Western blot analyses were performed on 444 serum specimens: 40 sera from microfilaraemic individuals, 10 sera from elephantiasis patients, 24 treated individuals, 50 sera from residents of endemic areas without anti-filarial IgG4 antibodies (endemic normals), 20 sera from amicrofilaraemic individuals with high anti-filarial IgG4 antibodies, 200 sera from healthy city-dwellers (non-endemic samples), and 100 sera from soil-transmitted helminth-infected individuals. Phast electrophoresis system was used to electrophorese Brugia malayi soluble adult worm antigen on 10-15% SDS-PAGE gradient gels followed by electrophoretic transfer onto PVDF membranes. Membrane strips were then successively incubated with blocking solution, human sera, and monoclonal anti-human IgG4 antibody-HRP, with adequate washings done in between each incubation step. Luminol chemiluminescence detection was then used to develop the blots. An antigenic band with the MW of approximately 37 kDa was found to be consistently present in the Western blots of all microfilaraemic sera, all amicrofilaraemic sera with high titres of anti-filarial IgG4 antibodies, some treated patients, and some elephantiasis patients. The antigen did not occur in immunoblots of individuals with other helminthic infections, normal endemic individuals, and city dwellers. Therefore the B. malayi antigen of with the MW of approximately 37 kDa demonstrated specific reactions with sera of B. malayi-infected individuals and thus may be useful for diagnostic application.
In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.
We are reporting a case of an eye lesion caused by an adult Brugia malayi. The patient was a 3-year-old Chinese boy from Kemaman District, Terengganu, Peninsular Malaysia. He presented with a one week history of redness and palpebral swelling of his right eye. He claimed that he could see a worm in his right eye beneath the conjunctiva. He had no history of traveling overseas and the family kept dogs at home. He was referred from Kemaman Hospital to the eye clinic of Hospital Tengku Ampuan Afzan, Kuantan, Pahang, Malaysia. On examination by the ophthalmologist, he was found to have a subconjunctival worm in his right eye. Full blood count revealed eosinophilia (10%). Four worm fragments, each about 1 cm long were removed from his right eye under general anesthesia. A thick blood smear stained with Giemsa was positive for microfilariae of Brugia malayi. A Brugia Rapid test done was positive. He was treated with diethylcarbamazine.
Study site: Opthamolagy clinic, Hospital Tengku Ampuan Afzan
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.
Accurate identification of filarial parasites in mosquitoes poses a major problem for the coordination of filariasis control programs. Traditional methods are tedious, and some are not specific enough to give satisfactory results. Amplification of specific gene sequences by primer-directed polymerase chain reaction (PCR) has been increasingly utilized as a diagnostic tool. However, current protocols for the extraction of parasite DNA from mosquito samples are tedious and could lead to failure of PCR amplification. We demonstrate that the use of Chelex is an efficient method for DNA extraction from mosquitoes and the parasite and that PCR amplification with primers specific for Brugia malayi yields a band of the expected size. The PCR products were transferred to a nylon membrane with Southern blotting, and a B. malayi-specific digoxigenin-labeled probe confirmed the sequence similarity of the PCR-amplified fragment and increased the sensitivity of the PCR assay. Use of this probe enabled us to detect PCR-amplified product from B. malayi even when a product was not visible on an ethidium bromide-stained agarose gel. This increased sensitivity allowed us to detect the parasite in the heads of mosquitoes.
Malaria and filariasis still continue to pose public health problems in developing countries of the tropics. Although plans are in progress for the elimination of both these parasitic vector borne diseases, we are now faced with a daunting challenge as we have a fifth species, Plasmodium knowlesi a simian malaria parasite affecting humans. Similarly in peninsular Malaysia, filariasis was mainly due to Brugia malayi. However, we now see cases of Wuchereria bancrofti in immigrant workers coming into the country. In order to successfully eliminate both these diseases we need to know the vectors involved and introduce appropriate control measures to prevent the diseases occurring in the future. As for knowlesi malaria it is still uncertain if human to human transmission through mosquito bites is occurring. However, P. knowlesi in human is not a rare occurrence anymore and has all the characteristics of a pathogen spreading due to changes in the ecosystem, international travel, and cross border migration. This has created a more complex situation. In order to overcome these challenges we need to revamp our control measures. This paper reviews the vectors of malaria and filariasis in Southeast Asia with special emphasis on P. knowlesi and W. bancrofti in Malaysia and their control strategies.
Wuchereria bancrofti, Brugia malayi and Brugia timori are the causative agents of lymphatic filariasis in Indonesia but in some endemic areas, B malayi is more commonly found. Diagnosis of filariasis is normally based on clinical, parasitological and immunological examinations but those methods have limitations. The discovery of monoclonal antibodies is expected to provide a new dimension to the efforts in the development of specific and sensitive immunological tests for the various stages of filariasis infection. This preliminary report, using monoclonal antibodies and dot-blot assay in human lymphatic filariasis showed that 75% of sera from microfilaremic patients with clinical signs, 40% of sera from amicrofilaraemic patients with clinical signs, 88.8% of sera from microfilaremic patients without clinical signs and 19.6% of sera from amicrofilaremic patients without clinical signs have circulating antigens.
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.
BmR1 recombinant antigen has previously been shown to demonstrate high sensitivity and specificity in the serological diagnosis of brugian filariasis in humans. In this study, the pattern of recognition of antibody to BmR1 during Brugia malayi infection was investigated by employing Meriones unguiculatus as the experimental model. Thirty two gerbils were infected subcutaneously with 120 L(3); and two control groups each comprising 25 animals were employed. ELISA using BmR1 was used to detect filaria-specific IgG antibodies elicited by the gerbils; using sera collected from the day 1 until day 150 post-inoculation (p.i.). The results showed that BmR1 detected B. malayi infection in gerbils harboring adult worms irrespective of the presence of circulating microfilaria, and was exemplified by positive ELISA results in nine a microfilaraemic animals that harbored live adult worms. The initial time of the antibody recognition was at day 8 p.i. and the antibody titre showed some correlation with adult worm burden.
The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.
In the absence of a suitable Brugia malayi antigen detection assay, PCR remains one of the more sensitive alternatives to Giemsa-stained thick blood films for B. malayi detection. The need for refrigerated storage and transportation of blood has limited the use of PCR for large-scale epidemiology studies in remote endemic areas. Here we report simple finger-prick blood-spot collection, a one-tube DNA template extraction method and the development of a B. malayi-specific nested PCR assay. The assay was tested on 145 field samples and was positive for all 30 microscopy-positive samples and for an additional 13 samples which were microscopy-negative.
The periodicity of the microfilariae of Brugia malayi was studied in 9 human carriers from Shin-san Ri, Seong-san Myon, Cheju Island. The periodicity pattern was markedly nocturnal and the peaks were observed between 21:30 p.m. and 5:30 a.m. The average peak count was 1:30 a.m. and the percentage of peak count at this time was 95.3. The ratio of minimum of the average peak count percentage to the maximum was 8.3. The periodicity pattern of B.malayi in Cheju Island was compared with that in Inland and no differences were found between two forms. From the above observations, it was concluded that the periodicity of B. malayi in Korea is markedly nocturnal and closely resembles that in the strain of Penang, Malaya.