There is geographical variation in the morphology and genetics of Wuchereria bancrofti, the major cause of human lymphatic filariasis. This study aims to compare morphological and genetic variation of W. bancrofti microfilariae recovered from carriers in Lao PDR, Myanmar and Thailand. Six morphological parameters (body length, cephalic space length and width, length of head to nerve ring, body width at nerve ring, Innenkȍrper length and number of column nuclei between the cephalic space and nerve ring) were evaluated from microfilariae in Giemsa-stained thick blood films. A portion of the cytochrome c oxidase subunit 1 gene of mitochondrial DNA was sequenced and analysed. Wuchereria bancrofti microfilariae showed a wide variation in their morphology and morphometry among three countries. Phylogenetic analysis confirmed that all microfilariae belonged to W. bancrofti. Higher mutation frequencies were observed in samples from Myanmar, relative to Thailand and Lao PDR. This study highlights the morphological disparities of microfilariae and genetic variability within W. bancrofti among three geographical locations. We found that reported morphometric differences between localities were less clear-cut than previously thought. Further studies are needed to determine the microfilarial periodicity in Lao PDR.

Efficacious therapeutic strategies against lymphatic filariasis are always sought after. However, natural products are a promising resource for developing effective antifilarial agents. Azadirachtin, a significant tetranortriterpenoid phytocompound found in Azadirachta indica, was evaluated in vitro for antifilarial potential against the filarial parasite Setaria cervi. Dye exclusion and MTT assay confirmed the antifilarial potential of azadirachtin against S. cervi with a median lethal dose (LC50) of 6.28 μg/ml for microfilariae (mf), and 9.55 μg/ml for adult parasites. Morphological aberrations were prominent in the histological sections of the azadirachtin-exposed parasites. Moreover, alterations in the reactive oxygen species (ROS) parameters in treated parasites were evident. Induction of apoptosis in treated parasites was confirmed by DNA laddering, acridine orange (AO)/ethidium bromide (EtBr) double staining and in situ DNA fragmentation. The downregulation of anti-apoptotic CED-9 and upregulation of proapoptotic EGL-1, CED-4 and CED-3 at both the transcription and translation levels confirmed apoptosis execution at the molecular level. Changes in the gene expressions of nuc-1, cps-6 and crn-1 further clarified the molecular cause of DNA degradation. Furthermore, azadirachtin was found to be non-toxic in both in vitro and in vivo toxicity analyses. Therefore, the experimental evidence detailed the pharmacological effectiveness of azadirachtin as a possible therapeutic agent against filariasis.

Monitoring vectors is relevant to ascertain transmission of lymphatic filariasis (LF). This may require the best sampling method that can capture high numbers of specific species to give indication of transmission. Gravid anophelines are good indicators for assessing transmission due to close contact with humans through blood meals. This study compared the efficiency of an Anopheles gravid trap (AGT) with other mosquito collection methods including the box and the Centres for Disease Control and Prevention gravid, light, exit and BioGent-sentinel traps, indoor resting collection (IRC) and pyrethrum spray catches across two endemic regions of Ghana. The AGT showed high trapping efficiency by collecting the highest mean number of anophelines per night in the Western (4.6) and Northern (7.3) regions compared with the outdoor collection methods. Additionally, IRC was similarly efficient in the Northern region (8.9) where vectors exhibit a high degree of endophily. AGT also showed good trapping potential for collecting Anopheles melas which is usually difficult to catch with existing methods. Screening of mosquitoes for infection showed a 0.80–3.01% Wuchereria bancrofti and 2.15–3.27% Plasmodium spp. in Anopheles gambiae. The AGT has shown to be appropriate for surveying Anopheles populations and can be useful for xenomonitoring for both LF and malaria.

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of B. malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing B. malayi third-stage larvae (L3) in endothelial basal media supplemented with 20% fetal bovine serum and 75 µm ascorbic acid in a temperature- and humidity-controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behaviour studies and drug screening applications for disease eradication.

Practical diagnostic tools of sufficient sensitivity to detect levels of infection that can lead to transmission have been identified as a critical component of successful disease elimination programmes. In this review we describe the diagnostic tests currently available for six neglected tropical diseases that have been targeted for elimination; assess their performance in the light of the requirements for surveillance, certification of elimination and post-elimination surveillance; consider the unmet need for diagnostic tests for these diseases; and review recent technical developments that could meet these needs.

Lymphatic filariasis (LF), which is highly endemic in 73 countries worldwide, is targeted for elimination by 2020. The strategy for achieving this goal is based on 4 sequential programmatic steps: mapping, Mass drug administration (MDA) implementation, post-MDA surveillance and verification of LF elimination. All 4 stages of the implementation process are dependent on the availability of user friendly and highly sensitive rapid diagnostic tools. By the end of 2012, 59 countries had completed mapping for LF and Eritrea was the only country yet to start the process. Rolling out new diagnostic tools to facilitate the mapping process will enable an accelerated shrinking of the LF map to zero endemic countries by 2020. When the Global Programme to Eliminate Lymphatic Filariasis was launched in 2000, diagnostic tools for LF were limited to clinical examination, detection of microfilaria (MF) by microscopy in night blood samples and detection of antibodies to native-antigen preparations. There has been a significant improvement in the traditional LF diagnostic methods in recent years and some new tools are now available. This paper provides an update on the human diagnostic tests available for LF and their current applications as tools in mapping and transmission monitoring. The values of entomological indicators and parasite detection and speciation methods applied to vector populations are also discussed.

Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4–6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA ‘endgame scenarios’, where test and treat strategies become more cost effective and deliverable.

Lymphatic filariasis (LF), a common parasitic infection in tropical countries, causes lymphoedema of limbs, hydrocele and acute attacks of dermato-lymphangio-adenitis. Recent advances in diagnosis have helped to recognize that LF infection is often acquired in childhood. Newly available diagnostic techniques like sensitive antigen and antibody assays, Doppler ultrasonography and lymphoscintigraphy have helped to understand the subclinical pathology caused by this infection, which was hitherto generally believed to be irreversible. Recent studies indicate that drugs used in the mass drug administration (MDA) programme under GPELF are capable of reversing the sub-clinical lymphatic damage in children and provide benefits other than interruption of transmission. Albendazole and ivermectin used in MDA are effective against soil-transmitted helminthic infections common in children in LF endemic areas. Thus MDA had other ‘beyond LF’ benefits in treated children including increased appetite, weight gain, greater learning ability and concentration, better school attendance and prevention of anaemia. MDA should no longer be viewed as a measure for interrupting transmission alone. Recent findings of reversibility of early lymphatic pathology in treated children indicate that both MDA and ‘foot-hygiene’ measures are effective strategies in preventing and managing morbidity. Programme managers should effectively utilize this information to strengthen their advocacy efforts to achieve high and sustainable coverage in MDA.

The effect of an alkaloidal fraction from the stem bark of Artabotrys odoratissimus R. Br. on larvae of the mosquito vector of lymphatic filariasis, Culex quinquefasciatus Say, was investigated. Concentrations of 10, 20, 40, 80, 160 and 320 ppm were tested in the laboratory against larvae of various ages. A general increase in lethal concentration 50 (LC50) values from the second to fourth instars was observed. Based on overlapping confidence intervals (CIs), the LC50 value following 12 h of exposure was lowest for the second instar (59.2 ppm) and was significantly different from that recorded for the fourth instar (110.4 ppm). LC50 values decreased significantly with time (from 12 to 24 h after treatment) in all the instars. These results indicate the potential utility of the A. odoratissimus extract as a mosquito larvicidal agent, and further research is recommended.

Objective. Maternal infection has been considered to be a risk factor for filarial infection in offspring. In order to examine the influence of maternal infection in neonates, we have determined the prevalence of circulating filarial antigen (CFA) and anti-filarial antibodies in 119 maternal and corresponding cord blood samples collected from an area endemic for bancroftian filariasis. Method. Prevalence of antigenaemia was detected using Og4C3 circulating filarial antigen enzyme-linked immunosorbent assay. The presence of microfilariae was determined by filtration of a 1 ml sample through a Nuclepore membrane. Antibody isotypes (IgG, IgM, and IgE) to filarial antigen (Setaria digitata antigenic extract) were determined by enzyme linked immunosorbent assay (ELISA). Results. Microfilariae were detected in 14 cases (11·8%), whereas the Og4C3 assay could detect filarial antigen in 44·5% of pregnant mothers. Interestingly, 24·5% of samples born from CFA-positive mothers were found positive for CFA. None of the cord samples from CFA-negative mothers were found positive for CFA. No significant difference was observed in prevalence of filarial-specific IgG, IgM and IgE antibodies in CFA-positive and negative mothers. IgG antibody was detected in 60·5% of maternal and 21·8% of cord samples. IgG antibody in the cord does not differ with the antigen status of the mother. In contrast IgM and IgE antibody prevalence was significantly higher in cord from infected mothers than non-infected mothers (11·3% vs 0 for IgM, 24·5% vs 3·03% for IgE). Conclusion. Our study demonstrates the transplacental transfer of circulating filarial antigen from mother to cord. Filaria-specific IgM and IgE antibodies were higher in cord blood from infected mothers than from non-infected mothers. The findings of the study provide additional circumstantial evidence for pre-natal sensitization to filarial antigens developed in utero.

This paper uses simple mathematical models and statistical estimation techniques to analyse the frequency distribution of microfilariae (mf) in blood samples from human populations which are endemic for lymphatic filariasis. The theoretical analysis examines the relationship between microfilarial burdens and the prevalence of adult (macrofilarial) worms in the human host population. The main finding is that a large proportion of observed mf-negatives may be ‘true’ zeros, arising from the absence of macrofilarial infections or unmated adult worms, rather than being attributable to the blood sampling process. The corresponding mf distribution should then follow a Poisson mixture, arising from the sampling of mf positives, with an additional proportion of ‘true’ mf-zeros. This hypothesis is supported by analysis of observed Wuchereria bancrofti mf distributions from Southern India, Japan and Fiji, in which zero-truncated Poisson mixtures fit mf-positive counts more effectively than distributions including the observed zeros. The fits of two Poisson mixtures, the negative binomial and the Sichel distribution, are compared. The Sichel provides a slightly better empirical description of the mf density distribution; reasons for this improvement, and a discussion of the relative merits of the two distributions, are presented. The impact on observed mf distributions of increasing blood sampling volume and extraction efficiency are illustrated via a simple model, and directions for future work are identified.

Cats were repeatedly inoculated with infective larvae of Brugia pahangi. On parasitological grounds they could be divided into 5 groups. Group I – most cats (some 70%) became microfilaraemic (mf+) and retained high levels of microfilariae (mf) in their blood for over 2 years. In some Group I cats mf counts stabilized at high levels whilst in others mf counts continued to increase. Large numbers of fecund adult worms were recovered from their lymphatics. Adult counts were not made on the cats in the current experiments but over 100 adults have been recovered from ‘super-susceptible’ cats. Large amounts of B. pahangi adult antigen were consistently present in the serum of all Group I cats. About 30% of cats became amicrofilaraemic (mf–). In these cats the peak mf levels were seldom above 10000 mf/ml. Group II – these cats had less than 10 000 mf/ml and low antigen levels. After more than 1 year of being repeatedly infected B. pahangi adult antigen slowly declined and eventually could no longer be detected in their serum and the number of mf declined very slowly after the fall in antigen levels. This shows that in Group II cats the adult worms die and as the cats are resistant to the development of the continuing weekly inoculation of L3 no new adults can develop. Group III – these cats became mf – during the first year of infection but remained B. pahangi antigen-positive for many weeks after this and, at autopsy, had living adults in their lymphatics. Group IV – in these cats there was a sudden decline in the number of mf, usually in the first year and after they have received a relatively small number of re-infections. Circulating adult B. pahangi antigen disappeared from the serum within a short time of the loss of mf and at autopsy no adult female worms were found. Group V – these cats do not become mf+ when infected. These groups are compared to the parasitological groups seen in areas endemic for infection with Wuchereria bancrofti.

This study examines the effects of host age and sex on the frequency distribution of Brugia malayi infections in the human host. Microfilarial (mf) counts for a large data base on the epidemiology of brugian filariasis in Shertallai, Kerala, South India are analysed. Frequency distributions of microfilarial counts partitioned by age are successfully described by zero-truncated negative binomial distributions, fitted by maximum likelihood. This analysis provides estimates of the proportion of mf-positive individuals who are identified as negative due to sampling errors, allowing the construction of corrected mf age–prevalence curves, which indicate that the observed prevalence may under-estimate the true figures by between 18 and 47%. There is no evidence from these results for a decrease in the degree of over-dispersion of parasite frequency distributions with host age, such as might be produced by the acquired immunity to infection. This departure from the pattern in bancroftian filariasis (where there is evidence of such decreases in over-dispersion; Das et al. 1990) is discussed in terms of the long history of filariasis control (and consequently low infection prevalence) in Shertallai.

This paper estimates the global burden of lymphatic filariasis based on a review of the published literature on infection and disease surveys. A method for aggregating and projecting prevalence data from individual studies to national, regional and global levels, which also facilitates the estimation of gender and age-specific burdens, is presented. The method weights in favour of the larger, and hence presumbably more reliable, studies and relies on estimated empirical relationships between gender, age, infection and disease in order to correct studies with incomplete data. The results presented here suggest that although the overall prevalence of filariasis cases is 2·0% globally (approximately totalling 119 million cases), the disease continues to be of considerable local importance, particularly in India and Sub-Saharan Africa. Estimates by age and gender clearly show that, unlike other helminth infections, filariasis is mainly a disease of the adult and older age-classes and appears to be more prevalent in males. This work suggests that the derivation of more accurate estimates of the burden of filariasis will require a better understanding of both the epidemiology and the spatial aspects of infection and disease. It also suggests that filariasis is preventable based on a geographically targeted strategy for control.

This paper examines the effects of host age and sex on the frequency distribution of Wuchereria bancrofti infections in the human host. Microfilarial counts from a large data base on the epidemiology of bancroftian filariasis in Pondicherry, South India are analysed. Frequency distributions of microfilarial counts divided by age are successfully described by zero-truncated negative binomial distributions, fitted by maximum likelihood. Parameter estimates from the fits indicate a significant trend of decreasing overdispersion with age in the distributions above age 10; this pattern provides indirect evidence for the operation of density-dependent constraints on microfilarial intensity. The analysis also provides estimates of the proportion of mf-positive individuals who are identified as negative due to sampling errors (around 5% of the total negatives). This allows the construction of corrected mf age–prevalence curves, which indicate that the observed prevalence may underestimate the true figures by between 25% and 100%. The age distribution of mf-negative individuals in the population is discussed in terms of current hypotheses about the interaction between disease and infection.

LYMFASIM is a simulation model for lymphatic filariasis transmission and control. We quantified its parameters to simulate Wuchereria bancrofti transmission by Anopheles mosquitoes in African villages, using a wide variety of reported data. The developed model captures the general epidemiological patterns, but also the differences between communities. It was calibrated to represent the relationship between mosquito biting rate and the prevalence of microfilariae (mf) in the human population, the age-pattern in mf prevalence, and the relation between mf prevalence and geometric mean mf intensity. Explorative simulations suggest that the impact of mass treatment depends strongly on the mosquito biting rate and on the assumed coverage, compliance and efficacy. Our sensitivity analysis showed that some biological parameters strongly influence the predicted equilibrium pre-treatment mf prevalence (e.g. the lifespan of adult worms and mf). Other parameters primarily affect the post-treatment trends (e.g. severity of density dependence in the mosquito uptake of infection from the human blood, between-person variability in exposure to mosquito bites). The longitudinal data, which are being collected for evaluation of ongoing elimination programmes, can help to further validate the model. The model can help to assess when ongoing elimination activities in African populations can be stopped and to design surveillance schemes. It can be a valuable tool for decision making in the Global Programme to Eliminate Lymphatic Filariasis.

The role of acquired immunity in lymphatic filariasis is uncertain. Assuming that immunity against new infections develops gradually with accumulated experience of infection, models predict a decline in prevalence after teenage or early adulthood. A strong indication for acquired immunity was found in longitudinal data from Pondicherry, India, where Mf prevalence was highest around the age of 20 and declined thereafter. We reviewed published studies from India and Subsaharan Africa to investigate whether their age-prevalence patterns support the models with acquired immunity. By comparing prevalence levels in 2 adult age groups we tested whether prevalence declined at older age. For India, comparison of age groups 20–39 and 40+ revealed a significant decline in only 6 out of 53 sites, whereas a significant increase occurred more often (10 sites). Comparison of older age groups provided no indication that a decline would start at a later age. Results from Africa were even more striking, with many more significant increases than declines, irrespective of the age groups compared. The occurrence of a decline was not related to the overall Mf prevalence and seems to be a chance finding. We conclude that there is no evidence of a general age-prevalence pattern that would correspond to the acquired immunity models. The Pondicherry study is an exceptional situation that may have guided us in the wrong direction.

This paper investigates a cohort of 2187 laboratory reared Culex quinquefasciatus fed on 69 human volunteers, including 59 persons with different levels of Wuchereria bancrofti microfilariae and 10 without microfilaria. Mosquitoes were followed until death. Mosquito survival was analysed in relation to the level of microfilaria in the human and larval count in the dead mosquito. Vector mortality during the extrinsic incubation period (12 days post-engorgement) was significantly higher in mosquitoes fed on microfilaraemic volunteers (50%) than in those fed on amicrofilaraemics (29%). Both the percentage infected and the geometric mean parasite density was significantly higher among mosquitoes which died before 13 days (45% infected and 10 larvae per infected mosquito) than those surviving beyond 13 days (39% and 2·2), suggesting a parasite loss of more than 80% during the extrinsic incubation period. A large proportion (62%) of the mosquitoes that died during the early of phase of parasite development were infected (36% in low, 26% in medium and 90% in high human Mf-density). Survival analysis showed that the parasite load in mosquitoes and the human Mf-density for a given parasite load are independent risk factors of vector survival. Overall, the hazard of dying was found to be 11–15 times higher among mosquitoes fed on microfilaraemic volunteers than those fed on amicrofilaraemics. The hazard doubles for every increase of about 60–70 parasites in the vector. As a consequence of the parasite-induced reduction in vector survival, the transmission success of the parasite is reduced. The implication of the results on control/elimination of lymphatic filariasis using mass-drug administration is discussed.

This paper presents a model-based analysis of longitudinal data describing the impact of integrated vector management on the intensity of Wuchereria bancrofti infection in Pondicherry, India. The aims of this analysis were (1) to gain insight into the dynamics of infection, with emphasis on the possible role of immunity, and (2) to develop a model that can be used to predict the effects of control. Using the LYMFASIM computer simulation program, two models with different types of immunity (anti-L3 larvae or anti-adult worm fecundity) were compared with a model without immunity. Parameters were estimated by fitting the models to data from 5071 individuals with microfilaria-density measurement before and after cessation of a 5-year vector management programme. A good fit, in particular of the convex shape of the age-prevalence curve, required inclusion of anti-L3 or anti-fecundity immunity in the model. An individual's immune-responsiveness was found to halve in ~10 years after cessation of boosting. Explanation of the large variation in Mf-density required considerable variation between individuals in exposure and immune responsiveness. The mean life-span of the parasite was estimated at about 10 years. For the post-control period, the models predict a further decline in Mf prevalence, which agrees well with observations made 3 and 6 years after cessation of the integrated vector management programme.

This review of the safety of the co-administration regimens to be used in programmes to eliminate lymphatic filariasis (albendazole+ivermectin or albendazole+diethylcarbamazine [DEC]) is based on 17 studies conducted in Sri Lanka, India, Haiti, Ghana, Tanzania, Kenya, Ecuador, the Philippines, Gabon, Papua New Guinea, and Bangladesh. The total data set comprises 90635 subject exposures and includes individuals of all ages and both genders. Results are presented for hospital-based studies, laboratory studies, active surveillance of microfilaria-positive and microfilaria-negative individuals, and passive monitoring in both community-based studies and mass treatment programmes of individuals treated with albendazole (n=1538), ivermectin (9822), DEC (576), albendazole+ivermectin (7470), albendazole+DEC (69020), or placebo (1144). The most rigorous monitoring, which includes haematological and biochemical laboratory parameters pre- and post-treatment, provides no evidence that consistent changes are induced by any treatment; the majority of abnormalities appear to be sporadic, and the addition of albendazole to either ivermectin or DEC does not increase the frequency of abnormalities. Both DEC and ivermectin show, as expected, an adverse event profile compatible with the destruction of microfilariae. The addition of albendazole to either single-drug treatment regimen does not appear to increase the frequency or intensity of events seen with these microfilaricidal drugs when used alone. Direct observations indicated that the level of adverse events, both frequency and intensity, was correlated with the level of microfilaraemia. In non microfilaraemic individuals, who form 80–90% of the ‘at risk’ populations to be treated in most national public health programmes to eliminate lymphatic filariasis (LF), the event profile with the compounds alone or in combination does not differ significantly from that of placebo. Data on the use of ivermectin+albendazole in areas either of double infection (onchocerciasis and LF), or of loiais (with or without concurrent LF) are still inadequate and further studies are needed. Additional data are also recommended for populations infected with Brugia malayi, since most data thus far derive from populations infected with Wuchereria bancrofti.