Recombinant antigens used as diagnostic tools for lymphatic filariasis.

Lymphatic filariasis (LF) is a parasitic disease caused by the worms Wuchereria bancrofti, Brugia malayi, or Brugia timori. It is a tropical and subtropical illness that affects approximately 67 million people worldwide and that still requires better diagnostic tools to prevent its spread and enhance the effectiveness of control procedures. Traditional parasitological tests and diagnostic methods based on whole protein extracts from different worms are known for problems related to sample time collection, sensitivity, and specificity. More recently, new diagnostic tools based on immunological methods using recombinant antigens have been developed. The current review describes the several recombinant antigens used as tools for lymphatic filariasis diagnosis in antigen and antibody capture assays, highlighting their advantages and limitations as well as the main commercial tests developed based on them. The literature chronology is from 1991 to 2021. First, it describes the historical background related to the identification of relevant antigens and the generation of the recombinant polypeptides used for the LF diagnosis, also detailing features specific to each antigen. The subsequent section then discusses the use of those proteins to develop antigen and antibody capture tests to detect LF. So far, studies focusing on antibody capture assays are based on 13 different antigens with at least six commercially available tests, with five proteins further used for the development of antigen capture tests. Five antigens explored in this paper belong to the SXP/RAL-2 family (BmSXP, Bm14, WbSXP-1, Wb14, WbL), and the others are BmShp-1, Bm33, BmR1, BmVAH, WbVAH, BmALT-1, BmALT-2, and Wb123. It is expected that advances in research with these antigens will allow further development of tests combining both sensitivity and specificity with low costs, assisting the Global Program to Eliminate Lymphatic Filariasis (GPELF).

Association of a PD-L2 Gene Polymorphism with Chronic Lymphatic Filariasis in a South Indian Cohort.

Lymphatic filariasis (LF) is a parasitic infection, caused by three closely related nematodes, namely Wuchereria bancrofti, Brugia malayi, and Brugia timori. Previously, we have shown that lysate from B. malayi microfilariae induces the expression of interleukin (IL)-10 and programmed death-ligand (PD-L) 1 on monocytes, which lead to inhibition of CD4+ T-cell responses. In this study, we investigated associations of IL-10 and programmed cell death (PD)-1 pathway gene polymorphisms with clinical manifestation in LF. We evaluated the frequency of alleles and genotypes of IL-10 (rs3024496, rs1800872), IL-10RA (rs3135932), IL-10RB (rs2834167), PD-1 (rs2227982, rs10204525), PD-L1 (rs4143815), PD-L2 (rs7854413), and single-nucleotide polymorphisms (SNPs) in 103 patients with chronic pathology (CP), such as elephantiasis or hydrocele and 106 endemic normal (EN) individuals from a South Indian population living in an area endemic for LF. Deviations from the Hardy-Weinberg equilibrium were tested, and we found a significant difference between the frequency of polymorphisms in PD-L2 (rs7854413; P < 0.001) and IL-10RB (rs2834167; P = 0.012) between the CP and the EN group, whereas there were no significant differences found among IL-10, IL-10RA, PD-1, and PD-L1 SNPs. A multivariate analysis showed that the existence of a CC genotype in PD-L2 SNP rs7854413 is associated with a higher risk of developing CP (OR: 2.942; 95% confidence interval [CI]: 0.957-9.046; P = 0.06). Altogether, these data indicate that a genetically determined individual difference in a non-synonymous missense SNP of PD-L2 might influence the susceptibility to CP.

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.

Brugia Rapid™ antibody responses in communities of Indonesia in relation to the results of 'transmission assessment surveys' (TAS) for the lymphatic filariasis elimination program.

BACKGROUND: The Global Programme to Eliminate Lymphatic Filariasis recommends the transmission assessment survey (TAS) as the preferred methodology for determining whether mass drug administration can be stopped in an endemic area. Because of the limited experience available globally with the use of Brugia Rapid™ tests in conducting TAS in Brugia spp. areas, we explored the relationship between the antibody test results and Brugia spp. infection as detected by microfilaremia in different epidemiological settings. METHODS: The study analyzes the Brugia Rapid™ antibody responses and microfilaremia in all ages at three study sites in: i) a district which was classified as non-endemic, ii) a district which passed TAS, and iii) a district which failed TAS. Convenience sampling was done in each site, in one to three purposefully selected villages with a goal of 500 samples in each district. RESULTS: A total of 1543 samples were collected from residents in all three study sites. In the site which was classified as non-endemic and where MDA had not been conducted, 5 % of study participants were antibody positive, none was positive for microfilaremia, and age-specific antibody prevalence peaked at almost 8 % in the 25-34 year-old age range, with no antibody-positive results found in children under eight years of age. In the site that had passed TAS, 1 % of participants were antibody positive and none was positive for microfilaremia. In the site which failed TAS, 15 % of participants were antibody positive, 0.2 % were microfilaremic, and age-specific antibody prevalence was highest in 6-7 year olds (30 %), but above 8 % in all age levels above 8 years old. CONCLUSIONS: These results from districts which followed the current WHO guidance for mapping, MDA, and implementing TAS, while providing antibody profiles of treated and untreated populations under programmatic settings, support the choice of antibody prevalence in the 6- and 7-year-old age group in TAS for making stopping MDA decisions. Since only one study participant was microfilaremic, no conclusions could be drawn about the relationship between antibodies and microfilaremia and further longitudinal studies are required to understand this relationship.

Immunology of lymphatic filariasis.

The immune responses to filarial parasites encompass a complex network of innate and adaptive cells whose interaction with the parasite underlies a spectrum of clinical manifestations. The predominant immunological feature of lymphatic filariasis is an antigen-specific Th2 response and an expansion of IL-10 producing CD4(+) T cells that is accompanied by a muted Th1 response. This antigen-specific T-cell hyporesponsiveness appears to be crucial for the maintenance of the sustained, long-standing infection often with high parasite densities. While the correlates of protective immunity to lymphatic filariasis are still incompletely understood, primarily due to the lack of suitable animal models to study susceptibility, it is clear that T cells and to a certain extent B cells are required for protective immunity. Host immune responses, especially CD4(+) T-cell responses clearly play a role in mediating pathological manifestations of LF, including lymphedema, hydrocele and elephantiasis. The main underlying defect in the development of clinical pathology appears to be a failure to induce T-cell hyporesponsiveness in the face of antigenic stimulation. Finally, another intriguing feature of filarial infections is their propensity to induce bystander effects on a variety of immune responses, including responses to vaccinations, allergens and to other infectious agents. The complexity of the immune response to filarial infection therefore provides an important gateway to understanding the regulation of immune responses to chronic infections, in general.

Laboratory detection of strongyloidiasis: IgG-, IgG4 - and IgE-ELISAs and cross-reactivity with lymphatic filariasis.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG4 and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG4 , IgG, IgE and IgG (IVD) assays were 76.9%, 84.6%, 7.7% and 84.6%, respectively, while the specificities were 92.7%, 81.8%, 100% and 83.6%, respectively. If filariasis samples were excluded, the specificities of the IgG4 -ELISA and both IgG-ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG- and IgG4 -ELISAs (r = 0.4828; P = 0.0125). IgG- and IgG- (IVD) ELISAs (r = 0.309) were positively correlated, but was not significant (P = 0.124). Meanwhile there was no correlation between IgG4 - and IgG- (IVD) ELISAs (r = 0.0042; P = 0.8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG4 and the optical densities of anti-Strongyloides IgG4 -ELISA (r = 0.4544, P = 0.0294). In conclusion, the detection of both anti-Strongyloides IgG4 and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.

Inter and intra-specific diversity of parasites that cause lymphatic filariasis.

Lymphatic filariasis is caused by three closely related nematode parasites: Wuchereria bancrofti, Brugia malayi and Brugia timori. These species have many ecological variants that differ in several aspects of their biology such as mosquito vector species, host range, periodicity, and morphology. Although the genome of B. malayi (the first genome sequenced from a parasitic nematode) has been available for more than five years, very little is known about genetic variability among the lymphatic dwelling filariae. The genetic diversity among these worms is not only interesting from a biological perspective, but it may have important practical implications for the Global Program to Eliminate Lymphatic Filariasis, as the parasites may respond differently to diagnostic tests and/or medical interventions. Therefore, better information on their genetic variability is urgently needed. With improved methods for nucleic acid extraction and recent advances in sequencing chemistry and instrumentation, this gap can be filled relatively inexpensively. Improved information on filarial genetic diversity may increase the chances of success for lymphatic filariasis elimination programs.

Duration of detection of anti-BmR1 IgG4 antibodies after mass-drug administration (MDA) in Sarawak, Malaysia.

The detection rates of brugian filariasis in three regions of Sarawak namely Central, North and South after three courses of mass drug administration (MDA) from year 2004 to 2006 was investigated. A recombinant BmR1 antigen-based IgG4 detection test, named Brugia Rapid and night blood smear for microfilaria (mf) detection were used. All three regions recorded a sharp fall in mf positive rates after a year post-MDA. Meanwhile Brugia Rapid positive rates declined more gradually to 3.8% and 5.6% of the pre-MDA levels in the Central and North regions, respectively. This study showed that in filariasis endemic areas in Sarawak, anti-filarial IgG4 antibodies to BmR1, as detected by the Brugia Rapid test, were positive for one to two years after mf disappearance.

A novel gene from Brugia sp. that encodes a cytotoxic fatty acid binding protein allergen recognized by canine monoclonal IgE and serum IgE from infected dogs.

Brugia pahangi infection of dogs is a well characterized model of human lymphatic filariasis in which sera consistently show IgG or IgE reactivity to a 35-kDa antigen. Using dog lymph node B cells, we previously established a heterohybridoma cell line producing canine monoclonal IgE (cmAb 2.39) that activates and degranulates canine mast cells, and specifically recognizes a 35-kDa B. pahangi antigen. By affinity purification and sequencing of the native protein from B. pahangi adults, a 19-amino acid sequence was obtained; the derived nucleotide sequence showed homology to a Brugia malayi and 2 related Onchocerca volvulus expressed sequence tag (EST) clones from the Filarial Genome Project database. Consensus primers amplified a 244-bp product from adult and infective larval stage cDNA libraries of B. malayi, O. volvulus, and Wuchereria bancrofti, but not from those of nonfilarial nematodes. The B. malayi EST clone only showed nucleotide sequence homology to O. volvulus EST sequences. A 684-bp region from the open reading frame was expressed as a glutathione S-transferase fusion protein designated BmAl-1. CmAb 2.39, as well as serum IgE from dogs infected with B. pahangi and canine filarial heartworm, Dirofilaria immitis, recognized BmAl-1 on enzyme-linked immunosorbent assay and Western blots. BmAl-1 showed high binding affinity for a fatty acid; however, a search for sequence homology with known fatty acid binding proteins indicated that BmAl-1 is a unique fatty acid binding protein. This 35-kDa protein seems to be highly conserved in different stages and species of filarids, and it represents a previously unknown allergen that is possibly involved in the pathogenesis of filarial disease.

Use of Brugia Rapid dipstick and ICT test to map distribution of lymphatic filariasis in the Democratic Republic of Timor-Leste.

The newly-introduced Brugia Rapid dipstick for filarial antibodies and ICT filarial antigen card test were used to confirm historical data on the distribution of lymphatic filariasis in the Republic of Timor-Leste. Twelve out of thirteen districts were confirmed as being endemic. Brugian filariasis predominates, with an average prevalence of 11.6%. The average prevalence of Bancroftian filariasis was 1.1%. The study demonstrated that the Brugia Rapid test can provide useful information about the distribution of Brugian filariasis in circumstances where it is difficult or impossible to obtain night blood samples for microfilariae.

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.

Lymphatic filariasis and Brugia timori: prospects for elimination.

Brugia timori is a pathogenic filarial nematode of humans, replacing the closely related species Brugia malayi on some islands in eastern Indonesia. Recent studies on Alor island show that, locally, B. timori is still of great public health importance, causing mainly acute filarial fever and chronic lymphedema. PCR-based assays to detect parasite DNA, in addition to assays for detecting specific antibodies that have been originally developed for B. malayi, can be used efficiently as diagnostic tools for B. timori. In the framework of the Global Program to Eliminate Lymphatic Filariasis, a single annual dose of diethylcarbamazine, in combination with albendazole, was found to reduce the prevalence and density of microfilaraemia persistently. Therefore, elimination of B. timori appears to be achievable.

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).

The role of endosymbiotic Wolbachia bacteria in filarial disease.

In this review, we describe the pathogenic role of Wolbachia endosymbiotic bacteria in filarial diseases, focusing on the host innate immune responses to filarial and Wolbachia products. A description of the host pathogen recognition and early inflammatory responses including TLR4-mediated signalling, chemokine and cytokine responses and inflammatory cell recruitment is provided from human studies and from animal models of filarial disease. Finally, the impact of the discovery and characterization of Wolbachia on filarial research and treatment programmes is discussed.

Immunity to filarial nematodes.

Mosquito-borne filarial nematodes cause the severe, debilitating disease of human lymphatic filariasis. In areas endemic for this disease, differential responses range from putative immunity through asymptomatic microfilaraemic infection to chronic pathology. Current research in mouse models of infection is elucidating the immunological mechanisms that can lead to immunity against this disease. In this review, the importance of different immunological pathways are discussed in relation to their role in human disease and in terms of their ability to kill separate developmental stages of the filarial parasite.

The third-stage larva (L3) of Brugia: its role in immune modulation and protective immunity.

In this review, we focus on the role of the L3 (third-stage larva) of lymphatic filarial nematodes in immunomodulation and in the development of protective immunity. Studies in the mouse models of Brugia have been fundamental to our understanding of the mechanisms by which infection with L3 results in Th2 responses and the active suppression of Th1 responses. The relevance of these phenomena to the human infection is discussed.

Adverse reactions following diethylcarbamazine (DEC) intake in 'endemic normals', microfilaraemics and elephantiasis patients.

This paper reports on adverse reactions following a 12-day course of 6 mg/kg diethylcarbamazine (DEC) therapy in brugian filariasis patients in Indonesia. Microfilaria-positive individuals (n = 26), 'endemic normals' (n = 12) and elephantiasis patients (n = 17) were included in the study. Fever, headache and body aches started between 2 and 24 h after DEC intake. Adverse reactions were categorized into 'no or mild', 'moderate' or 'severe' depending on the total reaction score. Four microfilaraemic individuals (15.4%) suffered from severe adverse reactions and their pre-treatment microfilarial levels (geometric mean, GM = 3060 mf/10 mL) were significantly higher than in the 5 microfilaraemic individuals (19.2%) suffering from moderate reactions (GM = 1268 mf/10 mL) and in the 17 microfilaraemic patients (65.4%) who experienced no or mild reactions (GM = 6 mf/10 mL)(P < 0.001 and P < 0.001, respectively). Endemic normals showed no or mild adverse reactions. No or mild adverse reactions were also recorded in all but 2 elephantiasis patients after DEC intake. Two elephantiasis patients with moderate reactions had high levels of circulating microfilariae at pre-treatment (2097 and 7375 mf/10 mL). Concentrations of DEC were measured in plasma, but could not explain the differences in the severity of adverse reactions.

The L3 of Brugia induces a Th2-polarized response following activation of an IL-4-producing CD4-CD8- alphabeta T cell population.

Lymphatic filariasis in man is characterized by a profound bias in the immune response. Parasite-specific Th1 responses are dramatically down-regulated while Th2 responses dominate. We have used the infective larval stage of the nematode parasite Brugia pahangi, a potent Th2 inducer in naive mice, to examine cytokine production during the initiation phase of the response. For comparative purposes, the early cytokine transcription pattern elicited by microfilariae (mf), another life cycle stage of the parasite known to induce a primary Th1 response, was analysed in parallel. At 24 h post-infection (p.i.) a burst of IL-4 transcription was detected in the draining popliteal lymph node of L3-infected animals. IL-4 was the only cytokine transcript detectable at this early time point and was not present in mf-infected mice. From day 4 p.i. onwards, the L3 elicited a Th2 response as defined at the level of cytokine mRNA and protein production by CD4+ cells. In contrast, mf stimulate high levels of IFN-gamma mRNA at day 4 p.i. in the absence of IL-4 or IL-10 induction. Cell selection analysis indicated that IL-4 produced at 24 h derived from a population of CD4-CD8- alphabeta T cells. These results suggest that triggering of an unusual double-negative T cell population to secrete IL-4 at the very outset of infection with the L3 of B. pahangi may be the critical factor favouring the development of antigen-specific Th2 cells in response to this stage of the parasite.