X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

Portable infrared imaging for longitudinal limb volume monitoring in patients with lymphatic filariasis.

BACKGROUND: The Global Programme to Eliminate Lymphatic Filariasis (LF) emphasizes hygiene, exercise, and other measures to reduce morbidity and disability related to LF. We recently reported that a portable, three-dimensional, infrared imaging system (3DIS) provides accurate limb volume measurements in patients with filarial lymphedema. To assess the practical utility of repeated 3DIS measurements for longitudinal lymphedema management, we examined intraday and day-to-day leg volume changes in adults with filarial lymphedema in southern Sri Lanka. METHODOLOGY AND PRINCIPAL FINDINGS: We assessed 41 participants with lower extremity lymphedema (stages 1-6) in their homes in the mornings (6:00-9:00 AM) and afternoons (2:00-6:00 PM) of three days within one calendar week. Two examiners performed replicate 3DIS volume measurements at each visit. Median coefficient of variation among replicate volume measurements was 1.7% (IQR 1.1% - 2.3%) for left legs and 2.2% (IQR 1.6% - 2.8%) for right legs. Median intraday volume increase was 3.0%. Range among daily volume measurements tended to be lower for afternoon measurements (median 2.25%, IQR 1.4%- 5.4%) than for morning measurements (median 3.0%, IQR 1.4% - 8.4%). CONCLUSIONS AND SIGNIFICANCE: Limb volume measurements by 3DIS are accurate and reproducible, and this technique is feasible for use in patients' homes. We have developed practical suggestions for optimal outcomes with 3DIS. Duplicate measurements should be performed and repeat assessments should be done at approximately the same time of day to minimize bias. Duplicate measures that vary by more than 8% should prompt review of scanning technique with a repeat measurement. With proper training and attention to technique, 3DIS can be a valuable tool for healthcare workers who work with lymphedema patients.

Systematic sampling of adults as a sensitive means of detecting persistence of lymphatic filariasis following mass drug administration in Sri Lanka.

BACKGROUND: Sri Lanka's Anti-Filariasis Campaign conducted 5 annual rounds of mass drug administration (MDA) with diethylcarbamazine (DEC) plus albendazole to eliminate lymphatic filariasis (LF) in all endemic districts between 2002 and 2006. Post-MDA surveillance has consistently documented Wuchereria bancrofti microfilaremia (Mf) rates below 1% in all sentinel and spot check sites since that time, and all implementation units easily satisfied WHO's target for school-based transmission assessment surveys (school-TAS) in 2013. However, more detailed studies have identified foci of persistent infection in the large coastal evaluation unit (EU) (population about 0.6 million) in Galle district. Therefore, the purpose of this study was to assess the sensitivity and feasibility of community-based TAS in adults (adult-TAS) and to compare results obtained by adult-TAS with prior school-TAS and molecular xenomonitoring (MX, molecular detection of filarial DNA in systematically sampled mosquitoes) results in this known problem area. METHODOLOGY AND PRINCIPAL FINDINGS: Two cluster surveys were performed in independent samples of 30 evaluation areas (EAs) in the coastal Galle EU in 2015. Each survey tested approximately 1,800 adults for circulating filarial antigenemia (CFA) with the Alere Filariasis Test Strip. The CFA prevalence for all persons tested (N = 3,612) was 1.8% (CI 1.4-2.2), and this was significantly higher than the CFA rate of 0.4% obtained by school-TAS in 2013. CFA prevalences in the two samples were similar [1.5% (CI 1.0-2.2), and 2.0% (CI 1.4-2.7)]. Antigenemia prevalence in sampled EUs was highly variable (range 0-11%), and it exceeded 5% in 6 EAs. The 30 EAs sampled in one of our adult-TAS surveys had recently been assessed for persistent filariasis by molecular xenomonitoring (MX). CFA prevalence in adults and filarial DNA prevalence in mosquitoes in these EAs were significantly correlated (r = 0.43; P = 0.02). CONCLUSIONS: Community based adult-TAS provided a reproducible measure of persistent W. bancrofti infection in a large evaluation unit in Sri Lanka that has low-level persistence of LF following multiple rounds of MDA. In addition, adult-TAS and MX results illustrate the focality of persistent LF in this setting. Adult-TAS may be more sensitive than school-TAS for this purpose. Adult-TAS and MX are potential options for post-MDA and post-validation surveillance programs to identify problem areas that require mop-up activities. Adult-TAS should also be useful for remapping areas with uncertain LF endemicity for possible inclusion in national LF elimination programs.

Comprehensive Assessment of a Hotspot with Persistent Bancroftian Filariasis in Coastal Sri Lanka.

The Sri Lankan Anti-Filariasis campaign distributed five rounds of mass drug administration (MDA with diethylcarbamazine plus albendazole) to some 10 million people in eight districts between 2002 and 2006. Sri Lanka was recognized by the WHO for having eliminated lymphatic filariasis (LF) as a public health problem in 2016. However, recent studies by our group documented pockets with persistent LF in coastal Sri Lanka, especially in Galle district. The present study was performed to reexamine an area previously identified as a potential hotspot for persistent LF (Balapitiya Public Health Inspector area, population 17,500). A community survey documented high rates for circulating filarial antigenemia (3%, confidence interval [CI]: 1.8-4.9) and microfilaremia (1%, CI: 0.5-2.5%). Circulating filarial antigenemia rates were 2.8-fold higher in males than females. High prevalence was also observed for anti-filarial antibodies in young children (5.7%, CI: 3.7-8.4%) and for filarial DNA in vector mosquitoes (5.2%, CI: 4.2-6.3%). Spatial data showed that persistent LF was dispersed across the entire study area. Other studies showed that persistent LF was not limited to Balapitiya and not solved by additional rounds of MDA. Molecular xenomonitoring studies conducted in 2016 in 22 of 168 Public Health Midwife areas in the coastal Galle evaluation unit (approximate population 600,000) found that 179 of 660 (27%) pools of Culex collected from all areas were positive for Wuchereria bancrofti DNA by quantitative polymerase chain reaction; the estimated infection rate in mosquitoes was 1.26%, CI: 1.0-1.5%. Interventions other than routine MDA will be required to remove LF hotspots in Balapitiya and in other areas in coastal Sri Lanka.

Use of a Novel Portable Three-Dimensional Imaging System to Measure Limb Volume and Circumference in Patients with Filarial Lymphedema.

The World Health Organization's Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional imaging system (3DIS) against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of imaging system measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0-6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). 3DIS measurements correlated nearly perfectly with WD (r2 = 0.9945) and TMLC values (r2 > 0.9801). The median time required to acquire imaging system measurements for both legs was 2.1 minutes, compared with 17, 7, and 29 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5-2.1%) for WD and 1.7% (IQR 1.2-2.4%) for the 3DIS. CVs for circumference measurements were 1.4% (IQR 0.8-2.4%) by TMLC and 1.3% (0.8-1.9%) by 3DIS. Median interexaminer CV for STU was 13.7% (IQR 8.5-21.3%). The portable imaging system noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Reassessment of areas with persistent Lymphatic Filariasis nine years after cessation of mass drug administration in Sri Lanka.

BACKGROUND: Sri Lanka was one of the first countries to initiate a lymphatic filariasis (LF) elimination program based on WHO guidelines. The Anti-Filariasis Campaign provided 5 annual rounds of mass drug administration (MDA) with diethylcarbamazine plus albendazole in all 8 endemic districts from 2002-2006. Microfilaremia (Mf) prevalences have been consistently <1% in all sentinel and spot-check sites since 2006, and all evaluation units passed school-based transmission assessment surveys (TAS) in 2013. We previously reported results from comprehensive surveillance studies conducted in 2011-2013 that documented low-level persistence of Wuchereria bancrofti in 19 high risk areas in 8 endemic districts. We now present results from repeat surveys conducted 3 to 4 years later in 6 areas that had the strongest LF signals in the prior study. METHODOLOGY AND PRINCIPAL FINDINGS: The surveys assessed prevalence of filarial antigenemia (CFA) and Mf in communities, CFA and anti-filarial antibody in school children (ages 6-8), and filarial DNA in Culex mosquitoes (molecular xenomonitoring, MX). Three study areas had significantly improved infection parameters compared to the prior study, but three other areas had little change. MX was more sensitive for detecting W. bancrofti persistence, and it was a better predictor than other parameters. Adult males accounted for more than 80% of infections detected in the study. CONCLUSIONS: These results suggest that W. bancrofti transmission was near the break point in some of the areas studied in 2011-13. LF is likely to decline to zero without further intervention in these areas, while other areas may require further intervention. Long term surveillance may be needed to verify W. bancrofti elimination in areas like Sri Lanka with efficient transmission by Culex. Test and treat or other programs targeting adult males plus bed net promotion may be more effective than MDA for clearing remaining hotspots of transmission in Sri Lanka.

Analysis of age-dependent trends in Ov16 IgG4 seroprevalence to onchocerciasis.

BACKGROUND: Diagnostics provide a means to measure progress toward disease elimination. Many countries in Africa are approaching elimination of onchocerciasis after successful implementation of mass drug administration programs as well as vector control. An understanding of how markers for infection such as skin snip microfilaria and Onchocerca volvulus-specific seroconversion perform in near-elimination settings informs how to best use these markers. METHODS: All-age participants from 35 villages in Togo were surveyed in 2013 and 2014 for skin snip Onchocerca volvulus microfilaria and IgG4 antibody response by enzyme-linked immunosorbent assay (ELISA) to the Onchocerca volvulus-specific antigen Ov16. A Gaussian mixture model applying the expectation-maximization (EM) algorithm was used to determine seropositivity from Ov16 ELISA data. For a subset of participants (n = 434), polymerase chain reaction (PCR) was performed on the skin snips taken during surveillance. RESULTS: Within the 2,005 participants for which there was Ov16 ELISA data, O. volvulus microfilaremia prevalence and Ov16 seroprevalence were, 2.5 and 19.7 %, respectively, in the total population, and 1.6 and 3.6 % in children under 11. In the subset of 434 specimens for which ELISA, PCR, and microscopy data were generated, it was found that in children under 11 years of age, the anti-Ov16 IgG4 antibody response demonstrate a sensitivity and specificity of 80 and 97 %, respectively, against active infections as determined by combined PCR and microscopy on skin snips. Further analysis was performed in 34 of the 35 villages surveyed. These villages were stratified by all-age seroprevalence into three clusters: < 15 %; 15-20 %; and > 20 %. Age-dependence of seroprevalence for each cluster was best reflected by a two-phase force-of-infection (FOI) catalytic model. In all clusters, the lower of the two phases of FOI was associated with a younger age group, as reflected by the seroconversion rates for each phase. The age at which transition from lower to higher seroconversion, between the two phases of FOI, was found to be highest (older) for the cluster of villages with < 15 % seroprevalence and lowest (younger) for the cluster with the highest all-age seroprevalence. CONCLUSIONS: The anti-Ov16 IgG4 antibody response is an accurate marker for active infection in children under 11 years of age in this population. Applying Ov16 surveillance to a broader age range provides additional valuable information for understanding progression toward elimination and can inform where targeted augmented interventions may be needed. Clustering of villages by all-age sero-surveillance allowed application of a biphasic FOI model to differentiate seroconversion rates for different age groups within the village cluster categories.

Programmatic Use of Molecular Xenomonitoring at the Level of Evaluation Units to Assess Persistence of Lymphatic Filariasis in Sri Lanka.

BACKGROUND: Sri Lanka's Anti Filariasis Campaign distributed 5 rounds of mass drug administration (MDA with DEC plus albendazole) to all endemic regions in the country from 2002-2006. Post-MDA surveillance results have generally been encouraging. However, recent studies have documented low level persistence of Wuchereria bancrofti in Galle district based on comprehensive surveys that include molecular xenomonitoring (MX, detection of filarial DNA in mosquitoes) results. The purposes of this study were to demonstrate the use of MX in large evaluation units (EUs) and to field test different mosquito sampling schemes. METHODOLOGY/PRINCIPAL FINDINGS: Galle district (population 1.1 million) was divided into two EUs. These included a coastal EU with known persistent LF and an inland EU with little persistent LF. Mosquitoes were systematically sampled from ~300 trap locations in 30 randomly selected clusters (health administrative units) per EU. Approximately 28,000 Culex quinquefasciatus were collected with gravid traps and tested for filarial DNA by qPCR. 92/625 pools (14.7%) from the coastal EU and 8/583 pools (1.4%) from the inland EU were positive for filarial DNA. Maximum likelihood estimates (MLE) for filarial DNA rates were essentially the same when the same number of mosquito pools were collected and tested from 75, 150, or 300 trap sites (range 0.61-0.78% for the coastal EU and 0.04-0.07% for the inland EU). The ability to use a smaller number of trap sites reduces the cost and time required for mosquito sampling. CONCLUSIONS/SIGNIFICANCE: These results suggest there is widespread persistence of W. bancrofti infection in the coastal Galle EU 8 years after the last round of MDA in 2006, and this is consistent with other data from the district. This study has shown that MX can be used by national programs to assess and map the persistence of W. bancrofti at the level of large EUs in areas with Culex transmission.

A comparison of two tests for filarial antigenemia in areas in Sri Lanka and Indonesia with low-level persistence of lymphatic filariasis following mass drug administration.

BACKGROUND: Filarial antigen tests are key tools for mapping the distribution of bancroftian filariasis and for detecting areas with persistent infections following mass drug administration (MDA). A recent study showed that the new Alere Filariasis Test Strip (FTS) has better analytical sensitivity than the BinaxNOW Filariasis card test (Card Test) for detecting circulating filarial antigen, and the FTS detected more positive results than the Card Test in a field study performed in a highly endemic area in Liberia. METHODS: The present study compared the performance of the FTS and the Card Test in community surveys that were conducted in southern Sri Lanka and in Indonesia (Central Java) in areas with low-level persistence of LF following multiple rounds of MDA with diethylcarbamazine plus albendazole. The studies were performed in densely populated semi-urban areas where Wuchereria bancrofti is transmitted by Culex quinquefasciatus. RESULTS: Antigenemia rates by FTS were 138% higher in the Sri Lanka study (43/852 vs. 18/852) and 21% higher in the Indonesia study (50/778 vs. 41/778) than antigenemia rates by Card Test. Antigenemia rates were significantly higher in males than in females and higher in adults than in children in both study sites. Although overall antigenemia rates and test scores were significantly higher by FTS than by Card Test in both study areas, rates in young children were similar with both tests in both areas. CONCLUSIONS: These results extend the previously reported superior sensitivity of the FTS to areas with low residual infection rates following MDA, and this could affect mapping and post-MDA survey results in adults. However, our findings suggest that results of transmission assessment surveys (TAS) performed in school-aged children are likely to be similar with both tests.

A comprehensive assessment of lymphatic filariasis in Sri Lanka six years after cessation of mass drug administration.

BACKGROUND: The Sri Lankan Anti-Filariasis Campaign conducted 5 rounds of mass drug administration (MDA) with diethycarbamazine plus albendazole between 2002 and 2006. We now report results of a comprehensive surveillance program that assessed the lymphatic filariasis (LF) situation in Sri Lanka 6 years after cessation of MDA. METHODOLOGY AND PRINCIPAL FINDINGS: Transmission assessment surveys (TAS) were performed per WHO guidelines in primary school children in 11 evaluation units (EUs) in all 8 formerly endemic districts. All EUs easily satisfied WHO criteria for stopping MDA. Comprehensive surveillance was performed in 19 Public Health Inspector (PHI) areas (subdistrict health administrative units). The surveillance package included cross-sectional community surveys for microfilaremia (Mf) and circulating filarial antigenemia (CFA), school surveys for CFA and anti-filarial antibodies, and collection of Culex mosquitoes with gravid traps for detection of filarial DNA (molecular xenomonitoring, MX). Provisional target rates for interruption of LF transmission were community CFA <2%, antibody in school children <2%, and filarial DNA in mosquitoes <0.25%. Community Mf and CFA prevalence rates ranged from 0-0.9% and 0-3.4%, respectively. Infection rates were significantly higher in males and lower in people who denied prior treatment. Antibody rates in school children exceeded 2% in 10 study sites; the area that had the highest community and school CFA rates also had the highest school antibody rate (6.9%). Filarial DNA rates in mosquitoes exceeded 0.25% in 10 PHI areas. CONCLUSIONS: Comprehensive surveillance is feasible for some national filariasis elimination programs. Low-level persistence of LF was present in all study sites; several sites failed to meet provisional endpoint criteria for LF elimination, and follow-up testing will be needed in these areas. TAS was not sensitive for detecting low-level persistence of filariasis in Sri Lanka. We recommend use of antibody and MX testing as tools to complement TAS for post-MDA surveillance.

Integrated school-based surveillance for soil-transmitted helminth infections and lymphatic filariasis in Gampaha district, Sri Lanka.

We explored the practicality of integrating surveillance for soil-transmitted helminthiasis (STH, assessed by Kato-Katz) with transmission assessment surveys for lymphatic filariasis (LF) in two evaluation units (EUs) in Gampaha district, Sri Lanka (population 2.3 million). The surveys were performed 6 years after five annual rounds of mass drug administration with diethylcarbamazine and albendazole. Each transmission assessment survey tested children (N = 1,462 inland EU; 1,642 coastal EU) sampled from 30 primary schools. Low filarial antigenemia rates (0% and 0.1% for the inland and coastal EUs) suggest that LF transmission is very low in this district. The STH rates and stool sample participation rates were 0.8% and 61% (inland) and 2.8% and 58% (coastal). Most STH detected were low or moderate intensity Trichuris trichiura infections. The added cost of including STH testing was ∼$5,000 per EU. These results suggest that it is feasible to integrate school-based surveillance for STH and LF.

Transmission assessment surveys (TAS) to define endpoints for lymphatic filariasis mass drug administration: a multicenter evaluation.

BACKGROUND: Lymphatic filariasis (LF) is targeted for global elimination through treatment of entire at-risk populations with repeated annual mass drug administration (MDA). Essential for program success is defining and confirming the appropriate endpoint for MDA when transmission is presumed to have reached a level low enough that it cannot be sustained even in the absence of drug intervention. Guidelines advanced by WHO call for a transmission assessment survey (TAS) to determine if MDA can be stopped within an LF evaluation unit (EU) after at least five effective rounds of annual treatment. To test the value and practicality of these guidelines, a multicenter operational research trial was undertaken in 11 countries covering various geographic and epidemiological settings. METHODOLOGY: The TAS was conducted twice in each EU with TAS-1 and TAS-2 approximately 24 months apart. Lot quality assurance sampling (LQAS) formed the basis of the TAS survey design but specific EU characteristics defined the survey site (school or community), eligible population (6-7 year olds or 1(st)-2(nd) graders), survey type (systematic or cluster-sampling), target sample size, and critical cutoff (a statistically powered threshold below which transmission is expected to be no longer sustainable). The primary diagnostic tools were the immunochromatographic (ICT) test for W. bancrofti EUs and the BmR1 test (Brugia Rapid or PanLF) for Brugia spp. EUs. PRINCIPAL FINDINGS/CONCLUSIONS: In 10 of 11 EUs, the number of TAS-1 positive cases was below the critical cutoff, indicating that MDA could be stopped. The same results were found in the follow-up TAS-2, therefore, confirming the previous decision outcome. Sample sizes were highly sex and age-representative and closely matched the target value after factoring in estimates of non-participation. The TAS was determined to be a practical and effective evaluation tool for stopping MDA although its validity for longer-term post-MDA surveillance requires further investigation.

Using existing drugs as leads for broad spectrum anthelmintics targeting protein kinases.

As one of the largest protein families, protein kinases (PKs) regulate nearly all processes within the cell and are considered important drug targets. Much research has been conducted on inhibitors for PKs, leading to a wealth of compounds that target PKs that have potential to be lead anthelmintic drugs. Identifying compounds that have already been developed to treat neglected tropical diseases is an attractive way to obtain lead compounds inexpensively that can be developed into much needed drugs, especially for use in developing countries. In this study, PKs from nematodes, hosts, and DrugBank were identified and classified into kinase families and subfamilies. Nematode proteins were placed into orthologous groups that span the phylum Nematoda. A minimal kinome for the phylum Nematoda was identified, and properties of the minimal kinome were explored. Orthologous groups from the minimal kinome were prioritized for experimental testing based on RNAi phenotype of the Caenorhabditis elegans ortholog, transcript expression over the life-cycle and anatomic expression patterns. Compounds linked to targets in DrugBank belonging to the same kinase families and subfamilies in the minimal nematode kinome were extracted. Thirty-five compounds were tested in the non-parasitic C. elegans and active compounds progressed to testing against nematode species with different modes of parasitism, the blood-feeding Haemonchus contortus and the filarial Brugia malayi. Eighteen compounds showed efficacy in C. elegans, and six compounds also showed efficacy in at least one of the parasitic species. Hypotheses regarding the pathway the compounds may target and their molecular mechanism for activity are discussed.

Effects of doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo.

BACKGROUND: Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi. METHODS: Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles. RESULTS AND DISCUSSION: Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion). CONCLUSIONS: Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.

A multicenter evaluation of diagnostic tools to define endpoints for programs to eliminate bancroftian filariasis.

Successful mass drug administration (MDA) campaigns have brought several countries near the point of Lymphatic Filariasis (LF) elimination. A diagnostic tool is needed to determine when the prevalence levels have decreased to a point that MDA campaigns can be discontinued without the threat of recrudescence. A six-country study was conducted assessing the performance of seven diagnostic tests, including tests for microfilariae (blood smear, PCR), parasite antigen (ICT, Og4C3) and antifilarial antibody (Bm14, PanLF, Urine SXP). One community survey and one school survey were performed in each country. A total of 8,513 people from the six countries participated in the study, 6,443 through community surveys and 2,070 through school surveys. Specimens from these participants were used to conduct 49,585 diagnostic tests. Each test was seen to have both positive and negative attributes, but overall, the ICT test was found to be 76% sensitive at detecting microfilaremia and 93% specific at identifying individuals negative for both microfilariae and antifilarial antibody; the Og4C3 test was 87% sensitive and 95% specific. We conclude, however, that the ICT should be the primary tool recommended for decision-making about stopping MDAs. As a point-of-care diagnostic, the ICT is relatively inexpensive, requires no laboratory equipment, has satisfactory sensitivity and specificity and can be processed in 10 minutes-qualities consistent with programmatic use. Og4C3 provides a satisfactory laboratory-based diagnostic alternative.

Evaluation of rectal dose during high-dose-rate intracavitary brachytherapy for cervical carcinoma.

High-dose-rate intracavitary brachytherapy (HDR-ICBT) for carcinoma of the uterine cervix often results in high doses being delivered to surrounding organs at risk (OARs) such as the rectum and bladder. Therefore, it is important to accurately determine and closely monitor the dose delivered to these OARs. In this study, we measured the dose delivered to the rectum by intracavitary applications and compared this measured dose to the International Commission on Radiation Units and Measurements rectal reference point dose calculated by the treatment planning system (TPS). To measure the dose, we inserted a miniature (0.1 cm(3)) ionization chamber into the rectum of 86 patients undergoing radiation therapy for cervical carcinoma. The response of the miniature chamber modified by 3 thin lead marker rings for identification purposes during imaging was also characterized. The difference between the TPS-calculated maximum dose and the measured dose was <5% in 52 patients, 5-10% in 26 patients, and 10-14% in 8 patients. The TPS-calculated maximum dose was typically higher than the measured dose. Our study indicates that it is possible to measure the rectal dose for cervical carcinoma patients undergoing HDR-ICBT. We also conclude that the dose delivered to the rectum can be reasonably predicted by the TPS-calculated dose.

Sesame oil exhibits synergistic effect with anti-diabetic medication in patients with type 2 diabetes mellitus.

BACKGROUND & AIMS: Recently, studies have reported that sesame oil lowered blood pressure and improved antioxidant status in hypertensive and diabetic-hypertensive patients. The aim of this study was to evaluate the effectiveness of sesame oil with anti-diabetic (glibenclamide) medication as combination therapy in mild-to moderate diabetic patients. METHODS: This open label study included sixty type 2 diabetes mellitus patients divided into 3 groups, receiving sesame oil (n = 18), 5 mg/day (single dose) of glibenclamide (n = 20), or their combination (n = 22). The patients were supplied with sesame oil [BNB Sesame oil(TM)] except glibenclamide group, and instructed to use approximately 35 g of oil/day/person for cooking, or salad preparation for 60 days. 12 h-fasting venous blood samples were collected at baseline (0 day) and after 60 days of the experiment for various biochemical analysis. RESULTS: As compared with sesame oil and glibenclamide alone, combination therapy showed an improved anti-hyperglycemic effect with 36% reduction of glucose (P < 0.001 vs before treatment, P < 0.01 vs sesame oil monotherapy, P < 0.05 vs glibenclamide monotherapy) and 43% reduction of HbA(1c) (P < 0.001 vs before treatment, P < 0.01 vs sesame oil monotherapy, P < 0.05 vs glibenclamide monotherapy) at the end point. Significant reductions in the plasma TC, LDL-C and TG levels were noted in sesame oil (20%, 33.8% and 14% respectively vs before treatment) or combination therapies (22%, 38% and 15% respectively vs before treatment). Plasma HDL-C was significantly improved in sesame oil (15.7% vs before treatment) or combination therapies (17% before treatment). Significant (P < 0.001) improvement was observed in the activities of enzymatic and non-enzymatic antioxidants in patients treated with sesame oil and its combination with glibenclamide. CONCLUSIONS: Sesame oil exhibited synergistic effect with glibenclamide and can provide a safe and effective option for the drug combination that may be very useful in clinical practice for the effective improvement of hyperglycemia.

Purification and characterization of cysteine protease from germinating cotyledons of horse gram.

BACKGROUND: Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. To study the mechanism of protein mobilization, undertaken the task of purifying and characterizing proteases, which occur transiently in germinating seeds of horse gram. RESULTS: Cysteine protease (CPRHG) was purified to homogeneity with 118 fold by four step procedure comprising Crude extract, (NH4)2SO4 fractionation, DEAE-Cellulose and CM-sephacel chromatography from the 2 day germinating cotyledons of horse gram (Macrotyloma uniflorum (Lam.) Verdc.). CPRHG is a monomer with molecular mass of 30 k Da, was determined by SDS-PAGE and gel filtration. The purified enzyme on IEF showed two isoforms having pI values of 5.85 and 6.1. CPRHG composed of high content of aspartic acid, glutamic acid and serine. The enzyme activity was completely inhibited by pCMB, iodoacetate and DEPC indicating cysteine and histidine residues at the active site. However, on addition of sulfhydryl reagents (cysteine, dithiothreitol, glutathione and beta-ME) reverse the strong inhibition by pCMB. The enzyme is fairly stable toward pH and temperature. Immunoblot analysis shows that the enzyme synthesized as zymogen (preproenzyme with 81 kDa) and processed to a 40 kDa proenzyme which was further degraded to give 30 kDa active enzyme. CONCLUSION: It appears that the newly synthesized protease is inactive, and activation takes place during germination. CPRHG has a broad substrate specificity and stability in pH, temperature, etc. therefore, this protease may turn out to be an efficient choice for the pharmaceutical, medicinal, food, and biotechnology industry.

A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA.

The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.

Brugia malayi: Effects of nitazoxanide and tizoxanide on adult worms and microfilariae of filarial nematodes.

There is an urgent need for safe and effective antifilarials. Prior studies have shown that the nitazoxanide (NTZ) exhibits broad activity against anaerobic bacteria, protozoa, and certain intestinal helminths. We examined the effects of NTZ and tizoxanide (TZ) on Brugia malayi nematodes in vitro and in vivo. In vitro, NTZ and TZ reduced worm motility and viability in a dose-dependent manner. Worm viability was reduced by 50% with both compounds at 2.5 and 20 microg/ml killed adult worms. NTZ or TZ (5 microg/ml) significantly reduced microfilaria release. These compounds blocked worm's embryogenesis, and decreased microfilarial motility and viability. Treated worms had damaged cuticles and abnormal mitochondria. Wolbachia were not cleared by NTZ or TZ treatment. Neither NTZ nor TZ cleared adult worms or microfilariae in infected gerbils. These results show that NTZ and TZ have potent effects on B. malayi nematodes in vitro. However, they were not effective in vivo.