Tropical pulmonary eosinophilia (TPE) is considered to be a variant of human filarial infection. The pulmonary manifestations of TPE have been well described. Extra-pulmonary features of the disease, although not commonly seen, have been reported previously. A 9-year-old Malay girl with a history of recurrent cough and wheezing was admitted because of cardiac failure. Physical examination revealed a very sick girl with tachypnoea, central cyanosis, finger clubbing, elevated jugular venous pulse, generalized crackles and rhonchi in the chest, a loud second heart sound and hepatosplenomegaly. A chest radiograph showed cardiomegaly and right pleural effusion. Laboratory investigations revealed hypochromic, microcytic anaemia with persistent blood eosinophilia (absolute eosinophil counts varied from 1.9 to 5.5 x 10(9)/1). The ELISA test for antifilarial IgG antibodies was strongly positive. She responded promptly to treatment with diethylcarbamazine. In summary, this is a patient with TPE who presented with cor pulmonale, probably due to late-stage interstitial pulmonary fibrosis. In order to prevent the long term morbidity of cardiorespiratory disability, the early signs of TPE should be recognized and the infection treated.
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.
Microscopic detection of active phase of lymphatic filariasis is indicated by the presence of microfilaria in whole blood. This method is not sensitive and requires relatively large amount of blood sample. PCR allows very sensitive detection of the parasite DNA using a smaller amount of blood; and the use of dried blood spots facilitates sample transportation. Nevertheless, limited studies have been reported on PCR using dried blood spot for detection of Brugia malayi. In this study, we investigated the effects of concentrating whole blood genomic DNA sample and the amplification methods [conventional PCR (C-PCR) and real-time PCR] on the detection of B. malayi DNA from dried blood spots from a very low endemic area in Malaysia. Both C-PCR and real-time PCR detected 2 out of 18 (11%) samples as positive from non-concentrated genomic DNA preparations. After the DNA samples were pooled and concentrated, both C-PCR and realtime PCR detected B. malayi DNA amplifications in 7 out of 18 (39%) samples. However one sample which showed faint band in C-PCR was detected as highly positive in real-time PCR. In conclusion, both C-PCR and real-time PCR using dried blood spots from a low endemic area demonstrated equal sensitivity for detection of B. malayi DNA.
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.
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.
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.
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.
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.
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.
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.
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.
Sera from fifty subjects with different presentations of Brugian filariasis and from common soil-transmitted helminth infections were tested for specific anti-filarial IgG and its subclasses. Anti-filarial IgG, IgG1 and IgG3 showed cross-reactivities with soil-transmitted helminthic infections and no significant differences in optical densities among the various groups of filarial patients. In comparison with other groups of subjects, IgG4-ELISA of sera from microfilaraemic patients and some previously microfilaraemic patients showed a significant increase in optical density readings, while IgG2-ELISA showed elevated optical density readings in sera of patients with chronic elephantiasis. Therefore IgG2-ELISA is potentially useful in the diagnosis of brugian chronic elephantiasis while IgG4-ELISA may be beneficial for follow-up diagnosis of treated microfilaraemic patients.
The study compared the effectiveness of a single dose of diethylcarbamazine (DEC) (6mg/kg) with the standard regimen of 6 doses (total 36 mg/kg) in mass chemotherapy for the control of brugian filariasis. Mass chemotherapy with single-dose DEC was instituted in one area and standard dose in the other and treatment was repeated after one year. Parasitological surveys were conducted before, and 3, 7 and 12 months after treatment. Pretreatment characteristics were not significantly different between the 2 treatment areas. There was a significant reduction in microfilaraemia prevalence rate from 24.7% to 14.7% at 12 months and to 6.8% at 19 months in the single dose area and from 22.8% to 9.6% at 12 months and to 2.7% at 19 months with the standard dose. Maximum reduction was at 7 months after treatment with both regimens. There was also significant progressive reduction in mean microfilarial density from 4.39 +/- 20.37 to 0.89 +/- 4.16 per 60 microL in the single-dose area and from 4.43 +/- 17.31 to 0.75 +/- per 60 microL in the standard dose area. There was a greater reduction of both microfilarial prevalence and density using the standard regimen but it was not statistically significant. Thus, a single dose of DEC is as effective as the standard dose in controlling periodic brugian filariasis.
A clinical trial on the efficacy of a single oral dose of ivermectin at 20, 50, 100, and 200 micrograms/kg was carried out in 40 subjects with subperiodic Brugia malayi microfilaremia. There was no significant difference in the clearance of microfilaremia in the four treatment groups, and the lowest geometric mean microfilarial count (GMC) achieved in the 40 subjects was 8.8/ml or 8.3% of the initial count (106.1/ml), at two weeks post-treatment. The GMC started to increase at one month post-treatment and by six months was 22.2% of the initial GMC. Only 27.5%, 23.1%, 15.0%, and 18.9% of subjects were amicrofilaremic at two, four, 12, and 24 weeks post-treatment, respectively. Mild fever in 35% of the subjects was the primary side reaction and was more common in those with microfilarial counts > or = 500/ml (85.7%) than in those with counts < 500/ml (32%). The clearance of B. malayi microfilaremia by ivermectin was less rapid than that reported for Wuchereria bancrofti. The smaller number of side reactions encountered in the present study compared with those reported for bancroftian filariasis is probably related to the lower microfilarial density in the present subjects. Since ivermectin at a single oral dose of 20-200 micrograms/kg can reduce the GMC to less than 10% at two weeks and maintain it below 25% of the initial level even at six months post-treatment, it is recommended that the drug be seriously evaluated for use in the control of brugian filariasis.
We conducted a field study of a rapid test (Brugia Rapid) for detection of Brugia malayi infection to validate its sensitivity and specificity under operational conditions. Seven districts in the state of Sarawak, Malaysia, which are endemic for brugian filariasis, were used to determine the test sensitivity. Determination of specificity was performed in another state in Malaysia (Bachok, Kelantan) which is non-endemic for filariasis but endemic for soil-transmitted helminths. In Sarawak both the rapid test and thick blood smear preparation were performed in the field. The rapid test was interpreted on site, whereas blood smears were taken to the district health centres for staining and microscopic examination. Sensitivity of Brugia Rapid dipstick as compared with microscopy of thick blood smears was 87% (20/23; 95% CI: 66.4-97.2) whereas the specificity was 100% (512/512). The lower sensitivity of the test in the field than in laboratory evaluations (> or =95%), was probably due to the small number of microfilaraemic individuals, in addition to difficulties in performing the test in remote villages by field personnel. The overall prevalence of brugian filariasis as determined by the dipstick is 9.4% (95% CI: 8.2-0.5) while that determined by microscopy is 0.90% (95% CI: 0.5-1.3) thus the dipstick detected about 10 times more cases than microscopy. Equal percentages of adults and children were found to be positive by the dipstick whereas microscopy showed that the number of infected children was seven times less than infected adults. The rapid dipstick test was useful as a diagnostic tool for mapping and certification phases of the lymphatic filariasis elimination programme in B. malayi-endemic areas.
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.
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.
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.