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.

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.

Isozymic patterns of lactate dehydrogenase (E.C. 1.1.1.27) by polyacrylamide gel electrophoresis (PAGE) were observed m various categories of filariasis and controls, i.e. asymptomatic microfilaraemia and symptomatic amicrofilaraemia, endemic normal and non-endemic normal. Lactate dehydrogenase (LDH) activity was also observed amongst the above categories of patients. An increase in enzyme activity and change in the isozymic pattern was observed in the above categories of filaria infected serum. LDH activity doubled in asymptomatic microfilaraemia whereas in symptomatic amicrofilaraemia the increase in LDH activity was thirtyfold. The isozymic pattern of microfilaraemia cases showed the presence of three bands B4, A1B3, A2 B2, which are quite thick as compared to normal healthy subjects, whereas the patients with symptomatic amicrofilaraemia showed marked elevation of serum LDH-4 or A3B1. The LDH was partially purified by combined treatment of (NH4)2SO4 fractionation and gel filtration. The isozymic pattern of purified LDH showed a similar pattern.

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.

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.

A low molecular weight (15 kDa) surface antigen of the cattle filarial nematode, Setaria digitata, was earlier shown to be specifically recognized by the antibodies from human bancroftian filarial (Mf positive) patients' sera (Theodore & Kaliraj, 1990). The filarial specific antibodies bound to a 15 kDa peptide in preparative Western blots were eluted and employed in screening of candidate antigens expressed in the genomic library of Wuchereria bancrofti at the IgG4 subclass antibody level. A recombinant clone (λ WbG7) reacting strongly with filarial sera but poorly with sera from patients infected with non-filarial helminths was selected for further studies. The 2 kb DNA insert of the clone λ WbG7 was recloned into a pMAL vector and the recombinant clone pGT7 thus obtained was over-expressed and affinity purified. The purified 105 kDa fusion protein of the clone pGT7 was specific and was not recognized by the non-filarial sera at the IgG4 level. All microfilaraemic individuals were positive by IgG4 assay. However, similar attempts to diagnose by filarial-specific IgE assays failed to recognize microfilaraemic individuals. Moreover, by filarial-specific IgG4 assays, the endemic normals were distinctly divided into two groups, showing higher and lower recognition for this antigen indicating current and past/no infection. Among the filarial-IgG4 (assay)-positive ‘endemic normals’, 14% showed ‘microfilariae’ during repeated peripheral night blood examination, confirming the validity of the recombinant antigen, pGT7 based assay.

The vector competences of 6 geographic strains of Aedes polynesiensis for Wuchereria bancrofti were studied using two types of experimental infections. Experimental infection of laboratory-bred mosquitoes fed on the carriers' forearms with different levels of microfilaraemia showed that microfilariae (mf) uptake was directly proportional to the carrier's mf density and, as mf densities decreased, concentration capacity of Ae. polynesiensis increased. It was also shown that infection has an important effect on mosquito mortality, and that the mortality rate differed among mosquito strains. In infections using artificial feeders, the mf uptake was closely regulated, thus showing differences in the vectorial efficiency of Ae. polynesiensis related to the geographic origin of the mosquito strain. The mosquitoes from the Society archipelago were more efficient intermediate hosts than geographically distant strains when infected with W. bancrofti from an island within the archipelago (Tahiti). Mosquito strains from the Society archipelago developed the highest proportion of infective-stage larvae and exhibited the lowest mortality rate when infected with sympatric Tahitian W. bancrofti.

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.

Malaria and filaria infection rates were determined for anopheline mosquitoes collected whilst biting and resting in village houses in Papua New Guinea. The number of anophelines infected with both parasites was greater than expected from the infection rates of each parasite and this difference was significant in resting collections. The excess of multiply infected mosquitoes is probably a result of a vector population composed of individuals with differing numbers of opportunities to become infected. Malaria-positive, Anopheles punctulatus from resting catches had a significantly greater number of Stage 3 Wuchereria bancroftilarvae than malaria-negative mosquitoes. However, multiply infected mosquitoes appear to suffer greater mortality than non-infected or singly infected mosquitoes when the filarial worm reaches the third stage. Any potential increase in transmission resulting from multiple infections is thereby offset by a greater mortality rate in these mosquitoes.

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.

Currently, annual mass treatments with albendazole (ABZ) plus ivermectin (IVM) or diethylcarbamazine (DEC) are administered under the Global Programme to Eliminate Lymphatic Filariasis (GPELF). Drug resistance against both ABZ and IVM is prevalent in nematodes of veterinary importance, raising awareness that if anthelmintic resistance were to develop among Wuchereria bancrofti populations, this would jeopardize GPELF's goals. Genetic structure was incorporated into an existing transmission dynamics model for lymphatic filariasis (LF) to investigate the potential development of concurrent resistance to ABZ and IVM. The resultant models explore the impact of different inheritance modes of resistance to ABZ and IVM on the likely risk of treatment failure under our model assumptions. Results indicate that under ABZ+IVM combination, selection for resistance to one drug is enhanced if resistance to the other drug is already present. Excess parasite homozygosity may increase selection for dominant IVM resistance via enhancing the frequency of recessive ABZ resistance. The model predicts that if multiple resistance genes are associated with different efficacy properties of a drug combination, then examining changes at single loci may be misleading. Sampling schemes in genetic epidemiological surveys investigating the frequency of an allele under selection should consider host age, as individuals of different ages may acquire parasites at different rates.

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) intends to achieve its aims through yearly mass treatments with albendazole (ABZ) combined with ivermectin (IVM) or diethylcarbamazine (DEC). The use of ABZ and IVM separately to combat parasites of veterinary importance has, on many occasions, resulted in widespread drug resistance. In order to help predict the spread of potential ABZ resistance alleles through a population of Wuchereria bancrofti, we have developed a mathematical model that incorporates population genetics into EPIFIL, a model which examines the transmission dynamics of the parasite. Our model considers the effect of the combined treatments on the frequency of a recessive allele, which confers ABZ resistance. The model predicts that after 10 yearly treatments with ALB and DEC, 85% coverage and an initial resistance allele frequency of 5%, the frequency of the resistance genotype will increase from 0·25 to 12·7%. If non-random mating is assumed, the initial genotype frequency will be 2·34% and will increase to 62·7%. ABZ and IVM combination treatment may lead to weaker selection for this genotype. Treatment coverage, initial allele frequencies and number of treatments also affect the rate of selection.

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.

An ultrastructural study of microfilariae of Wuchereria bancrofti was performed after in vitro treatment with diethylcarbamazine. One of the first morphological alterations produced by treatment with 5, 10 or 50 μg/ml of DEC was the loss of microfilarial sheaths. Drastic effects of DEC were only detected when microfilariae were exsheathed. Microfilariae treated with 5, 10 or 50 μg/ml DEC showed severely affected organelles, formation of several vacuoles mainly in the hypodermis, and cytolysis. Some microfilariae showed extreme cellular disorganization with abundance of electron-dense degenerating organelles, numerous large vacuoles, and nuclear condensation. Lamellar bodies probably related to an assembly of endoplasmic reticulum membranes were observed in some damaged microfilariae. Thus, in vitro treatment with concentrations of DEC similar to therapeutic conditions had direct effect by causing a loss of the microfilarial sheath of W. bancrofti with subsequent damage of organelles and apoptosis.

The natural history of human filarial infections leading to development of disease has been a subject of intense debate. The models proposed so far have largely been based on cross-sectional data on microfilariae (Mf) and disease prevalence in filariasis endemic areas. In an attempt to study the parasitological and clinical consequences of filarial infection in Beldal (Orissa, India), an area endemic for Bancroftian filariasis, cohorts of 59 asymptomatic Mf carriers (AS) and 187 asymptomatic and amicrofilaraemic subjects or ‘endemic normals’ (‘EN’), were followed-up and a fraction (73% and 46% respectively) re-examined after 13 years to monitor (a) Mf prevalence, (b) Mf density, (c) circulating filarial antigen (CFA) and (d) chronic disease manifestations. The Mf prevalence and density were also monitored in Mf carriers after 1 and 4 years. Both Mf prevalence and density decreased progressively in the cohort of Mf carriers over a period of 13 years in Beldal. Only 37% of them continued to be microfilaraemic and the Mf density in these subjects was only 10% of the original level. However, loss of circulating Mf in this cohort did not result in loss of CFA and 95% remained CFA positive regardless of Mf status. About 23% of males in the ‘EN’ cohort developed hydrocoele while only 5·7% of male Mf carriers, who were not treated with DEC, had developed hydrocoele after 13 years. A cohort of Mf carriers in another area, Jatni, was also examined after 10 years to study the parasitological and clinical outcome. In this area, about 59% of the Mf carriers continued to be microfilaraemic after 10 years. These results reveal that in Mf carriers adult filarial worms persist for several years and that loss of circulating Mf with or without chemotherapy with DEC (single 12-day course) does not influence adult worm survival. The findings have been discussed in the context of ‘static’ and ‘dynamic’ models describing the relationship between infection and disease in human filariasis.

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.

This detailed review of the published studies underlying ivermectin's recent registration for use in lymphatic filariasis (LF) demonstrates the drug's single-dose efficacy (over the range of 20–400μg/kg) in clearing microfilaraemia associated with both Wuchereria bancrofti and Brugia malayi infections of humans. While doses as low as 20μg/kg could effect transient microfilarial (mf) clearance, higher dosages induced greater and more sustained mf reduction. The single dose of 400μg/kg yielded maximal responses, but a number of practical considerations suggest that either 400μg/kg or 200μg/kg doses would be acceptable for use in LF control programmes. Associated safety assessments indicate that adverse events, which occur commonly following treatment of microfilaraemic individuals, develop not because of drug toxicity but because of host inflammatory responses to dying microfilariae killed by the ivermectin treatment. Ivermectin is, therefore, a highly effective and generally well tolerated microfilaricide that may soon become an essential component of many public health initiatives to interrupt transmission of lymphatic filarial infection in an effort to eliminate LF globally.

DEC-fortified salt has been used successfully as a principal public health tool to eliminate lymphatic filariasis (LF) in China and, less extensively, in several other countries. Studies from 1967 to the present conducted in Brazil, Japan, Tanzania, India, China, and Taiwan involving administration of DEC salt for 18 days to 1 year, have shown this intervention to be effective for both bancroftian and brugian filariasis, as measured by reductions in both microfilarial density and positivity, and in some studies through reduction in mosquito positivity rates as well. Furthermore, studies suggest specific advantages from using DEC salt, including lack of side effects, particularly for bancroftian filariasis, and ability to reduce prevalence below 1% when used in conjunction with standard regimens of DEC tablets. However, use of DEC salt as a control tool suffers from a concern that health authorities might find it difficult to manage a programme involving a commodity such as salt. In the past decade, the very successful global efforts to eliminate iodine deficiency through universal salt iodization have demonstrated that partnership with the salt industry can be both successful and effective as a public health tool. Use of DEC salt can be most successfully implemented in areas in which (a) there is adequate governmental support for its use and for elimination of filariasis, (b) filariasis-endemic areas are clearly defined, (c) political leaders, health officials and the salt industry agree that DEC salt is an appropriate intervention, (d) the salt industry is well-organized and has known distribution patterns, (e) a successful national salt iodization effort exists, (f) a monitoring system exists that ensures adequacy of salt iodine content during production and that can also measure household coverage, and (g) measurement of impact on transmission of LF with the new antigen or filarial DNA detection methods can be established. There are advantages and disadvantages of using DEC-fortified salt compared with other interventions for LF elimination programmes, but rather than being considered as a ‘competing’ intervention, DEC salt should be seen as an additional option. Indeed, it is likely that many countries will derive maximal benefit from the synergistic effects of combining different intervention strategies in their national programmes to eliminate lymphatic filariasis.