An open label, randomized clinical trial to compare the tolerability and efficacy of ivermectin plus diethylcarbamazine and albendazole vs. diethylcarbamazine plus albendazole for treatment of brugian filariasis in Indonesia.

Improved treatments for lymphatic filariasis (LF) could accelerate the global elimination program for this disease. A triple drug combination of the anti-filarial drugs ivermectin, diethylcarbamazine (DEC) and albendazole (IDA) has been shown to be safe and effective for achieving sustained clearance of microfilariae (Mf) of the filarial parasite Wuchereria bancrofti from human blood. However, the triple drug combination has not been previously been evaluated for treatment of brugian filariasis, which accounts for about 10% of the global LF burden. This hospital-based clinical trial compared the safety and efficacy of IDA with that of the standard treatment (DEC plus albendazole, DA) in persons with Brugia timori infections on Sumba island, Indonesia. Fifty-five asymptomatic persons with B. timori Mf were treated with either a single oral dose of IDA (28 subjects) or with DEC plus albendazole (DA, 27 subjects). Participants were actively monitored for adverse events (AE) for two days after treatment by nurses and physicians who were masked regarding treatment assignments. Passive monitoring was performed by clinical teams that visited participant's home villages for an additional five days. Microfilaremia was assessed by membrane filtration of 1 ml night blood at baseline, at 24h and one year after treatment. IDA was more effective than DA for completely clearing Mf at 24 hours (25/28, 89% vs. 8/27, 30%, P < 0.001). By 12 months after treatment, only one of 27 IDA recipients had Mf in their blood (4%) vs. 10 of 25 (40%) in persons treated with DA (P = 0.002). Approximately 90% of participants had antibodies to recombinant filarial antigen BmR1 at baseline. Antibody prevalence decreased to approximately 30% in both treatment groups at 12 months. About 45% of persons in both treatment groups experienced AE such as fever, muscle aches, lower back, joint and abdominal pain. These were mostly mild and most common during the first two days after treatment. No participant experienced a severe or serious AE. This study showed that IDA was well-tolerated and significantly more effective for clearing B. timori Mf from the blood than DA. Larger studies should be performed to further assess the safety and efficacy of IDA as a mass drug administration regimen to eliminate brugian filariasis. Trial Registration: NCT02899936.

Morphological characteristics of microfilariae in blood smears of the common treeshrew Tupaia glis (Mammalia: Scandentia) in Gemas, Negeri Sembilan, Malaysia.

Some filarial nematodes, such as Wuchereria bancrofti, Brugia malayi, and Brugia timori, cause lymphatic diseases in humans in the tropics, whereas other filarial parasites from wild animals cause zoonotic diseases in humans worldwide. To elucidate the prevalence and diversity of filarial parasites in Malaysia, we investigated the filarial parasites from wild animals in Gemas, Negeri Sembilan. To find adult filarial parasites, we dissected 26 animals, which included five frogs, one skink, one snake, two birds, six common treeshrews, and 11 rats. Then, we examined microfilariae in the blood smears and skin snips obtained from each animal. We found two types of microfilariae in the blood smears of common treeshrews: one was very similar to Malayfilaria sofiani and the other closely resembled Brugia tupaiae. These findings indicate an additional distribution of these filarial parasites in Gemas.

Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes.

BACKGROUND: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack. CONCLUSIONS/SIGNIFICANCE: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

Cervical Lymphatic Filariasis in a Pediatric Patient: Case Report and Database Analysis of Lymphatic Filariasis in the United States.

Lymphatic filariasis is a mosquito-borne parasitic infection caused by Wuchereria bancrofti and Brugia spp. Commonly seen in tropical developing countries, lymphatic filariasis occurs when adult worms deposit in and obstruct lymphatics. Although not endemic to the United States, a few cases of lymphatic filariasis caused by zoonotic Brugia spp. have been reported. Here we present a case of an 11-year-old female with no travel history who was seen in our clinic for a 1-year history of painless left cervical lymphadenopathy secondary to lymphatic filariasis. We review the literature of this infection and discuss the management of our patient. Using the National Inpatient Sample (NIS), the largest publicly available all-payer inpatient care database in the United States, we also examine the demographics of this infection. Our results show that chronic lymphadenopathy in the head and neck is the most common presenting symptoms of domestic lymphatic filariasis. Diagnosis is often made after surgical lymph node excision. Examination of the NIS from 2000 to 2014 revealed 865 patients admitted with a diagnosis of lymphatic filariasis. Most patients are in the mid to late sixties and are located on the eastern seaboard. Eight hundred and twenty six cases (95.5%) were likely due to zoonotic Brugia spp. and 39 (4.5%) due to W. bancrofti. Despite being rare, these data highlight the need to consider filariasis in patients presenting with chronic lymphadenopathy in the United States.

Human infection with sub-periodic Brugia spp. in Gampaha District, Sri Lanka: a threat to filariasis elimination status?

BACKGROUND: Post-mass drug administration (MDA) surveillance during the lymphatic filariasis (LF) elimination program in Sri Lanka, revealed the re-emergence of brugian filariasis after four decades. This study was done with the objectives of investigating the epidemiology and age-specific vulnerability to infection. Surveillance was done using night blood smears (NBS) and the Brugia rapid test (BRT), to detect microfilaria (MF) and anti-Brugia IgG4 antibodies in blood samples collected from an age-stratified population enrolled from two high-risk study areas (SA)s, Pubudugama and Wedamulla in the Gampaha District. The periodicity of the re-emergent Brugia spp. was characterized by quantitative estimation of MF in blood collected periodically over 24 h using nucleopore-membrane filtration method. RESULTS: Of 994 participants [Pubudugama 467 (47.9%) and Wedamulla 527 (53%)] screened by NBS, two and zero cases were positive for MF at Pubudugama (MF rate, 0.43) and Wedamulla (MF rate, 0), respectively, with an overall MF rate of 0.2. Of the two MF positives, one participant had a W. bancrofti while the other had a Brugia spp. infection. Of 984 valid BRT test readings [Pubudugama (n = 461) and Wedamulla (n = 523)], two and seven were positive for anti-brugia antibodies by BRT at Pubudugama (antibody rate 0.43) and Wedamulla (antibody rate 1.34), respectively, with an overall antibody rate of 0.91. Both MF positives detected from SAs and two of three other Brugia spp. MF positives detected at routine surveillance by the National Anti-Filariasis Campaign (AFC) tested negative by the BRT. Association of Brugia spp. infections with age were not evident due to the low case numbers. MF was observed in the peripheral circulation throughout the day (subperiodic) with peak counts occurring at 21 h indicating nocturnal sub-periodicity. CONCLUSIONS: There is the low-level persistence of bancroftian filariasis and re-emergence of brugian filariasis in the Gampaha District, Sri Lanka. The periodicity pattern of the re-emergent Brugia spp. suggests a zoonotic origin, which causes concern as MDA may not be an effective strategy for control. The importance of continuing surveillance is emphasized in countries that have reached LF elimination targets to sustain programmatic gains.

Pyrosequencing Using SL and 5S rRNA as Molecular Markers for Identifying Zoonotic Filarial Nematodes in Blood Samples and Mosquitoes.

BACKGROUNDS: Lymphatic filariasis is principally caused by Wuchereria bancrofti, and Brugia malayi. The other two filarial nematode species, Brugia pahangi and Dirofilaria immitis, possibly cause human zoonotic diseases. METHODS: We propose the development of a PCR assay linked with DNA pyrosequencing as a rapid tool to identify W. bancrofti, B. malayi, B. pahangi, and D. immitis in blood samples and mosquitoes. Primers targeting the fragment of the 5S ribosomal RNA and spliced leader sequences were newly designed and developed to identify these four filarial nematodes. Analytical sensitivity and specificity were evaluated. RESULTS: Pyrosequencing determination of nucleotide variations within 36 nucleotides for B. malayi and B. pahangi, and 32 nucleotides for W. bancrofti and D. immitis is sufficient for differentiation of those filarial nematodes, and for detection of intraspecies genetic variation of B. malayi. This analysis could detect a single B. malayi, B. pahangi, W. bancrofti, and D. immitis microfilaria in blood samples. CONCLUSIONS: Overall, the PCR-linked pyrosequencing-based method was faster than direct sequencing and less expensive than real-time PCR or direct sequencing. This is the possibility of choice that can be applied in a high-throughput platform for identification and surveillance of reservoirs and vectors infected with lymphatic filaria in endemic areas.

Transmission assessment surveys (TAS) to define endpoints for lymphatic filariasis mass drug administration: a multicenter evaluation.

BACKGROUND: Lymphatic filariasis (LF) is targeted for global elimination through treatment of entire at-risk populations with repeated annual mass drug administration (MDA). Essential for program success is defining and confirming the appropriate endpoint for MDA when transmission is presumed to have reached a level low enough that it cannot be sustained even in the absence of drug intervention. Guidelines advanced by WHO call for a transmission assessment survey (TAS) to determine if MDA can be stopped within an LF evaluation unit (EU) after at least five effective rounds of annual treatment. To test the value and practicality of these guidelines, a multicenter operational research trial was undertaken in 11 countries covering various geographic and epidemiological settings. METHODOLOGY: The TAS was conducted twice in each EU with TAS-1 and TAS-2 approximately 24 months apart. Lot quality assurance sampling (LQAS) formed the basis of the TAS survey design but specific EU characteristics defined the survey site (school or community), eligible population (6-7 year olds or 1(st)-2(nd) graders), survey type (systematic or cluster-sampling), target sample size, and critical cutoff (a statistically powered threshold below which transmission is expected to be no longer sustainable). The primary diagnostic tools were the immunochromatographic (ICT) test for W. bancrofti EUs and the BmR1 test (Brugia Rapid or PanLF) for Brugia spp. EUs. PRINCIPAL FINDINGS/CONCLUSIONS: In 10 of 11 EUs, the number of TAS-1 positive cases was below the critical cutoff, indicating that MDA could be stopped. The same results were found in the follow-up TAS-2, therefore, confirming the previous decision outcome. Sample sizes were highly sex and age-representative and closely matched the target value after factoring in estimates of non-participation. The TAS was determined to be a practical and effective evaluation tool for stopping MDA although its validity for longer-term post-MDA surveillance requires further investigation.

Impact of six rounds of mass drug administration on Brugian filariasis and soil-transmitted helminth infections in eastern Indonesia.

BACKGROUND: The lymphatic filarial parasite Brugia timori occurs only in eastern Indonesia where it causes high morbidity. The absence of an animal reservoir, the inefficient transmission by Anopheles mosquitoes and the high sensitivity to DEC/albendazole treatment make this species a prime candidate for elimination by mass drug administration (MDA). METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the effect of MDA using DEC and albendazole on B. timori and soil transmitted helminths (STH) in a cross-sectional study of a sentinel village on Alor Island annually over a period of 10 years. Pre-MDA the microfilaria (MF) prevalence was 26% and 80% of the residents had filaria-specific IgG4 antibodies. In 2010, 34 months after the 6(th) round of MDA, MF and antibody rates were only 0.17% and 6.4%, respectively. The MDA campaign had also a beneficial effect on STH. Baseline prevalence rates for Ascaris, hookworm and Trichuris were 34%, 28%, and 11%, respectively; these rates were reduced to 27%, 4%, and 2% one year after the 5(th) round of MDA. Unfortunately, STH rates rebounded 34 months after cessation of MDA and approached pre-MDA rates. However, the intensity of STH infection in 2009 was still reduced, and no heavy infections were detected. CONCLUSIONS/SIGNIFICANCE: MDA with DEC/albendazole has had a major impact on B. timori MF and IgG4 antibody rates, providing a proof of principle that elimination is feasible. We also documented the value of annual DEC/albendazole as a mass de-worming intervention and the importance of continuing some form of STH control after cessation of MDA for filariasis.

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

The effects of size and synthesis methods of gold nanoparticle-conjugated MαHIgG4 for use in an immunochromatographic strip test to detect brugian filariasis.

This study describes the properties of colloidal gold nanoparticles (AuNPs) with sizes of 20, 30 and 40 nm, which were synthesized using citrate reduction or seeding-growth methods. Likewise, the conjugation of these AuNPs to mouse anti-human IgG(4) (MαHIgG(4)) was evaluated for an immunochromatographic (ICG) strip test to detect brugian filariasis. The morphology of the AuNPs was studied based on the degree of ellipticity (G) of the transmission electron microscopy images. The AuNPs produced using the seeding-growth method showed lower ellipticity (G ≤ 1.11) as compared with the AuNPs synthesized using the citrate reduction method (G ≤ 1.18). Zetasizer analysis showed that the AuNPs that were synthesized using the seeding-growth method were almost monodispersed with a lower polydispersity index (PDI; PDI≤0.079), as compared with the AuNPs synthesized using the citrate reduction method (PDI≤0.177). UV-visible spectroscopic analysis showed a red-shift of the absorbance spectra after the reaction with MαHIgG(4), which indicated that the AuNPs were successfully conjugated. The optimum concentration of the BmR1 recombinant antigen that was immobilized on the surface of the ICG strip on the test line was 1.0 mg ml(-1). When used with the ICG test strip assay and brugian filariasis serum samples, the conjugated AuNPs-MαHIgG(4) synthesized using the seeding-growth method had faster detection times, as compared with the AuNPs synthesized using the citrate reduction method. The 30 nm AuNPs-MαHIgG(4), with an optical density of 4 from the seeding-growth method, demonstrated the best performance for labelling ICG strips because it displayed the best sensitivity and the highest specificity when tested with serum samples from brugian filariasis patients and controls.

Midgut barrier imparts selective resistance to filarial worm infection in Culex pipiens pipiens.

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro.

Elimination of lymphatic filariasis in Southeast Asia.

Approximately 15 million people with lymphatic filariasis (LF) live in Southeast Asia. Wuchereria bancrofti (transmitted by the Mansonia and Anopheles vectors), Brugia malayi and Brugia timori (both transmitted by Culex quinquefasciatus) are the filarial species in this region. The endemic countries are: Cambodia, Lao People's Democratic Republic, the Philippines, Indonesia, Thailand and Timor-Leste, which have all agreed to eliminate transmission of the disease by 2020. The public health interventions with respect to LF are based on the 1997 World Health Assembly resolution (WHA 50.29) which recommends elimination of the disease through mass drug administration (MDA) using diethylcarbamazine (DEC) and albendazole. The drugs are generally donated and as governments contribute 60-90% of the operational costs, MDA is deemed to be comparatively inexpensive for local administrations in relation to other public health programmes. So far, elimination has been accomplished only in the People's Republic of China (P.R. China) and this achievement is therefore described here in some detail. Resurgences have occurred but they have been successfully dealt with. Historically, the endemic areas in P.R. China covered 16,514 townships (or urban sub-districts), situated in 864 counties (or cities) in 14 provinces (or autonomous regions or municipalities). The total population at risk of infection in all endemic areas of P.R. China was originally 342 million.

Using an intravenous catheter to carry out abdominal lavage in the gerbil.

Abdominal lavage is used in laboratory rodents for a variety of applications but carries an inherent risk of abdominal organ laceration; therefore, personnel carrying out this procedure must have considerable expertise. In this paper, the authors describe an improved method for delivering sterile media to and collecting peritoneal fluids from dark-clawed Mongolian gerbils (Meriones unguiculatus) that had been peritoneally infected with filarial nematode parasites (genus Brugia). To carry out this gravity-assisted technique, the authors used a catheter to introduce sterile media into the peritoneal cavity of each gerbil and then to passively drain peritoneal fluid and larval worms for collection. Average fluid recovery was consistently greater when using this gravity-assisted method than when using aspiration. Larval parasites were recovered by both methods. To recover large volumes of fluid using the standard method of abdominal lavage, personnel typically must euthanize rodents. This gravity-assisted technique allows researchers to collect large numbers of parasite larvae without euthanizing gerbils.

Zoonotic deep cutaneous filariasis--three pediatric cases from Québec, Canada.

Three rare cases of pediatric Québec-based zoonotic filarial nematode deep skin infections were reviewed. These rare cases were processed at our pediatric hospital within the last 6-year period. Patient age, travel information, lesional characteristics, systemic findings, serology, histopathology, treatment, and follow-up were gathered from the submitting specimen and the treating physicians. Species identification was performed by the Parasitic Disease Branch, Division of Infectious and Tropical Diseases Pathology, AFIP, Washington, DC.

Estimation of the prevalence of lymphatic filariasis by a pool screen PCR assay using blood spots collected on filter paper.

The prevalence of lymphatic filariasis was estimated by PCR-based pool screening of night blood collected from 865 individuals living in ten areas endemic for Wuchereria bancrofti, Brugia malayi or B. timori in Indonesia. A total of 232 microfilaraemics were identified by filtration of 1 ml of blood. The microfilaria (mf) prevalence ranged from 6% to 54%, and the mf density in microfilaraemics ranged from 1 mf/ml to 6028 mf/ml. PCR assays both for W. bancrofti or Brugia spp. detected a single mf present on a 30 microl dried filter paper blood spot. One hundred and seventy-eight pools of five blood spots in each pool (pool-5) were tested by PCR and 101 (57%) pools were positive. When pool size was increased to 10 spots per pool (pool-10), 65 (70%) of 93 pools were positive. Pearson's correlation and linear regression showed a strong correlation between filtration and pool screen PCR results for pool-10 (r=0.835) and pool-5 (r=0.695). Based on the determination coefficient (R), the results of pool-10 PCR (R=0.697) gave a better prediction compared with pool-5 PCR (R=0.483). This study suggests that pool screen PCR may be a useful tool for monitoring the Global Program to Eliminate Lymphatic Filariasis.

Detection of filaria-specific IgG4 antibodies and filarial DNA, for the screening of blood spots for Brugia timori.

The establishment of simple, sensitive and specific tools for the diagnosis of brugian lymphatic filariasis is a prerequisite for a successful intervention to control the disease. In the simple and rapid Brugia Rapid (BR) test, an immunochromatographic dipstick is used to detect IgG(4) antibodies that are reactive with a recombinant Brugia malayi antigen. When sera from 109 individuals with Brugia microfilaraemias (12 with B. malayi and 97 with B. timori) were investigated using the BR test, all were found positive. In contrast, all of the 150 sera from individuals with Onchocerca volvulus or Mansonella infections investigated were found negative in BR tests. Some unwelcome cross-reactions were observed, however, with sera from individuals infected with Wuchereria bancrofti (three of 12 test-positive) and Dirofilaria (one of nine test-positive). In an attempt to facilitate sample collection and detect any cross-reactions, the BR dipstick was used to screen blood spots, that had been allowed to dry on filter paper, for B. timori microfilariae, before the dipstick-positive samples were tested with a PCR-based assay. Of the 66 individuals so tested, 37 (56%) were found positive by the BR test used on dry blood spots and eight (22%) by the filtration of fresh blood samples. Only nine of the 37 dipstick-positive samples were found PCR-positive. The combined use of BR tests and PCR-based assays, for testing blood spots in areas where brugian filariasis is endemic, appears to be a promising method not only for post-treatment monitoring but also for the certification activities planned within the framework of the Global Programme to Eliminate Lymphatic Filariasis.

Detection of IgG antibodies of Brugian filariasis with crude male and female antigens of Dirofilaria immitis.

Crude antigens from male and female Dirofilaria immitis were used to detect antibody to Brugian filariasis in humans by indirect ELISA. Both antigens were tested with 42 cases of Brugian filariasis, 131 cases of 20 heterologous infections and 35 healthy controls. The results--using male and female antigens--showed sensitivity of 88.1% and 88.1%, and specificities of 64.1% and 51.8%, respectively. Cross-reaction from other helminthic infections using crude male antigen gave false-positives with 48 sera from 13 heterologous diseases at the threshold value of 0.180, while the female antigen gave 63 sera from 15 diseases, at 0.309. Serum antibodies from patients with other helminthic infections--gnathostomiasis, strongyloidiasis, hookworm infections, trichinellosis, capillariasis, angiostrongyliasis, ascariasis, trichuriasis, toxocariasis, neurocysticercosis, cystic echinococcosis, taeniasis and opisthorchiasis--resulted in false-positives with both male and female antigens. One each of sparganosis and paragonimiasis heterotremus sera cross-reacted with only crude female antigen and their OD values were close to the threshold value. Although crude male antigen showed better specificity than crude female antigen, both female and male worms are sources of antigens needed for further purification. This study provides baseline data for further serodiagnosis of Brugian filariasis using dirofilaria antigen.

Detection of filaria-specific IgG4 antibodies using Brugia Rapid test in individuals from an area highly endemic for Brugia timori.

The filarial parasite Brugia timori is of great public health importance in some islands of Eastern Indonesia. To establish a simple serological test for the identification and post-treatment monitoring of areas endemic for B. timori, a rapid immunochromatographic dipstick test (Brugia Rapid, BR) was evaluated on microfilaraemic and amicrofilaraemic individuals. This test is based on the detection of anti-filarial IgG4 antibodies that react with a recombinant Brugia malayi antigen (BmR1). In our study area on Alor island the prevalence of microfilaraemia was 26%. With the BR test, 100% of 196 sera from microfilaraemic persons and 76% of 563 sera from amicrofilaraemic persons, either symptomatic or asymptomatic, reacted positive. All 50 control sera from areas non-endemic for lymphatic filariasis gave negative BR test results. This study showed that the BR test can be also used to detect antibodies against B. timori. Due to the high prevalence of IgG4 antibodies as detected by the BR test (81%), no significant correlation with the prevalence of microfilaraemia could be detected within the endemic village. The BR test also shows great promise to be employed as a monitoring tool for B. timori in the framework of the Global Program to Eliminate Lymphatic Filariasis (GPELF).

Comparison of two IgG4 assay formats (ELISA and rapid dipstick test) for detection of brugian filariasis.

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.