or both methods) was 10.6% (n=43) (95% CI 7.9-13.9%). Of these 43 women,22 (51.1%) had positive results by both methods. Seven (16.3%) and 14 (32.6%) mothers were positive only according to WHO and IADPSG criteria respectively. Out of 29 mothers who fulfilled WHO criteria, only one had an abnormal fasting plasma glucose, but 28 had abnormal 2 hour values. A total of 170 (42.0%) participants had at least one risk factor or early indicator of GDM. A risk based approach would have detected only 22 GDM patients according to IADPSG criteria, missing 14 cases (38.9%) who did not have any of the risk factors for GDM. CONCLUSIONS The risk factor based approach misses more than one third of GDM cases. Urgent revision of current GDM screening guidelines is recommended.

Duration of exclusive breastfeeding; validity of retrospective assessment at nine months of age

BackgroundIn cross sectional, case control and retrospective cohort studies, duration of Exclusive Breastfeeding (EBF) usually depends on maternal recall. Retrospective data are often subjected to recall bias and could lead to a potential for exposure misclassification. The purpose of the present paper is to assess the validity of maternal recall of EBF duration during infancy, after cessation of EBF and to evaluate the two methods to collect retrospective data on EBF.MethodsA cohort study was carried out in Naula Medical Officer of Health (MOH) area. Study cohort included all infants born during the months of February to April 2008 and currently residing in Naula MOH area. Baseline data collection was carried out using the pregnancy record, the child health development record and by using an interviewer administered structured questionnaire. Data extraction from the pregnancy record and the child health development record were carried out by public health midwives. The interviewer administered structured questionnaire was administered by the MOH during the follow-up visits. Duration of EBF was assessed in three ways; based on prospective data since birth: Retrospective data based on an event calendar: and the Mother reported EBF duration.ResultsA total of 114 mother-infant pairs were recruited and followed up. Proportion of infants receiving EBF up to the completion of the sixth month by the three methods were; data since birth (actual EBF rate) - 23.9%; mother reported data - 77.7% and event calendar method - 41.3%. Median duration of EBF reported in the three methods was 5, 6, and 5 respectively. A statistically significant difference was observed in these differences from Kaplan-Meire Survival analysis (Log rank test - Chi square-63.4, p < 0.001). Validity of retrospective methods was analysed using data since birth as the gold standard. Sensitivity of both methods to detect exclusively breastfed babies were 100.0%. Specificity of mother recall data was 26.2% (95%CI-17.9, 36.8%) compared to 75.0% (95% CI-64.5, 83.2%) in the event calendar method.ConclusionsRetrospective evaluation methods systematically overestimate the duration of EBF. Maternal recall data provide highly unspecific data whereas use of an event calendar provided more valid data. Reporting of data accrual methods in breastfeeding studies will allow the readers to interpret findings accurately and the use of event calendars rather than direct questioning as a valid method of determining EBF is recommended.