Global and local genetic diversity at two microsatellite loci in Plasmodium vivax parasites from Asia, Africa and South America.

BACKGROUND: Even though Plasmodium vivax has the widest worldwide distribution of the human malaria species and imposes a serious impact on global public health, the investigation of genetic diversity in this species has been limited in comparison to Plasmodium falciparum. Markers of genetic diversity are vital to the evaluation of drug and vaccine efficacy, tracking of P. vivax outbreaks, and assessing geographical differentiation between parasite populations. METHODS: The genetic diversity of eight P. vivax populations (n=543) was investigated by using two microsatellites (MS), m1501 and m3502, chosen because of their seven and eight base-pair (bp) repeat lengths, respectively. These were compared with published data of the same loci from six other P. vivax populations. RESULTS: In total, 1,440 P. vivax samples from 14 countries on three continents were compared. There was highest heterozygosity within Asian populations, where expected heterozygosity (He) was 0.92-0.98, and alleles with a high repeat number were more common. Pairwise FST revealed significant differentiation between most P. vivax populations, with the highest divergence found between Asian and South American populations, yet the majority of the diversity (~89%) was found to exist within rather than between populations. CONCLUSIONS: The MS markers used were informative in both global and local P. vivax population comparisons and their seven and eight bp repeat length facilitated population comparison using data from independent studies. A complex spatial pattern of MS polymorphisms among global P. vivax populations was observed which has potential utility in future epidemiological studies of the P. vivax parasite.

Uncertain etiologies of proteinuric-chronic kidney disease in rural Sri Lanka.

The global prevalence of chronic kidney disease (CKD) of uncertain etiology may be underreported. Community-level epidemiological studies are few due to the lack of national registries and poor focus on the reporting of non-communicable diseases. Here we describe the prevalence of proteinuric-CKD and disease characteristics of three rural populations in the North Central, Central, and Southern Provinces of Sri Lanka. Patients were selected using the random cluster sampling method and those older than 19 years of age were screened for persistent dipstick proteinuria. The prevalence of proteinuric-CKD in the Medawachchiya region (North Central) was 130 of 2600 patients, 68 of 709 patients in the Yatinuwara region (Central), and 66 of 2844 patients in the Hambantota region (Southern). The mean ages of these patients with CKD ranged from 44 to 52 years. Diabetes and long-standing hypertension were the main risk factors of CKD in the Yatinuwara and Hambantota regions. Age, exceeding 60 years, and farming were strongly associated with proteinuric-CKD in the Medawachchiya region; however, major risk factors were uncertain in 87% of these patients. Of these patients, 26 underwent renal biopsy; histology indicated tubulointerstitial disease. Thus, proteinuric-CKD of uncertain etiology is prevalent in the North Central Province of Sri Lanka. In contrast, known risk factors were associated with CKD in the Central and Southern Provinces.

Pre-elimination stage of malaria in Sri Lanka: assessing the level of hidden parasites in the population.

BACKGROUND: With the dramatic drop in the transmission of malaria in Sri Lanka in recent years, the country entered the malaria pre-elimination stage in 2008. Assessing the community prevalence of hidden malaria parasites following several years of extremely low transmission is central to the process of complete elimination. The existence of a parasite reservoir in a population free from clinical manifestations, would influence the strategy for surveillance and control towards complete elimination. METHODS: The prevalence of hidden parasite reservoirs in two historically malaria endemic districts, Anuradhapura and Kurunegala, previously considered as high malaria transmission areas in Sri Lanka, where peaks of transmission follow the rainy seasons was assessed. Blood samples of non-febrile individuals aged five to 55 years were collected from randomly selected areas in the two districts at community level and a questionnaire was used to collect demographic information and movement of the participants. A simple, highly sensitive nested PCR was carried out to detect both Plasmodium falciparum and Plasmodium vivax, simultaneously. RESULTS: In total, 3,023 individuals from 101 villages participated from both districts comprising mostly adults between the ages 19-55 years. Out of these, only about 1.4% of them (n = 19) could recall having had malaria during the past five years. Analysis of a subset of samples (n = 1322) from the two districts using PCR showed that none of the participants had hidden parasites. DISCUSSION: A reservoir of hidden parasites is unlikely to be a major concern or a barrier to the ongoing malaria elimination efforts in Sri Lanka. However, as very low numbers of indigenous cases are still recorded, an island-wide assessment and in particular, continued alertness and follow up action are still needed. The findings of this study indicate that any future assessments should be based on an adaptive sampling approach, involving prompt sampling of all subjects within a specified radius, whenever a malaria case is identified in a given focus.

Models for short term malaria prediction in Sri Lanka.

BACKGROUND: Malaria in Sri Lanka is unstable and fluctuates in intensity both spatially and temporally. Although the case counts are dwindling at present, given the past history of resurgence of outbreaks despite effective control measures, the control programmes have to stay prepared. The availability of long time series of monitored/diagnosed malaria cases allows for the study of forecasting models, with an aim to developing a forecasting system which could assist in the efficient allocation of resources for malaria control. METHODS: Exponentially weighted moving average models, autoregressive integrated moving average (ARIMA) models with seasonal components, and seasonal multiplicative autoregressive integrated moving average (SARIMA) models were compared on monthly time series of district malaria cases for their ability to predict the number of malaria cases one to four months ahead. The addition of covariates such as the number of malaria cases in neighbouring districts or rainfall were assessed for their ability to improve prediction of selected (seasonal) ARIMA models. RESULTS: The best model for forecasting and the forecasting error varied strongly among the districts. The addition of rainfall as a covariate improved prediction of selected (seasonal) ARIMA models modestly in some districts but worsened prediction in other districts. Improvement by adding rainfall was more frequent at larger forecasting horizons. CONCLUSION: Heterogeneity of patterns of malaria in Sri Lanka requires regionally specific prediction models. Prediction error was large at a minimum of 22% (for one of the districts) for one month ahead predictions. The modest improvement made in short term prediction by adding rainfall as a covariate to these prediction models may not be sufficient to merit investing in a forecasting system for which rainfall data are routinely processed.

Temporal correlation between malaria and rainfall in Sri Lanka.

BACKGROUND: Rainfall data have potential use for malaria prediction. However, the relationship between rainfall and the number of malaria cases is indirect and complex. METHODS: The statistical relationships between monthly malaria case count data series and monthly mean rainfall series (extracted from interpolated station data) over the period 1972 - 2005 in districts in Sri Lanka was explored in four analyses: cross-correlation; cross-correlation with pre-whitening; inter-annual; and seasonal inter-annual regression. RESULTS: For most districts, strong positive correlations were found for malaria time series lagging zero to three months behind rainfall, and negative correlations were found for malaria time series lagging four to nine months behind rainfall. However, analysis with pre-whitening showed that most of these correlations were spurious. Only for a few districts, weak positive (at lags zero and one) or weak negative (at lags two to six) correlations were found in pre-whitened series. Inter-annual analysis showed strong negative correlations between malaria and rainfall for a group of districts in the centre-west of the country. Seasonal inter-annual analysis showed that the effect of rainfall on malaria varied according to the season and geography. CONCLUSION: Seasonally varying effects of rainfall on malaria case counts may explain weak overall cross-correlations found in pre-whitened series, and should be taken into account in malaria predictive models making use of rainfall as a covariate.

Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in the Plasmodium vivax dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pvdhps) genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP). Monitoring these SNPs provide insights into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ) with primaquine (PQ) and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious against P. vivax infections, thus SP is rarely used and it is assumed that the prevalence of SNPs related to P. vivax SP resistance is low. However, this has not been assessed in Sri Lanka as in most other parts of Asia. This study describes the prevalence and distribution of SNPs related to P. vivax SP resistance across Sri Lanka. SUBJECTS AND METHODS: P. vivax-positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene and 383, 553 and 585 of the Pvdhps gene by applying PCR followed by a hybridization step using sequence specific oligonucleotide probes (SSOPs) in an ELISA format. RESULTS: In the study period, the government of Sri Lanka recorded 2,149 P. vivax cases from the nine districts out of which, 454 (21.1%) blood samples were obtained. Pvdhfr haplotypes could be constructed for 373 of these. The FSTS wild-haplotype was represented in 257 samples (68.9%), the double mutant LRTS haplotype was the most frequently observed mutant (24.4%) while the triple mutation (LRTN) was only identified once. Except for two samples of the single mutated Pvdhps GAV haplotype, the remaining samples were wildtype. Geographical differences were apparent, notably a significantly higher frequency of mutant Pvdhfr haplotypes was observed in the Northern districts. CONCLUSION: Since SP is rarely used in Sri Lanka, the high frequency and diversity of Pvdhfr mutations was unexpected indicating the emergence of drug resistant parasites despite a low level of SP drug pressure.

Malaria in Sri Lanka: one year post-tsunami.

One year ago, the authors of this article reported in this journal on the malaria situation in Sri Lanka prior to the tsunami that hit on 26 December 2004, and estimated the likelihood of a post-tsunami malaria outbreak to be low. Malaria incidence has decreased in 2005 as compared to 2004 in most districts, including the ones that were hit hardest by the tsunami. The malaria incidence (aggregated for the whole country) in 2005 followed the downward trend that started in 2000. However, surveillance was somewhat affected by the tsunami in some coastal areas and the actual incidence in these areas may have been higher than recorded, although there were no indications of this and it is unlikely to have affected the overall trend significantly. The focus of national and international post tsunami malaria control efforts was supply of antimalarials, distribution of impregnated mosquito nets and increased monitoring in the affected area. Internationally donated antimalarials were either redundant or did not comply with national drug policy, however, few seem to have entered circulation outside government control. Despite distribution of mosquito nets, still a large population is relatively exposed to mosquito bites due to inadequate housing. There were no indications of increased malaria vector abundance. Overall it is concluded that the tsunami has not negatively influenced the malaria situation in Sri Lanka.

Combined Effects of Pesticides and Trematode Infections on Hourglass Tree Frog Polypedates cruciger.

The impact of widespread and common environmental factors, such as chemical contaminants, on infectious disease risk in amphibians is particularly important because both chemical contaminants and infectious disease have been implicated in worldwide amphibian declines. Here we report on the lone and combined effects of exposure to parasitic cercariae (larval stage) of the digenetic trematode, Acanthostomum burminis, and four commonly used pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) at ecologically relevant concentrations on the survival, growth, and development of the common hourglass tree frog, Polypedates cruciger Blyth 1852. There was no evidence of any pesticide-induced mortality on cercariae because all the cercariae successfully penetrated each tadpole host regardless of pesticide treatment. In isolation, both cercarial and pesticide exposure significantly decreased frog survival, development, and growth, and increased developmental malformations, such as scoliosis, kyphosis, and also edema and skin ulcers. The combination of cercariae and pesticides generally posed greater risk to frogs than either factor alone by decreasing survival or growth or increasing time to metamorphosis or malformations. The exception was that lone exposure to chlorpyrifos had higher mortality without than with cercariae. Consistent with mathematical models that suggest that stress should increase the impact of generalist parasites, the weight of the evidence from the field and laboratory suggests that ecologically relevant concentrations of agrochemicals generally increase the threat that trematodes pose to amphibians, highlighting the importance of elucidating interactions between anthropogenic activities and infectious disease in taxa of conservation concern.

Maps of the Sri Lanka malaria situation preceding the tsunami and key aspects to be considered in the emergency phase and beyond.

BACKGROUND: Following the tsunami, a detailed overview of the area specific transmission levels is essential in assessing the risk of malaria in Sri Lanka. Recent information on vector insecticide resistance, parasite drug resistance, and insights into the national policy for malaria diagnosis and treatment are important in assisting national and international agencies in their control efforts. METHODS: Monthly records over the period January 1995-October 2004 of confirmed malaria cases were used to perform an analysis of malaria distribution at district spatial resolution. Also, a focused review of published reports and routinely collected information was performed. RESULTS: The incidence of malaria was only 1 case per thousand population in the 10 months leading up to the disaster, in the districts with the highest transmission. CONCLUSION: Although relocated people may be more exposed to mosquito bites, and their capacity to handle diseases affected, the environmental changes caused by the tsunami are unlikely to enhance breeding of the principal vector, and, given the present low parasite reservoir, the likelihood of a malaria outbreak is low. However, close monitoring of the situation is necessary, especially as December-February is normally the peak transmission season. Despite some losses, the Sri Lanka public health system is capable of dealing with the possible threat of a malaria outbreak after the tsunami. The influx of foreign medical assistance, drugs, and insecticides may interfere with malaria surveillance, and the long term malaria control strategy of Sri Lanka, if not in accordance with government policy.

Optimizing malarial epidemiological studies in areas of low transmission.

Malaria risk factor studies have traditionally used microscopy readings of blood slides as the measure of malaria infection in humans, although alternatives are available. There is the need for an assessment of how the use of these alternative diagnostic approaches will influence the efficiency and significance of epidemiological studies. In an area of Sri Lanka with known risk factors for malaria, two cross-sectional surveys were done at the start and at the peak of transmission season. Microscopy was compared with enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR). The major risk factor in this area was the location of houses relative to confirmed vector breeding sites. At the peak of the transmission season, the results pointed in the same direction, irrespective of the diagnostic method used. However, the importance of distance from the breeding site was not statistically significant when microscopy was used, which can be explained by the lower prevalence of microscopy positivity in comparison to the prevalence of ELISA- and PCR-positivity. This study suggests that in low-transmission areas, such as Sri Lanka, smaller sample sizes can be used for epidemiological research studies using PCR instead of microscopy to estimate parasite prevalence. This efficiency gain has to be weighed against the higher cost and complexity of the PCR. PCR cannot replace microscopy as the standard diagnostic procedure at the field level. ELISA is not directly comparable with microscopy and PCR but it can also be a useful tool in malaria epidemiological studies. This study indicates that cross-sectional surveys are only efficient if they take place during peak transmission season. Cross sectional surveys currently implemented by the Sri Lankan government in response to local malaria outbreaks can form the basis for valid epidemiological studies and be used for the generation of malaria risk maps if samples were also analyzed using PCR.

Multiple Origins of Mutations in the mdr1 Gene--A Putative Marker of Chloroquine Resistance in P. vivax.

BACKGROUND: Chloroquine combined with primaquine has been the recommended antimalarial treatment of Plasmodium vivax malaria infections for six decades but the efficacy of this treatment regimen is threatened by chloroquine resistance (CQR). Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene, Pvmdr1 are putative determinants of CQR but the extent of their emergence at population level remains to be explored. OBJECTIVE: In this study we describe the prevalence of SNPs in the Pvmdr1 among samples collected in seven P. vivax endemic countries and we looked for molecular evidence of drug selection by characterising polymorphism at microsatellite (MS) loci flanking the Pvmdr1 gene. METHODS: We examined the prevalence of SNPs in the Pvmdr1 gene among 267 samples collected from Pakistan, Afghanistan, Sri Lanka, Nepal, Sudan, São Tomé and Ecuador. We measured and diversity in four microsatellite (MS) markers flanking the Pvmdr1 gene to look evidence of selection on mutant alleles. RESULTS: SNP polymorphism in the Pvmdr1 gene was largely confined to codons T958M, Y976F and F1076L. Only 2.4% of samples were wildtype at all three codons (TYF, n = 5), 13.3% (n = 28) of the samples were single mutant MYF, 63.0% of samples (n = 133) were double mutant MYL, and 21.3% (n = 45) were triple mutant MFL. Clear geographic differences in the prevalence of these Pvmdr mutation combinations were observed. Significant linkage disequilibrium (LD) between Pvmdr1 and MS alleles was found in populations sampled in Ecuador, Nepal and Sri Lanka, while significant LD between Pvmdr1 and the combined 4 MS locus haplotype was only seen in Ecuador and Sri Lanka. When combining the 5 loci, high level diversity, measured as expected heterozygosity (He), was seen in the complete sample set (He = 0.99), while He estimates for individual loci ranged from 0.00-0.93. Although Pvmdr1 haplotypes were not consistently associated with specific flanking MS alleles, there was significant differentiation between geographic sites which could indicate directional selection through local drug pressure. CONCLUSIONS: Our observations suggest that Pvmdr1 mutations emerged independently on multiple occasions even within the same population. In Sri Lanka population analysis at multiple sites showed evidence of local selection and geographical dispersal of Pvmdr1 mutations between sites.

Towards a risk map of malaria for Sri Lanka: the importance of house location relative to vector breeding sites.

BACKGROUND: In Sri Lanka, the major malaria vector Anopheles culicifacies breeds in pools formed in streams and river beds and it is likely that people living close to such breeding sites are at higher risk of malaria than people living further away. This study was done to quantify the importance of house location relative to vector breeding sites for the occurrence of malaria in order to assess the usefulness of this parameter in future malaria risk maps. Such risk maps could be important tools for planning efficient malaria control measures. METHODS: In a group of seven villages in north central Sri Lanka, malaria cases were compared with community controls for distance from house to breeding sites and a number of other variables, including type of housing construction and use of anti-mosquito measures. The presence of An. culicifacies in bedrooms was determined by indoor insecticide spray collections. RESULTS: People living within 750 m of the local stream, which was the established vector-breeding site, were at much higher risk for malaria than people living further away (odds ratio adjusted for confounding by other variables 5.93, 95% CI: 3.50-8.91). Houses close to the stream also had more adult An. culicifacies in the bedrooms. Poor housing construction was an independent risk factor for malaria. CONCLUSIONS: Risk maps of malaria in Sri Lanka can be based on the location of houses relative to streams and rivers that are potential breeding sites for the malaria vector An. culicifacies. A distance of 750 m is suggested as the cut-off point in defining low- and high-risk villages.

Generalized seasonal autoregressive integrated moving average models for count data with application to malaria time series with low case numbers.

INTRODUCTION: With the renewed drive towards malaria elimination, there is a need for improved surveillance tools. While time series analysis is an important tool for surveillance, prediction and for measuring interventions' impact, approximations by commonly used Gaussian methods are prone to inaccuracies when case counts are low. Therefore, statistical methods appropriate for count data are required, especially during "consolidation" and "pre-elimination" phases. METHODS: Generalized autoregressive moving average (GARMA) models were extended to generalized seasonal autoregressive integrated moving average (GSARIMA) models for parsimonious observation-driven modelling of non Gaussian, non stationary and/or seasonal time series of count data. The models were applied to monthly malaria case time series in a district in Sri Lanka, where malaria has decreased dramatically in recent years. RESULTS: The malaria series showed long-term changes in the mean, unstable variance and seasonality. After fitting negative-binomial Bayesian models, both a GSARIMA and a GARIMA deterministic seasonality model were selected based on different criteria. Posterior predictive distributions indicated that negative-binomial models provided better predictions than Gaussian models, especially when counts were low. The G(S)ARIMA models were able to capture the autocorrelation in the series. CONCLUSIONS: G(S)ARIMA models may be particularly useful in the drive towards malaria elimination, since episode count series are often seasonal and non-stationary, especially when control is increased. Although building and fitting GSARIMA models is laborious, they may provide more realistic prediction distributions than do Gaussian methods and may be more suitable when counts are low.

Malformations and mortality in the Asian Common Toad induced by exposure to pleurolophocercous cercariae (Trematoda: Cryptogonimidae).

Malformations and increased mortality due to infection by the digenetic trematode, Riberioa ondatrae have been reported for many species of amphibians. Severe malformations have also been reported in the Common Hourglass Tree Frog, Polypedates cruciger induced by pleurolophocercous cercariae in Sri Lanka in addition to the changes in the behaviour, development and survival of the host. We exposed pre-limb bud stage tadpoles (Gosner stages 25-26) of the Asian Common Toad, Duttaphrynus melanostictus to the same pleurolophocercous type cercariae under laboratory conditions. Molecular and morphological identification showed that these cercariae belonged Acanthostomum burminis infecting freshwater snakes as definitive hosts. These cercariae induced malformations (27.8%) and reduced survival to metamorphosis (53.8%). The magnitude of the effects increased with the dose of cercariae. Types of malformations were mainly axial, such as scoliosis and kyphosis. Severe limb malformations such as extra or missing limbs as reported for amphibians exposed to R. ondatrae were not observed in the D. melanostictus. Same authors reported a higher percentage of malformations previously when P. cruciger was exposed to the cercariae A. burminis compared to D. melanostictus. However, tadpoles of D. melanostictus, which are smaller compared to those of P. cruciger, experienced higher mortality than P. cruciger tadpoles. Trematode induced malformations and mortality in amphibians are highly variable and depend on multiple factors such as host species differences such as resistance to infection and tolerance, life-history characteristics such as size at metamorphosis and length of the metamorphosis period, and other factors such as size of the amphibian at the time of trematode exposure.

Analysis of polymorphisms in the merozoite surface protein-3α gene and two microsatellite loci in Sri Lankan Plasmodium vivax: evidence of population substructure in Sri Lanka.

The geographical distribution of genetic variation in Plasmodium vivax samples (N = 386) from nine districts across Sri Lanka is described using three markers; the P. vivax merozoite surface protein-3α (Pvmsp-3α) gene, and the two microsatellites m1501 and m3502. At Pvmsp-3α, 11 alleles were found with an expected heterozygosity (H(e)) of 0.81, whereas at m1501 and m3502, 24 alleles (H(e) = 0.85) and 8 alleles (H(e) = 0.74) were detected, respectively. Overall, 95 unique three locus genotypes were detected among the 279 samples positive at all three loci (H(e) = 0.95). Calculating the pairwise fixation index (F(ST)) revealed statistically significant population structure. The presence of identical 2-loci microsatellite genotypes in a significant proportion of samples revealed local clusters of closely related isolates contributing to strong linkage disequilibrium between marker alleles. The results show evidence of high genetic diversity and possible population substructure of P. vivax populations in Sri Lanka.

Geographic structure of Plasmodium vivax: microsatellite analysis of parasite populations from Sri Lanka, Myanmar, and Ethiopia.

Genetic diversity and population structure of Plasmodium vivax parasites can predict the origin and spread of novel variants within a population enabling population specific malaria control measures. We analyzed the genetic diversity and population structure of 425 P. vivax isolates from Sri Lanka, Myanmar, and Ethiopia using 12 trinucleotide and tetranucleotide microsatellite markers. All three parasite populations were highly polymorphic with 3-44 alleles per locus. Approximately 65% were multiple-clone infections. Mean genetic diversity (H(E)) was 0.7517 in Ethiopia, 0.8450 in Myanmar, and 0.8610 in Sri Lanka. Significant linkage disequilibrium was maintained. Population structure showed two clusters (Asian and African) according to geography and ancestry. Strong clustering of outbreak isolates from Sri Lanka and Ethiopia was observed. Predictive power of ancestry using two-thirds of the isolates as a model identified 78.2% of isolates accurately as being African or Asian. Microsatellite analysis is a useful tool for mapping short-term outbreaks of malaria and for predicting ancestry.

Malaria seasonality and rainfall seasonality in Sri Lanka are correlated in space.

Correlation in space between seasonality of malaria and seasonality of rainfall was studied in Sri Lanka. A simple seasonality index was developed by making use of the bimodal seasonality of both malaria and rainfall. The malaria seasonality index was regressed against the rainfall seasonality index taking spatial autocorrelation into account. Despite the presence of spatial autocorrelation, the coefficient for the rainfall seasonality index in explaining the malaria seasonality index was found to be significant. The results suggest that rainfall is an important driver of malaria seasonality.