A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.
Quantitative understanding of the transmission dynamics of lymphatic filarial parasites is essential for the rational planning of control strategies. One of the most important determinants of transmission dynamics is the relationship between parasite yield, the success rate of ingested microfilariae (mf) becoming infective larvae in a mosquito vector, and mf density in the source of the human blood meal. Three types of relationship have been recognized in human filaria/mosquito couples--limitation, facilitation and proportionality; facilitation has hitherto been observed only in the couple Wuchereria bancrofti/Anopheles gambiae in Burkina Faso, in experimental studies on a high density mf carrier. The present paper demonstrates facilitation in W. bancrofti/An. gambiae and W. bancrofti/An. arabiensis in lower mf density carriers in The Gambia and Tanzania, and in W. bancrofti/An. funestus in Tanzania. Facilitation was not found in An. melas in The Gambia nor in An. merus in Tanzania. Analysis of published data shows limitation at low level mf densities in W. bancrofti/Culex quinquefasciatus in Sri Lanka, and in the same couple in India. Limitation also occurs in Brugia malayi/Aedes togoi in experimental cats; proportionality occurs in B. malayi/Mansonia bonneae in Malaysia. The epidemiological significance of these host/parasite relationships is discussed, and supporting evidence for its validity is presented from the published results of large-scale control programmes.
Ivermectin at single doses of 0.2-1.0 mg/kg body weight reduced the microfilarial counts of subperiodic Brugia malayi in Presbytis cristata by 59.9%-89.6% of initial counts, 4 weeks after treatment. Adult filaricidal activity was poor, live adult worms being recovered from all animals at autopsy. There was no serious side effect at these doses.
CGP 20376, a 5-methoxyl-6-dithiocarbamic-S- (2-carboxy-ethyl) ester derivative of benzothiazole was evaluated for its antifilarial properties and shown to be extremely effective against subperiodic Brugia malayi in the leaf-monkey, Presbytis cristata at oral doses of 20-100 mg/kg. The compound and/or its metabolites had complete micro- and microfilaricidal activities even when given at a single dose of 20 mg/kg. Lower doses had incomplete filaricidal action.
An intense global collaborative effort under the leadership of the Steering Committee of the Filariasis Scientific Working Group of the Tropical Diseases Research Programme, World Health Organization, has brought together researchers, pharmaceutical chemists and clinicians in the development and search for antifilarial compounds which are more effective and more convenient to administer than diethylcarbamazine citrate, the current drug of choice for lymphatic filariasis. The Brugia spp.-rodent model has been used extensively for the primary screening and B. pahangi infections in the dog or cat for the secondary screening, of potential filaricides. Recently, the leaf-monkey (Presbytis spp.) infected with subperiodic B. malayi or Wuchereria kalimantani has been used for the tertiary evaluation and pharmacokinetic studies of compounds which have shown effectiveness in the primary and secondary screens. Both P. cristata and P. melalophos are extremely susceptible to subperiodic B. malayi infection, but the former is a better host as a higher peak microfilaremia and adult worm recovery rate were obtained. Although more than 30 potential filaricides have been evaluated in the tertiary screen, only a few compounds have shown some promise against lymphatic filariasis. CGP 20376, a 5-methoxyl-6-dithiocarbamic-S-(2-carboxy-ethyl) ester derivative of benzothiazole, had complete adulticidal and microfilaricidal activities against the parasite at a single oral dose of 20 mg kg-1. However, as the compound or its metabolites caused hepatotoxicity, its clinical use in the present formulation is not recommended.(ABSTRACT TRUNCATED AT 250 WORDS)
DEC or ivermectin (IVM) in combination with albendazole (ALB) has been the recommended strategy of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) since 2000. Despite effective population coverage (> 65%) with several rounds of MDA with DEC or combination of DEC plus ALB, microfilariae persist in few individuals and they continue to be the source of infection for transmitting LF. We report an individual's variability in response to DEC by defining the response as complete absence of microfilaria (mf) (post-treatment mf count = 0) and non-response as presence of mf (post-treatment mf count ≥ 1). We analyzed follow-up data on individual's response to treatment from two randomized clinical trials in which 46 microfilaremic individuals were treated with single-dose DEC (6 mg/kg body weight). They were classified into low, medium, and high mf density categories based on their pre-treatment mf counts. Of the 46 individuals, 65.2% have not responded throughout the 12-month post-treatment period. Application of a logistic regression model with fixed (age, gender, mf density, post-treatment time, and their interactions) and random (individual's response over time) effects indicated that treatment response is independent of age, gender, and time. The overall treatment response increases in low and decreases in high mf density categories. Furthermore, the estimates for the random coefficients model showed that there is a greater variability in response between individuals over post-treatment time. The results substantiate that individual variation in response to DEC exists which indicate the importance of studying the parasite as well as host genetic factors associated with DEC action.
Advocacy and training on "Home care" for filarial lymphoedma (FLE) patients are provided through morbidity management and disability prevention (MMDP) clinic commonly known as filariasis clinic and clinical improvement is assessed by follow-up visits. While the physicians aim at reducing the recurrent ADL (coined as ADLA in 1997) episodes, the patients expect reduction in LE volume. The objective of the present study was to know whether the MMDP clinic serves the primary expectation of the FLE patients. LE patients who attended the clinic for at least four follow-up consultations and had LE volume measurements at three points of time during the one year period of observation were considered for analysis. Clinical assessment was done for LE grading and LE volume was measured by water displacement volumetry. Sixty-three patients who fulfilled the follow up criteria were included. It was observed that the median LE volume was 914ml (IQR 269 - 1935) at first visit of the observation period which reduced to 645ml (IQR 215- 1666) and 752ml (IQR 215 - 1720) at first and second follow-up visits respectively. Over all, in short span of one year, 21 of the 63 patients (33.3%) who visited MMDP clinic at least four times in a year were benefitted through the MMDP advocacy and the National filariasis control programme need to emphasise on the importance of follow up visits to FLE patients.
Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 109 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.
Studies on larval population densities and adult emergence rates of the Brugian filariasis vectors Mansonia bonneae Edwards and Ma. dives Schiner were conducted in freshwater swamp forest bordering the Sadong River, Serian District, Sarawak, East Malaysia, during 1984-85. Three species of aquatic host-plants in the Family Araceae were identified as supporting immature stages of the Ma. bonneae/dives complex. Proportions of positive plants were 4.7%, 6.5% and 3.4% with 6.4 +/- 2.6, 7.3 +/- 2.8 and 10.1 +/- 1.1 larvae per positive plant, respectively, for the plant species Homalomena cordata Schott, H. rostrata Griffiths and Hydrostemma motleyi (Hook. f.) Mabberley. These data indicate no significant preferences between the three types of host-plant. Detailed monitoring of the host-plant H. cordata revealed no significant monthly fluctuations in larval density per plant nor the proportion of positive plants. 11.6% of larvae were Ma. dives and 88.4% were Ma. bonneae. Mean daily yields of Ma. bonneae/dives adults per square metre of H. cordata vegetated water surface were 0.45 males plus 0.57 females during the wet season (December-February) compared with 0.2 males plus 0.31 females during the dry season (June-August). Thus output of adults per plant was approximately halved, and suitable breeding areas were further reduced, during the dry season. By extrapolation from these rates, a crude mean estimate for productivity of Ma. bonneae/dives females is 1.6 million per hectare per annum in swamp forest habitats vegetated with any of the host-plants studied.
Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.
Biotechnological tools are being used in malaria, filariasis and dengue research. The main emphasis has been on the production of reagents for immunodiagnosis and research. In this respect monoclonal antibodies (McAbs) against various species and stages of the above pathogens have been produced. It is hoped that these McAbs will be useful not only in immunodiagnosis but also for seroepidemiological applications. A DNA probe against Brugia malayi has been tested in Malaysia and was found to be sensitive and specific.
Lymphatic filariasis is the most widespread of human filarial infections, a group of vector-borne infestations. After the discovery of diethylcarbamazine (DEC), little advance was made in the development of new chemotherapeutic agents for the treatment of lymphatic filariasis until 1985. Since then, several new initiatives have occurred as the result of a global effort by the World Bank/UNDP/WHO Special Programme on Tropical Diseases and the Onchocerciasis Control Programme. Some of these global research initiatives are reviewed in this paper. Recent observations throw a new light on the rational use of DEC including its deployment as a medicated salt. Ivermectin, an established drug for the treatment of river-blindness is examined for its potential use in the treatment of lymphatic filariasis. Experimental results from two novel compounds out of several being developed by the WHO/OCP Macrofil project are considered in respect to their potential macrofilaricidal activity, particularly in relation to lymphatic filarial infections.
The genetics of human susceptibility to lymphatic filariasis, the genetic basis of filarial susceptibility in vector mosquitos, and the genetic constitution of human filarial parasites and their mosquito vectors are reviewed. It is evident that our present knowledge on the genetics of lymphatic filariasis is still very meagre. The need to study various genetic aspects of the disease is highlighted.
Successful colonization of Mansonia dives, the principal vector of subperiodic Brugia malayi was established in a field insectary. Mean egg clusters laid on Eichhornia crassipes, Pistia stratiotes, Homalomena cordata and polystyrofoam strips were 12.0, 10.4, 9.5 and 13.7 respectively. However, the mean number of first instar larvae hatched from each egg cluster laid by females on the three plant substrates (range 51.1 to 58.6) was higher than that laid on the polystyrofoam strips (41.8). There were no significant differences in the success pupation and adult emergence rates among the three host plants used as attachment substrates. Adult emergence occurred at a mean of 10.8 days. The first adult emergence was observed at the 25th day after hatching and continued till the 50th day. The 50% mortality rates for the adults were estimated as 8 days for the males and 14 days for the females. The mean gonotrophic cycle ranged from 3.8 to 4.3 days with a mean of 4.04 days. 63.6% of Ma. dives females oviposited in a medium of rat dung and water. The mean incubation period of eggs ranged from 5.2 to 6.5 days with a mean of 5.7 days. The biology of Ma. dives and Ma. bonneae is briefly compared.
Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in "Brugia Rapid". However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.
A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.
Conidiobolomycosis (also known as rhinoentomophthoramycosis) is a rare cutaneous/mucosal fungal infection seen mainly in the tropical rain forest regions of the world that can be associated with disfiguring facial elephantiasis, and rarely, death.
Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.
BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.