Western blot analysis of infective larvae (L3) antigen of Brugia malayi were performed on 200 sera from six groups of individuals: 36 samples from B. malayi microfilaremic individuals; 10 samples from individuals with elephantiasis; 50 and 20 samples from amicrofilaremic individuals in a B. malayi endemic area with no anti-filarial IgG4 antibodies (towards microfilaria and adult worm antigens) and samples with high titres of the anti-filarial IgG4 antibodies respectively; 50 samples from non-endemic normals and 34 samples from geohelminth-infected individuals. After protein transfer, PVDF membrane strips were successively incubated with blocking solution, human sera, monoclonal anti-human IgG4 antibody-HRP and developed with luminol chemiluminescence substrate. 28/36 (78%), 1/10 (10%) and 16/20(80%) of sera from individuals with microfilariae, elephantiasis and amicrofilaremic individuals with high titers of anti-filarial IgG4 antibodies respectively recognized L3 antigenic epitopes; the dominant and consistent antigenic bands were of approximately MW 43 kDa, 14 kDa, 15 kDa and 59 kDa. The rest of the sera were unreactive. This study showed that microfilaremics may or may not mount a notable antibody response to somatic L3 antigens, thus lending evidence that antibody response to this antigen is not protective against establishment of Brugia malayi infection.
The Filariasis Control Program was established more than 30 years ago in the country and the disease is still a public health problem in some states. Since 1983, a total of 17 filariasis control teams were formed throughout the country to carry out filariasis control work. The teams conduct house and population censuses, nocturnal mass blood surveys and treatment of microscopically confirmed cases. Individual case follow-up is being carried out after 3-5 months while the locality is resurveyed after about 2-3 years. During the years 1988 to 1990, there appeared to be a decreasing trend in the number of filariasis cases detected countrywide. In 1991, brugian filariasis accounted for 92% of the cases detected. The microfilaria rate (MFR) also showed a decreasing trend countrywide for the years 1988 (0.57%) to 1990 (0.35%) but there was an increase in 1991 although it remained well below the 5% MFR targeted in the program objective, In 1991, the filariasis control teams and the district multi-purpose teams collected a total of 167, 151 blood slides out of which 871 were found to be positive for microfilaria. To determine the true endemicity of filariasis in the country, the malaria district multi-purpose teams are also utilized to assist in probe surveys in new areas of the district. Two species of filarial worms, namely Brugia malayi and Wuchereria bancrofti, and the mosquito vectors belonging to the Anopheles and Mansonia genera are involved in the transmission of filariasis in Malaysia. Monkeys and domestic cats are the reservoir hosts for the subperiodic strain of B. malayi.
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.
Hematological changes were monitored in the leaf-monkey, Presbytis cristata, infected experimentally with 200 subperiodic Brugia malayi infective larvae. Prepatent periods were 54-86 days and peak microfilarial geometric mean counts (GMCs) were 1324 per ml blood. Total leukocyte and differential counts were measured at pre-infection, and then at weakly intervals before and during patency. Blood eosinophil level increased to about thrice the initial level at 3 weeks post-infection and this was maintained for the next 13 weeks before it started to rise again, increasing to more than 5 times the initial level at 20 weeks post-infection. The observed pattern of eosinophilia is probably related to the level of microfilaremia and the destruction of microfilariae in the spleen. There was no significant change in the total leukocyte counts during the period of observation.
The indirect immunofluorescence test using sonicated microfilariae of Brugia malayi has been evaluated on 173 sera from patients and persons exposed to Wuchereria bancrofti and B. malayi in endemic areas of Peninsular Malaysia. In the microfilaria-negative group, without signs and symptoms of filariasis 55/62 sera (89%) had titers of 1:16 and less. In the microfilaremic groups and in the amicrofilaremic cases with clinical filariasis, all the sera tested were positive, with the antibody titers ranging generally from 1:16 - 1:256. Cross-reaction tests were done on 16 samples of onchocerciasis sera from West Africa using sonicated antigen as well as antigen-coated CNB1-activated sepharose. Antibody titers were detected in all the sera. The usefulness of the sonicated microfilarial antigen in serodiagnosis of filariasis is discussed.
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.
Random amplification of polymorphic DNA (RAPD) was used to analyse genomic DNA from virgin females and males of Brugia malayi, with a view to identifying sex-specific differences predicted by an XX/XY system of chromosomal sex determination. A product of 2338 bp, amplified with the arbitrary primer 5' GTTGCGATCC 3', was obtained exclusively from males. Primers based on the sequence of this product amplified a DNA fragment of the expected size from each of two independent isolates of B. malayi (from Malaysia and Indonesia) by PCR. No reaction product was obtained from the closely related species Brugia pahangi. In a genetic cross between B. malayi males and B. pahangi females, F1 hybrid microfilariae were PCR-positive, indicating that the locus is paternally-inherited. Southern blotting demonstrated that the target sequence resides in the high molecular weight fraction of genomic DNA, confirming that it is of chromosomal, rather than mitochondrial, origin. Sequencing of the locus revealed significant similarity with members of a family of reverse transcriptase-like genes in Caenorhabditis elegans. In-frame stops indicate that the gene is non-functional, but multiple bands of hybridisation in Southern blots suggest that the RT sequence may be the relic of a transposable element. Multiple repeats of the dinucleotide AT occurred in another region of the sequence. These varied in number between the two isolates of B. malayi in the manner of a microsatellite, surprisingly the first to be described from the B. malayi genome. Because of its association with the Y chromosome, we have given the locus the acronym TOY (Tag On Y). Identification of this chromosome-specific marker confirms the XX/XY heterogametic karyotype in B. malayi and opens the way to elucidation of the role of Y in sex determination.
The dynamics of the transmission of subperiodic Brugia malayi in a typical endemic area in Malaysia was studied over a period of 4 years. Mass chemotherapeutic control with diethylcarbamazine citrate was found to be inefficient, new cases being detected even after the fifth treatment cycle of 6 mg/kg X 6 days per cycle. This is in marked contrast to the situation in periodic b. malayi areas where mass treatment efficiently controlled the infection. The disparity in results in these two areas is attributed to zoonotic transmission of subperiodic B. malayi from non-human primates where a mean infection rate of 76.3% was found.
Introduction: Collecting mosquitoes is essential for research in mosquito-borne diseases, but the light traps used for that purpose are expensive and often difficult to obtain around research fields. We designed a new 3D-printable mosquito light trap that can be made inexpensively anywhere where electricity is available (Hoshi et al, Scientific Reports, in press). In this study, we produced that trap in Sabah and demonstrated its usefulness in the field. Meth-ods: With a 3D printer, the main parts of the trap - body, lid, lamp stand and collection box - were printed in Kota Kinabalu using black polylactic acid (PLA) filaments purchased online. All other parts such as the computer fan and batteries were commercially available at local shops in Sabah. The parts were assembled into the complete units at Universiti Malaysia Sabah’s Rural Medical Education Centre (RMEC) in Sikuati, Kudat. Demonstration was performed at two sites in the Kudat district: RMEC campus and the premises of a local farm in Kampung Paradason. Results: The 3D traps collected 6 and 7 different species of mosquitoes at RMEC and Paradason sites, respectively. The numbers of mosquito species collected by the commercially-available CDC model-512 traps in parallel experiments were 2 (RMEC) and 10 (Paradason). The species collected by the 3D traps included Aedes albopictus (vector transmitting Dengue virus), Anopheles barbumbrosus (malaria), Culex quinquefasciatus (Wuchereria bancrofti, avian malaria, and arboviruses including Japanese encephalitis and Zika viruses) and Mansonia indiana (Brugia malayi). Conclu-sion: The 3D light trap which was produced in Sabah demonstrated its usefulness in the field tests performed in the Kudat district. This model can be used as an alternative to the rather expensive commercial light traps to collect the vector insects transmitting mosquito-borne diseases such as malaria, dengue, Japanese encephalitis, Zika fever and filariasis.
A case of double infection with Brugia pahangi and Dirofilaria immitis in a clouded leopard, Neofelis nebulosa, is presented. A brief review of filarial infections in both man and wild animals, and their medical importance is discussed.
Four Presbytis cristata were treated with oral ivermectin at the same time as the subcutaneous inoculation of 100 infective larvae monthly for three months. Two animals given 0.2 mg/kg monthly and two others given 0.3 mg/kg monthly as well as three control animals became patent for microfilaraemia. However, only 1% of the infective dose was recovered as adult worms from animals in the higher drug dosage group compared to 8.2% and 6.2% in the lower dosage and control groups respectively.
Lymphatic filariasis is a parasitic infection that causes a devastating public health and socioeconomic burden with an estimated infection of over 120 million individuals worldwide. The infection is caused by three closely related nematode parasites, namely, Wuchereria bancrofti, Brugia malayi, and B. timori, which are transmitted to human through mosquitoes of Anopheles, Culex, and Aedes genera. The species have many ecological variants and are diversified in terms of their genetic fingerprint. The rapid spread of the disease and the genetic diversification cause the lymphatic filarial parasites to respond differently to diagnostic and therapeutic interventions. This in turn prompts the current challenge encountered in its management. Furthermore, most of the chemical medications used are characterized by adverse side effects. These complications urgently warrant intense prospecting on bio-chemicals that have potent efficacy against either the filarial worms or thier vector. In lieu of this, we presented a review on recent literature that reported the efficacy of filaricidal biochemicals and those employed as vector control agents. In addition, methods used for biochemical extraction, screening procedures, and structure of the bioactive compounds were also presented.
Comparative studies of vector efficiency were done with the Liverpool and Malaysian strains of Aedes (Finlaya) togoi for subperiodic Brugia malayi and Brugia pahangi. The Malaysian strain of A. togoi was found to take in fewer microfilariae under the same experimental conditions than the Liverpool strain. Also, for various microfilarial densities in the host's peripheral blood, the Malaysian strain had less mean infective larvae per fed mosquito than the Liverpool strain. The microfilarial intake of A. togoi was not affected by the site of feeding on the host affected by the site of feeding on the host. Most of the mosquitoes took in fewer microfilariae than expected. It is concluded from these studies that the Malaysian strain of A. togoi is a susceptible and reasonably good vector for subperiodic B. malayi and B. pahangi. Further field studies should be carried out to determine its importance as a natural vector of Brugian filariasis.
A study was carried out to identify some of the cultural factors in the epidemiology of filariasis in an endemic community in Malaysia. The viewpoint of the community, data an responses on knowledge of illness and filariasis, host related factors, health examination, vector study were analysed and discussed. The observations noted on cultural factors were: Occupational pattern: Different agricultural occupations seemed to related to transmission in terms of body exposure. Activities not related to production of crops: Play groups in late afternoon, bathing of household members near and after sunset, congregations at prayer houses very much exposed the population to mosquito bites in different degree in terms of length of time spent outside the house. Knowledge of filariasis: Filariasis was understood in terms of elephantiasis, the chronic stage of the disease. Other signs of disease-adenolymphangitis, red lines running down one or both legs and abscess were generally recognized but not often annonated with specific disease. Attitude toward disease: Filariasis was not seen as a health problem and the idea of filarial worms was still hard to believe. Knowledge on disease causation: Biological causes were generally recognized, though the idea of bacteria was not widespread.
We have recently reported that a dipstick colloidal dye immunoassay (DIA) that detect parasite antigens in human serum is sensitive and specific for the diagnosis of active infection of lymphatic filariasis. Rabbit polyclonal antibodies (RbBmCAg) labelled with a commercial dye, palanil navy blue was used to detect filarial antigenemia among Indonesian and Bangladeshi immigrant workers (N= 630) at oil palm estates at Hulu Trengganu District, Peninsular Malaysia. Microfilaremia with Brugia malayi were detected in 51 (8.10 %) individuals, of which 42 (6.67 %) were among the Indonesians and 9 (1.98 %) among the Bangladeshis. Microfilaremia with Wuchereria bancrofti were detected in 33 (5.24 %) individuals of which 15 (2.38 %) were among the Indonesians and 18 (2.86 %) among the Bangladeshis workers. The DIA detected 96 (15.24 %) antigenemic cases which comprise of all the microfilaremic cases and 15 (2.38 %) amicrofilaremic cases. The amicrofilaremic cases with filarial antigenemia consisted of 9 (1. 43 %) Indonesians and 6 (0.95%) Bangladeshis. We have used 6 ul of the RbBmCAg and diluted (1:10) patients' sera per dipstick which make the DIA reagent conservative. The DIA is a rapid test and can be read in approximate 2 hours.. Additionally, coloured dots developed in the DIA can be qualitatively assessed visually for intensity. The DIA does not require sophisticated equipment or radioactivity, and therefore suitable for field application.
In 2011, we reported occurrence of natural human infections with Brugia pahangi, a filarial worm of dogs and cats, in a surburb of Kuala Lumpur, the capital city of Malaysia. Our preliminary entomological survey at that time suggested the mosquito species Armigeres subalbatus as the vector of the zoonotic infections. In this present report, we provide biological evidence to confirm our preliminary finding.
Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG4 and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG4 , IgG, IgE and IgG (IVD) assays were 76.9%, 84.6%, 7.7% and 84.6%, respectively, while the specificities were 92.7%, 81.8%, 100% and 83.6%, respectively. If filariasis samples were excluded, the specificities of the IgG4 -ELISA and both IgG-ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG- and IgG4 -ELISAs (r = 0.4828; P = 0.0125). IgG- and IgG- (IVD) ELISAs (r = 0.309) were positively correlated, but was not significant (P = 0.124). Meanwhile there was no correlation between IgG4 - and IgG- (IVD) ELISAs (r = 0.0042; P = 0.8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG4 and the optical densities of anti-Strongyloides IgG4 -ELISA (r = 0.4544, P = 0.0294). In conclusion, the detection of both anti-Strongyloides IgG4 and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.
Efforts to completely eradicate lymphatic filariasis from human population may be challenged by the emergence of Brugia pahangi as another zoonotic lymphatic filarial nematode. In this report, a genomic study was conducted to understand this species at molecular level.
An IgG4 ELISA based on a novel recombinant antigen was evaluated for detection of Brugia malayi infection, using 2487 sera from various institutions: 2031 samples from Universiti Sains Malaysia, 276 blinded sera from 2 other institutions in Malaysia, 140 blinded sera from India and 40 blinded sera from Thailand. These sera were from various groups of individuals, i.e., microfilaraemics, chronic patients, endemic normals, non-endemic normals and individuals with other parasitic and bacterial infections. Based on a cut-off optical density reading of 0.300, the IgG4 ELISA demonstrated specificity rates of 95.6-100%, sensitivity rates of 96-100%, positive predictive values of 75-100% and negative predictive values of 98.9-100%. These evaluation studies demonstrated the high specificity and sensitivity of this test for the detection of active B. malayi infection. Thus, the IgG4 ELISA would be very useful as a tool in diagnosis and in elimination programmes for brugian filariasis.
Five aqueous extracts from three plant species, i.e., dried husks (HX), dried seeds (SX) and dried leaves (LX) of Xylocarpus granatum (Meliaceae), dried stems (ST) of Tinospora crispa (Menispermaceae) and dried leaves (LA) of Andrographis paniculata (Acanthaceae) were tested in vitro against adult worms of subperiodic Brugia malayi. The relative movability (RM) value of the adult worms over the 24-h observation period was used as a measure of the antifilarial activity of the aqueous extracts. SX extract of X. granatum demonstrated the strongest activity, followed by the LA extract of A. paniculata, ST extract of T. crispa, HX extract and LX extract of X. granatum.