Echinococcus granulosus is a worldwide zoonotic infection that causes human cystic echinococcosis (CE) or hydatid disease. The present study describes the isolation and production of a monoclonal antibody against recombinant AgB protein using the developed Human AntibodY Disease ENhanced (HAYDEN)-Filariasis library. The DNA sequences of the isolated clones were analyzed, followed by gene analysis and binding assays. Clone E1 showed a full-length sequence and represents the IgHV5-LV3 antibody gene family. The antibody protein yield was satisfactory, and it reacted specifically against rAgB. The novel E1 protein is potentially useful for the development of an antigen detection assay for CE. The ability of the Brugia malayi immune antibody library to isolate antibodies against Echinococcus granulosus antigens highlights the broad coverage of immune antibody libraries.
Brugia malayi and Wuchereria bancrofti infections cause lymphatic filariasis in Malaysia. About 2.5 million people live in endemic areas of filariasis, of whom 5% have microfilaraemia and probably twice as many are infected. There is a wide clinical spectrum of response to the infection. While some have asymptomatic microfilaraemia, others have episodic attacks of fever, lymphadenitis, retrograde lymphangitis and lymphoedema. Elephantiasis is a late complication. Tropical pulmonary eosinophilia and other forms of occult filariasis are due to hyper allergic reactions to microfilarial antigens. Parasitological and serological tests aid in confirming the clinical diagnosis. The drug of choice is diethylcarbamazine citrate.
BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.
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.
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.
Developing and adult worms of the human lymphatic filarial parasites (Wuchereria bancrofti,
Brugia malayi, and Brugia timori) are located mainly in the lymphatic system and occasionally in aberrant sites like subcutaneous and conjunctival cysts. Lymphatic
pathology ranging from dilatation of lymphatic channels and lymphangiectasia are detected on ultrasonography in apparently healthy, amicrofilaraemic, but filarial antigen positive individuals in endemic areas. Microfilariae are distributed in various organs and may be associated with immune mediated pathology at these sites; tropical pulmonary eosinophilia is characterized by intense immune mediated destruction of microfilariae in the lung parenchyma. In the spleen and other sites, nodular granulomatous lesions can occur where microfilariae are trapped and destroyed. The finding of Wolbachia endosymbionts in all stages of lymphatic filarial parasites has provided new insight on the adverse reactions
associated with anti-filarial chemotherapy. Inflammatory molecules mainly lipopolysaccharide (LPS)-like molecules released from endosymbionts on death of the
parasites are largely responsible for the adverse reactions encountered during anti-filarial chemotherapy. Prenatal tolerance or sensitization to parasite derived molecules can immune-modulate and contribute to both pathology and susceptibility/resistance to infection. Pathological responses thus depend not only on exposure to filarial antigens/infection, but also on host-parasiteendosymbiont factors and to intervention with antifilarial treatment. Treatment induced or host mediated death of parasites are associated with various grades of inflammatory response, in which eosinophils and LPS from endosymbionts play prominent roles, leading to death of the parasite, granulomatous formation, organization and fibrosis. The non-human primate (Presbytis spp.) model of
Brugia malayi developed for the tertiary screening of anti-filarial compounds has provided unique opportunities for the longitudinal study of the pathology associated with lymphatic filariasis. The pathology in this non-human primate model closely follows that seen in
human lymphatic filarial infections and correlates with clinical evidence of lymphatic pathology as detected with ultrasonography. These studies also show that successful treatment as detected by loss of motility and calcification of worms on ultrasonography is associated with reversal of early dilatations of lymphatic channels.
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.
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.
Parasitological and serological investigations for lymphatic filariasis were performed on 450 immigrants detained at the lmmigration Centre at Semenyih, Selangor, West Malaysia. The country of origin of these immigrants were Indonesia, The Philippines, Myanmar, Bangladesh, India and Pakistan. Brugia malayi adult worm homogenate (BmAH) antigen was used for the detection of antiifilarial IgG. A monoclonal antibody-based ELISA (MAb.XC3-ELISA) specific for filarial circulating antigens and non-phosphorylcholine reactive was used to detect antigenemia in these immigrants. Parasitologically 67 (14.89 %) were positive for W. bancrofti and 54 (12.0%) for Brugia malayi. Serologically 63% had antifilarial IgG titre to the BmAH antigen. While Bancroftian filariasis is now unknown in Peninsular Malaysia, the potential of it to be reintroduced into Peninsular Malaysia by the immigrant population is discussed. KEYWORDS: Lymphatic filariasis, immigratits, antifilarial IgG, antigenemia
A rapid and selective high-performance liquid chromatographic assay for simultaneous quantitative determination of a new antifilarial drug (UMF-058, I) and mebendazole (MBZ) is described. After a simple extraction from whole blood, both compounds were analysed using a C18 Nova Pak reversed-phase column and a mobile phase of methanol-0.05 M ammonium dihydrogenphosphate (50:50, v/v) adjusted to pH 4.0, with ultraviolet detection at 291 nm. The average recoveries of I and MBZ over a concentration range of 25-250 ng/ml were 92.0 +/- 7.7 and 84.4 +/- 4.4%, respectively. The minimum detectable concentrations in whole blood for I and MBZ were 7 and 6 ng/ml, respectively. This method was found to be suitable for pharmacokinetic studies.
De novo approach was applied to design single chain fragment variable (scFv) for BmR1, a recombinant antigen from Bm17DIII gene which is the primary antigen used for the detection of anti-BmR1 IgG4 antibodies in the diagnostic of lymphatic filariasis. Three epitopes of the BmR1 was previously predicted form an ab initio derived three-dimensional structure. A collection of energetically favourable conformations was generated via hot-spot-centric approach. This resulted in a set of three different scFv scaffolds used to compute the high shape complementary conformations via dock-and-design approach with the predicted epitopes of BmR1. A total of 4227 scFv designs were generated where 200 scFv designs produced binding energies of less than -20 R.E.U with shape complementarity higher than 0.5. We further selected the design with at least one hydrogen bond and one salt bridge with the epitope, thus resulted in a total of 10, 1 and 19 sFv designs for epitope 1, 2 and 3, respectively. The results thus showed that de novo design can be an alternative approach to yield high affinity in silico scFv designs as a starting point for antibody or specific binder discovery processes.
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.
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.
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.
Introduction:Lymphatic filariasis (LF) is a neglected tropical disease that can cause significant morbidity. In Malay-sia, National Programme for the Elimination of Lymphatic Filariasis started in 2001 with the initial target of achiev-ing Lymphatic Filariasis elimination status by 2018 but it has been revised to year 2020. Mass Drug Administration (MDA) Programme was performed from 2004 to 2008 in all endemic areas (Red Implementation Unit, IU) in Malay-sia including Terengganu state to stop disease transmission. Transmission Assessment Surveys (TAS) were conducted later on and for Terengganu, they were done in 2011 (TAS 1), 2015 (TAS 2) and 2017 (TAS 3) and had passed all the surveys based on critical cut off (CCO) point given. Methods: A cross sectional analysis of 10-year Terengganu filariasis records (2009-2018) was initiated in June 2019 using data source from eVekpro and filariasis cases line-list-ing. Results: Majority of filariasis cases in Terengganu were among males (n=147, 76.6%) with the highest number among 30-39 year-old age group (n=35, 18.2%). Majority of cases were Malaysian citizens (n=162, 84.4%) with main filariasis species identified were Brugia Malayi (n=149, 77.6%). The number of cases diagnosed was slightly higher from Green Implementation Unit area (n=102, 53.1%) compared to Red Implementation Unit area. Conclu-sion: The number of lymphatic filariasis cases among Terengganu citizens was below critical cut off point after the accomplishment of MDA programme and in accordance with the aim of lymphatic filariasis elimination status in Malaysia by 2020.
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.
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.