Characterization of antigens of Enterobius vermicularis (pinworm) eggs.

Enterobiasis (pinworm infection) caused by Enterobius vermicularis is a common parasitic infection prevalent worldwide especially in children. Infection is diagnosed by microscopic detection of E. vermicularis eggs on perianal swabs. This study aimed to characterize the antigens of E. vermicularis eggs as a preliminary step towards identifying diagnostic targets for detection in infected individuals. The study was conducted between October 2019 and February 2020, following approval from Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC-19-034). E. vermicularis eggs were harvested from perianal swabs using acetone and purified with 1× PBS (pH 7.2). A portion of eggs was used for preparing antigen slides, while the rest were sonicated and vortexed with glass beads and inoculated subcutaneously (with weekly booster doses) into a Wistar rat for developing antibodies. Blood drawing from rat was done weekly for 5 weeks. Confirmation of the presence of antibodies was done by surface immunofluorescence against eggs on the antigen slides. Protein bands were determined using SDS-PAGE assay and immunogenic antigen bands were determined by reacting with antiserum after immunoblotting. The band sizes of the proteins were determined against corresponding bands of a protein ladder. Surface immunofluorescence was positive with serum obtained from day 14 post-inoculation from the Wistar rat as well as that obtained from a person with chronic enterobiasis. The most prominent and immunogenic protein bands identified from egg antigens were 21 kDa, 66 kDa, 83 kDa, 96 kDa, 112 kDa, 121 kDa, 140 kDa and 151 kDa. Methods used in this study were effective in obtaining E. vermicularis egg antigens which were immunogenic. Furthermore, surface antigens of intact eggs reacted with antibodies developed against crushed egg antigens. These findings may pave the way for the development of effective immunodiagnostics.

Dynamics of maternally transferred antibodies against measles, mumps, and rubella in infants in Sri Lanka.

BACKGROUND: Determining the dynamics of maternally transferred antibodies against measles, mumps, and rubella infections in infants is important for making evidence-based policy decisions regarding the timing of vaccination. METHODS: The levels of serum immunoglobulin G (IgG) developed against measles, mumps, and rubella infections were assessed using commercial ELISA kits in mother-newborn pairs (n = 294) and 6-12-month-old infants (n = 280) recruited from Colombo District, Sri Lanka. Antibody levels of mothers and their newborns were assessed with respect to sex and parity. Antibody levels and the protection conferred were assessed in a sample of infants who completed 6-12 months of age in relation to their age and sex. Antibody levels were compared between different age and sex groups using the Mann-Whitney U-test, and correlations of antibody titers were performed using the Spearman correlation test. RESULTS: The prevalence rates of seropositivity for measles, mumps, and rubella were 91.5%, 89%, and 88%, respectively, in mothers, and 95%, 91.5%, and 93%, respectively, in their newborns. The newborns had mean IgG levels exceeding those of the mothers (P < 0.001). Mothers with natural infections had higher antibody levels compared to vaccinated mothers, which resulted in a higher level of maternal transfer. All of the infants who were 9-10 months of age or older were seronegative for measles, all of those who were 10-11 months of age or older were seronegative for rubella, and all of those who were 11-12 months old were seronegative for mumps. CONCLUSIONS: The maternal transfer of antibodies to newborns is efficient and renders protection until the infants are 6-7 months old in the case of mumps and rubella and 7-8 months old in the case of measles. Hence infants remain vulnerable to infections before the first dose of the MMR vaccine.

Ion Transport Modulators Differentially Modulate Inflammatory Responses in THP-1-Derived Macrophages.

Ion transport modulators are most commonly used to treat various noncommunicable diseases including diabetes and hypertension. They are also known to bind to receptors on various immune cells, but the immunomodulatory properties of most ion transport modulators have not been fully elucidated. We assessed the effects of thirteen FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, omeprazole, pantoprazole, phenytoin, verapamil, drug X, and drug Y on superoxide production, nitric oxide production, and cytokine expression by THP-1-derived macrophages that had been stimulated with ethanol-inactivated Mycobacterium bovis BCG. Ambroxol HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, pantoprazole, phenytoin, verapamil, and drug Y had an inhibitory effect on nitric oxide production, while all the test drugs had an inhibitory effect on superoxide production. Amiloride HCl, diazoxide, digoxin, furosemide, phenytoin, verapamil, drug X, and drug Y enhanced the expression of IL-1ß and TNF-α. Unlike most immunomodulatory compounds currently in clinical use, most of the test drugs inhibited some inflammatory processes while promoting others. Ion pumps and ion channels could therefore serve as targets for more selective immunomodulatory agents which do not cause overt immunosuppression.

Ion Transport Modulators as Antimycobacterial Agents.

There is an urgent need for better and safer therapeutic interventions for tuberculosis (TB). We assessed the effects of FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide (HCTZ), metformin, omeprazole, pantoprazole, phenytoin, verapamil, and drug X and Y on the growth of free and intracellular Mycobacterium bovis BCG. Free and intracellular M. bovis BCG were cultured in the presence or absence of the test drugs for 3 to 9 days and then quantified. For both free and intracellular bacteria, cultures that were exposed to furosemide, phenytoin, or drug Y yielded lower bacteria counts compared to drug-free controls (p < 0.05). The same was observed with diazoxide, HCTZ, verapamil, and drug X, but only for intracellular M. bovis BCG (p < 0.05). To assess the effects of the drugs on bactericidal activity of rifampicin, free and intracellular M. bovis BCG were treated with rifampicin alone or in combination with each of the thirteen test drugs for 3 to 9 days. For extracellular bacteria, higher bacteria clearance rates were observed in cultures exposed to rifampicin in combination with amiloride HCl, diazoxide, digoxin, furosemide, HCTZ, metformin, pantoprazole, phenytoin, drug X, or drug Y than those exposed to rifampicin alone, indicating that rifampicin had a synergistic effect with these test drugs. Rifampicin was also synergistic with ambroxol HCl, diazoxide, digoxin, furosemide, HCTZ, omeprazole, pantoprazole, phenytoin, verapamil, and drug X against intracellular M. bovis BCG. The antimycobacterial properties exhibited by the ion transport modulators in this study make them viable candidates as adjuncts to the current anti-TB regimens.

Assessment of environmental variability on malaria transmission in a malaria-endemic rural dry zone locality of Sri Lanka: The wavelet approach.

Malaria is a global public health concern and its dynamic transmission is still a complex process. Malaria transmission largely depends on various factors, including demography, geography, vector dynamics, parasite reservoir, and climate. The dynamic behaviour of malaria transmission has been explained using various statistical and mathematical methods. Of them, wavelet analysis is a powerful mathematical technique used in analysing rapidly changing time-series to understand disease processes in a more holistic way. The current study is aimed at identifying the pattern of malaria transmission and its variability with environmental factors in Kataragama, a malaria-endemic dry zone locality of Sri Lanka, using a wavelet approach. Monthly environmental data including total rainfall and mean water flow of the "Menik Ganga" river; mean temperature, mean minimum and maximum temperatures and mean relative humidity; and malaria cases in the Kataragama Medical Officer of Health (MOH) area were obtained from the Department of Irrigation, Department of Meteorology and Malaria Research Unit (MRU) of University of Colombo, respectively, for the period 1990 to 2005. Wavelet theory was applied to analyze these monthly time series data. There were two significant periodicities in malaria cases during the period of 1992-1995 and 1999-2000. The cross-wavelet power spectrums revealed an anti-phase correlation of malaria cases with mean temperature, minimum temperature, and water flow of "Menik Ganga" river during the period 1991-1995, while the in-phase correlation with rainfall is noticeable only during 1991-1992. Relative humidity was similarly associated with malaria cases between 1991-1992. It appears that environmental variables have contributed to a higher incidence of malaria cases in Kataragama in different time periods between 1990 and 2005.

Health in rural Sri Lanka: A cross-sectional survey of three rural districts

Introduction: Sri Lanka has a predominantly rural population. However, there is a dearth of research on health and socioeconomic issues in this group. Objective: To describe basic socioeconomic characteristics and health profile in a rural population. Methods: A descriptive cross-sectional household survey was conducted in 1950 households in three rural districts, selected by a three-stage stratified cluster sampling method. Results: The population pyramid showed an ageing population (dependency ratio of 50%). Only 39% had completed GCE (ordinary level). Unemployment rates were high (25% males, 76% females). Agriculture and related work were main occupations. Most lacked amenities (e.g. 61% households lacked a refrigerator) and practiced inappropriate methods of waste disposal (e.g. open burning by 72%). Household illnesses were frequent: episodes of acute illness within two weeks, injuries within past year and chronic illness were reported from 35.9%, 14.9% and 48.3% households. The prevalence of chronic diseases in adults >20 years were high: diabetes 13.5%, hypertension 16.7% and overweight/obesity 28.2%. Of the males, 22.1% smoked and 12.3% took alcohol. Almost 25% adults chewed betel. Reports of snake bite, dog bites and suicide/attempted suicide were seen in 15.5%, 9.7% and 3.0% households respectively. Conclusions: This study shows a unique clustering of health-related problems in rural Sri Lanka. This was characterized by demographic transition, burden from snake bites, chronic diseases and acute illnesses. There were resource limitations and low levels of education. Cohort studies and comparisons with urban areas will enable further elucidation of determinants of health and other issues in rural Sri Lanka.

Effectiveness of targeting fathers for breastfeeding promotion: systematic review and meta-analysis.

BACKGROUND: Further research gaps exist in relation to the promotion of breastfeeding. Robust scientific evidence obtained by a meta-analysis would provide objectively summarized data while enabling the assessment of consistency of findings. This review includes the first documented meta-analysis done on the effectiveness of targeting fathers for promoting breastfeeding (BF). Assessments have been done for a primary outcome and for six more secondary outcomes. METHODS: PubMed, EMBASE, Google Scholar, CENTRAL databases and unpublished researches were searched. Selections of randomized-controlled trials and quasi-experimental studies were done in three rounds. Heterogeneity and potential publication bias were assessed. Eight studies were included in meta-analysis and others in narrative synthesis of the outcomes. Pooling was done with the Mental- Haenszel method using risk ratio (RR). Summary-of-Findings table was composed by Review-Manager (version 5.3) and GRADEproGDT applications. Subsequent sensitivity analysis was done. RESULTS: Selected eight interventional studies included 1852 families. Exclusive BF at six months was significantly higher (RR = 2.04, CI = 1.58-2.65) in the intervention groups. The RR at 4 months was 1.52 (CI = 1.14 to 2.03). Risk of full-formula-feeding (RR = 0.69, CI = 0.52-0.93) and the occurrence of lactation-related problems were lower in the intervention groups (RR = 0.24, CI = 0.10-0.57). More likelihood of rendering support in BF-related issues was seen in intervention groups (RR = 1.43, CI = 1.22-1.68). Increase of maternal knowledge and favorable attitudes on BF were higher in the intervention groups (P ≤; 0.001). The quality of evidence according to GRADE was "low" (for one outcome), "moderate" (for four outcomes), and "high" (for two outcomes). CONCLUSIONS: Targeting fathers in promotion of BF has provided favorable results for all seven outcomes with satisfactory quality of evidence. This review was registered in the PROSPERO-registry (ID: 2017-CRD42017076163) prior to its commencement.

Strain-specific protective effect of the immunity induced by live malarial sporozoites under chloroquine cover.

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2-4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2-5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites' circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both.

Lineage-specific positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium vivax and related simian malaria parasites.

BACKGROUND: The 200 kDa merozoite surface protein 1 (MSP-1) of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors). It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity. RESULTS: We performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1) from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species-specifically on msp1. CONCLUSIONS: The present results indicate that the msp1 locus of P. vivax and related parasite species has lineage-specific unique evolutionary history with positive selection. P. vivax and related simian malaria parasites offer an interesting system toward understanding host species-dependent adaptive evolution of immune-target surface antigen genes such as msp1.

Recent independent evolution of msp1 polymorphism in Plasmodium vivax and related simian malaria parasites.

The Plasmodium MSP-1 is a promising malaria vaccine candidate. However, the highly polymorphic nature of the MSP-1 gene (msp1) presents a potential obstacle for effective vaccine development. To investigate the evolutionary history of msp1 polymorphism in P. vivax, we construct phylogenetic trees of msp1 from P. vivax and related monkey malaria parasite species. All P. vivax msp1 alleles cluster in the P. vivax lineage and are not distributed among other species. Similarly, all P. cynomolgi msp1 alleles cluster in the P. cynomolgi lineage. This suggests that, in contrast to presumed ancient origin of P. falciparum msp1 polymorphism, the origin of P. vivax msp1 polymorphism is relatively recent. We observed positive selection in the P. vivax lineage but not in P. cynomolgi. Also, positive selection acts on different regions of msp1 in P. vivax and P. falciparum. This study shows that the evolutionary history of msp1 differs greatly among parasite lineages.

Numerous, robust genetic markers for Plasmodium chabaudi by the method of amplified fragment length polymorphism.

We have used the method of amplified fragment length polymorphism (AFLP) to identify genetic polymorphisms between two cloned isolates of the rodent malaria parasite Plasmodium chabaudi chabaudi. The method employs polymerase chain reaction (PCR)-amplification of genomic DNA fragments cut with specific combinations of restriction endonucleases; we used EcoRI and Tru1I (isoschizomer of MseI). We have identified 819 parasite clone-specific AFLPs between P. c. chabaudi clones AS and AJ. Of these, 403 fragments were specific to AS and 416 to AJ. In preparing blood stage parasites for DNA, nucleated host cells were removed by successive filtration of infected blood through powdered cellulose and Plasmodipur filters. This reduced nucleated host cell contamination to around 1-10 per million parasite nuclei and reduced host DNA to below the limit of detection by the AFLP method. Analysis of our results showed that the total number of PCR-amplified fragments of parasite DNA was consistent with the predicted number of EcoRI sites in the parasite genome. 19.4% of all amplified fragments were P. c. chabaudi clone-specific. From this figure we estimated that the diversity between clones AS and AJ, measured as the probability of a sequence difference, was between about 8 x 10(-3) and 4.6 x 10(-4) per base pair. This is consistent with the sequence diversity found between alleles of candidate drug resistance genes from P. c. chabaudi clones AS and AJ identified and sequenced in this laboratory.