Interruption of lymphatic filariasis transmission in Manaus, a former focus of Wuchereria bancrofti in the Western Brazilian Amazon / Interrupción de la transmisión de la filariasis linfática en Manaos, anteriormente un foco de Wuchereria bancrofti en la Amazonia occidental de Brasil / Interrupção da transmissão da filariose linfática em Manaus, anteriormente um foco de Wuchereria bancrofti na parte leste da Amazônia brasileira

ABSTRACT Objective. To confirm the absence of Wuchereria bancrofti autochthonous cases in Manaus, a former focus of lymphatic filariasis in the Western Brazilian Amazon. Methods. A field survey was carried out in 2016 using immunochromatographic rapid tests (ICT card) for the detection of circulating filarial antigens in blood. The sample included a group of 3 000 schoolchildren aged 6 to 10 years enrolled in schools from different urban areas of Manaus (including the former lymphatic filariasis focus in the city) and a group of 709 adolescents and adults, between the ages of 11 and 85 years, born and raised in different areas of Manaus. Results. All of the individuals tested negative for W. bancrofti antigen. Conclusions. Although Manaus was once considered endemic, this focus no longer seems to be active for lymphatic filariasis transmission. The results of this study could support the certification by the World Health Organization of the lymphatic filariasis transmission elimination exercise in Brazil.

RESUMEN Objetivo. Confirmar la ausencia de casos autóctonos de Wuchereria bancrofti en Manaos, anteriormente un foco de filariasis linfática en la Amazonia occidental de Brasil. Métodos. En el 2016 se llevó a cabo una encuesta en el terreno con pruebas rápidas inmunocromatográficas (tiras inmunocromatográficas) para detectar antígenos filáricos circulantes en sangre. La muestra constó de un grupo de 3 000 escolares de 6 a 10 años matriculados en escuelas de diferentes zonas urbanas de Manaos (incluida la zona que anteriormente era el foco de filariasis linfática en la ciudad) y de un grupo de 709 adolescentes y adultos, de edades comprendidas entre 11 y 85 años, nacidos y criados en diferentes áreas de Manaos. Resultados. Todas las personas dieron negativo en la prueba de antígeno de Wuchereria bancrofti. Conclusiones. Aunque hubo un tiempo en que Manaos se consideraba zona endémica, parece que este foco de transmisión de la filariasis linfática ya no está activo. Los resultados de este estudio podrían brindar apoyo a la certificación de la Organización Mundial de la Salud respecto de los esfuerzos realizados en Brasil para eliminar la transmisión de la filariasis linfática.

RESUMO Objetivo. Confirmar a ausência de casos autóctones de Wuchereria bancrofti em Manaus, anteriormente um foco da filariose linfática na parte leste da Amazônia brasileira. Métodos. Uma pesquisa de campo foi realizada em 2016 com o uso de teste rápido por imunocromatografia (cartão ICT) para detecção de antígenos de microfilárias circulantes no sangue. A amostra estudada consistiu de um grupo de 3 000 crianças escolares entre 6 e 10 anos de idade matriculados em escolas de diferentes áreas da zona urbana de Manaus (englobando a área anteriormente com o foco de filariose linfática) e um grupo de 709 adolescentes e adultos entre 11 e 85 anos de idade nascidos e crescidos em diferentes áreas de Manaus. Resultados. Todos os indivíduos pesquisados tiveram teste negativo para o antígeno da W. bancrofti. Conclusões. Apesar de Manaus ter sido anteriormente uma área endêmica, parece que não existe mais foco ativo de transmissão da filariose linfática na cidade. Os resultados deste estudo podem servir para embasar a certificação pela Organização Mundial da Saúde da eliminação da transmissão da filariose linfática no Brasil.

Standardisation of lymphatic filariasis microfilaraemia prevalence estimates based on different diagnostic methods: a systematic review and meta-analysis.

BACKGROUND: Lymphatic filariasis (LF) infection is generally diagnosed through parasitological identification of microfilariae (mf) in the blood. Although historically the most commonly used technique for counting mf is the thick blood smear based on 20 µl blood (TBS20), various other techniques and blood volumes have been applied. It is therefore a challenge to compare mf prevalence estimates from different LF-survey data. Our objective was to standardise microfilaraemia (mf) prevalence estimates to TBS20 as the reference diagnostic technique. METHODS: We first performed a systematic review to identify studies reporting on comparative mf prevalence data as measured by more than one diagnostic test, including TBS20, on the same study population. Associations between mf prevalences based on different diagnostic techniques were quantified in terms of odds ratios (OR, with TBS20 blood as reference), using a meta-regression model. RESULTS: We identified 606 articles matching our search strategy and included 14 in our analyses. The OR of the mf prevalences as measured by the more sensitive counting chamber technique (≥ 50 µl blood) was 2.90 (95% confidence interval (CI): 1.60-5.28). For membrane filtration (1 ml blood) the OR was 2.39 (95% CI: 1.62-3.53), Knott's technique it was 1.54 (95% CI: 0.72-3.29), and for TBS in ≥ 40 µl blood it was 1.37 (95% CI: 0.81-2.30). CONCLUSIONS: We provided transformation factors to standardise mf prevalence estimates as detected by different diagnostic techniques to mf prevalence estimates as measured by TBS20. This will facilitate the use and comparison of more datasets in meta-analyses and geographic mapping initiatives across countries and over time.

Development of a molecular xenomonitoring protocol to assess filariasis transmission.

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.

A Study on Serological Reactivity Profile of Different Antigen Preparations with Bancroftian filariasis Human Infection Sera.

BACKGROUND: Lymphatic Filariasis (LF) is one of the incapacitating and mosquito-borne sicknesses that on progression may prompt a few recognizable types of clutters like extreme lymphedema, hydrocele, and elephantiasis. METHODS: Antigenic preparations of B. malayi adult (BmA), S. cervi adult parasites and microfilariae (mf) total parasite extract were used to analyze the serological reactivity profile with human infectious sera collected from endemic areas of Bancroftian filariasis by performing Western blot and ELISA analysis. Sera from healthy human subjects were also included in the study to determine the variation incurred in the reactivity due to the filariasis infection. Gelelectrophoresis analysis of the crude-extract of BmA revealed seven protein bands while more than ten bands were recognized in S. cervi. RESULTS: our results represent a clear variation in protein patterns among the crude-antigens. ELISA results showed highest prevalence of IgG, IgM and IgG4 antibodies against all antigen preparations when recorded among microfilaraemic chronic infected patients. In both the antigenic preparations, the positive reactions were in the order of microfilaraemic>endemic normal>chronic>acute>nonendemic normal subjects. All sera of Mf+ patients were uniformly positive, while sera of both chronic and endemic normal subjects showed less reactivity. CONCLUSION: In the present study, we endeavoured to establish the extent of cross-reactivity of antigens derived from animal filarial parasites such as B. malayi and S. cervi with W. bancrofti filariasis sera of human patients. Besides, we further analyzed antibody-isotype profile of IgG, IgG4 and IgM in various human infection sera of bancroftian filarial subjects reactive to heterologous parasite antigens derived from adult worms of S. cervi from bovine and B. malayi from bovine and jirds.

Elimination or Resurgence: Modelling Lymphatic Filariasis After Reaching the 1% Microfilaremia Prevalence Threshold.

The low prevalence levels associated with lymphatic filariasis elimination pose a challenge for effective disease surveillance. As more countries achieve the World Health Organization criteria for halting mass treatment and move on to surveillance, there is increasing reliance on the utility of transmission assessment surveys (TAS) to measure success. However, the long-term disease outcomes after passing TAS are largely untested. Using 3 well-established mathematical models, we show that low-level prevalence can be maintained for a long period after halting mass treatment and that true elimination (0% prevalence) is usually slow to achieve. The risk of resurgence after achieving current targets is low and is hard to predict using just current prevalence. Although resurgence is often quick (<5 years), it can still occur outside of the currently recommended postintervention surveillance period of 4-6 years. Our results highlight the need for ongoing and enhanced postintervention monitoring, beyond the scope of TAS, to ensure sustained success.

Development of an Antigen Detection ELISA for Bancroftian Filariasis Using BmSXP-Specific Recombinant Monoclonal Antibody.

Lymphatic filariasis is a mosquito-borne parasitic disease responsible for morbidity and disability that affects 1.2 billion people worldwide, mainly the poor communities. Currently, filarial antigen testing is the method of choice for the detection of bancroftian filariasis, and to date, there are two commonly used tests. In the present study, a recently reported recombinant monoclonal antibody (5B) specific to BmSXP filarial antigen was used in developing an ELISA for the detection of circulating filarial antigen in sera of patients with bancroftian filariasis. The performance of the ELISA was evaluated using 124 serum samples. The ELISA was positive with all sera from microfilaremic bancroftian filariasis patients (n = 34). It also showed 100% diagnostic specificity when tested with sera from 50 healthy individuals and 40 patients with other parasitic diseases. The developed assay using the novel 5B recombinant monoclonal antibody could potentially be a promising alternative antigen detection test for bancroftian filariasis.

Prevalence of malaria and lymphatic filariasis in bateyes of the Dominican Republic.

BACKGROUND: The island of Hispaniola, shared by Haiti and the Dominican Republic (DR), is the only remaining malaria-endemic island in the Caribbean and accounts for 95% of the lymphatic filariasis (LF) burden in the Americas. Both countries aim to eliminate the diseases by 2020. Migration from Haiti, where both diseases are more prevalent, may promote transmission in the DR. Historically, Haitian migrant labourers live in rural Dominican agricultural 'company towns' called bateyes, many of which received mass drug administration (MDA) for LF elimination. This study sought to determine the prevalence of malaria and LF in bateyes of the DR and to describe related risk factors for disease. METHODS: From March to April 2016, a cross-sectional, cluster survey was conducted across Dominican bateyes stratified into three regions: southwest, north and east. A household questionnaire (n = 776), captured demographics, ethnic origin, mobility patterns, malaria intervention coverage, and knowledge, and recent fever and treatment-seeking. Two individuals per household (n = 1418) were tested for malaria parasites by microscopy and rapid diagnostic test (RDT) and LF antigen by filariasis test strip (FTS). Population-level estimates and confidence intervals (CI) were computed adjusting for the survey design. Two-sided t-tests compared differences in knowledge scores. RESULTS: No (0%) blood sample was Plasmodium-positive by microscopy or RDT. Six individuals were FTS-positive (0.5%; 95% CI: 0.2-1.5), but none (0%) of these were microfilariae-positive. Most batey residents were born in the DR (57.8%), documented (85.0%), and permanent residents (85.1%). Very few respondents (9.4%) reported travel to Haiti in the past year. Overall, half (53.8%) of respondents owned a bed net, and 82.3% of net owners reported using it the previous night. Indoor residual spraying (IRS) differed by region (range: 4.7%-61.2%). Most of those with recent fever sought care (56.0%), yet only 30.5% of those seeking care were tested for malaria. Compared to Dominican-born populations, Haitian-born respondents more frequently reported recent fever, did not seek care for the fever, had not heard of malaria, and could not name symptoms or prevention methods. CONCLUSIONS: Malaria and LF transmission appear absent or extremely low in Dominican bateyes, which are a mixture of Haitian and Dominican residents. Travel to Haiti is rare, meaning risk of malaria and LF importation is low. Addressing identified gaps in intervention coverage, malaria knowledge, treatment seeking and service delivery will improve the quality of surveillance for these diseases, particularly among marginalized populations and promote island-wide elimination.

Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis.

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (Brugia malayi and Litomosoides sigmodontis) and onchocerciasis (Onchocerca ochengi). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.

Integrated seroprevalence-based assessment of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test.

BACKGROUND: Mali has become increasingly interested in the evaluation of transmission of both Wuchereria bancrofti and Onchocerca volvulus as prevalences of both infections move toward their respective elimination targets. The SD Bioline Onchocerciasis/LF IgG4 Rapid Test was used in 2 evaluation units (EU) to assess its performance as an integrated surveillance tool for elimination of lymphatic filariasis (LF) and onchocerciasis. METHODOLOGY/PRINCIPAL FINDINGS: A cross sectional survey with SD Bioline Onchocerciasis/LF IgG4 Rapid Test was piggy-backed onto a transmission assessment survey (TAS) (using the immunochromatographic card test (ICT) Binax Filariasis Now test for filarial adult circulating antigen (CFA) detection) for LF in Mali among 6-7 year old children in 2016 as part of the TAS in two EUs namely Kadiolo-Kolondieba in the region of Sikasso and Bafoulabe -Kita-Oussoubidiagna-Yelimane in the region of Kayes. In the EU of Kadiolo- Kolondieba, of the 1,625 children tested, the overall prevalence of W. bancrofti CFA was 0.62% (10/1,625) [CI = 0.31-1.09]; while that of IgG4 to Wb123 was 0.19% (3/1,600) [CI = 0.04-0.50]. The number of positives tested with the two tests were statistically comparable (p = 0.09). In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of W. bancrofti CFA was 0% (0/1,700) and that of Wb123 IgG4 antibody was 0.06% (1/1,700), with no statistically significant difference between the two rates (p = 0.99). In the EU of Kadiolo- Kolondieba, the prevalence of Ov16-specific IgG4 was 0.19% (3/1,600) [CI = 0.04-0.50]. All 3 positives were in the previously O. volvulus-hyperendemic district of Kolondieba. In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of Ov16-specific IgG4 was 0.18% (3/1,700) [CI = 0.04-0.47]. These 3 Ov16 IgG4 positives were from previously O.volvulus-mesoendemic district of Kita. CONCLUSIONS/SIGNIFICANCE: The SD Bioline Onchocerciasis/LF IgG4 Rapid test appears to be a good tool for integrated exposure measures of LF and onchocerciasis in co-endemic areas.

Immunodiagnostic potential of Wuchereria bancrofti L1 antigen-based filarial immunoglobulin G4 detection assay.

Background: After mass drug administration to eliminate human lymphatic filariasis, there is a need for surveillance to detect the measurable endpoint of the program. Methods: An immunodominant seroreactive clone, WbL1, was identified through immunoscreening of a Wuchereria bancrofti L3 complementary DNA expression library. Recombinant WbL1 (rWbL1) was analysed with sera from W. bancrofti patients. Diagnostic evaluation was carried out by developing an enzyme-linked immunosorbent assay (ELISA) to detect the filarial-specific antibodies in various categories of filarial sera samples against recombinant WbL1 (rWbL1) protein. Results: Performance parameters of the test in terms of immunoglobulin G (IgG) and IgG4 detection displayed significant sensitivity and specificity values up to 77% and 100%, respectively. Our results showed filarial antibodies against rWbL1 to be highly reactive with microfilaremic and clinical filarial sera samples compared with the endemic and non-endemic control sera samples. Reasonably satisfactory performance of the test was also confirmed from the multicentric evaluation of an anti-WbL1 IgG4 detection ELISA. This test was found to be minimally reactive with other nematode parasites and protozoan infections. Conclusions: The anti-WbL1 IgG4 detection test can be considered as a field test for initial screening and epidemiological monitoring of filarial infections in filariasis-endemic areas.

Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis.

The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis.

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.

Seroprevalence of lymphatic filariasis among migrant workers in Peninsular Malaysia.

Background: Malaysia aims to eliminate lymphatic filariasis (LF) by the year 2020, thus the potential threat of LF from migrant workers needs to be investigated. Methods: Brugian and bancroftian filariasis among 484 migrant workers from six countries were investigated using rapid tests based on detection of specific IgG4 antibodies against BmR1 (Brugia Rapid) and BmSXP recombinant antigens. Results: The seroprevalence of brugian filariasis was very low; however, bancroftian filariasis was notable among workers from India, Nepal and Myanmar. Conclusion: Malaysia is not endemic for Wuchereria bancrofti, but harbors the vectors for the parasite, thus the results showed that migrant workers should be monitored for this infection.

Twelve-month longitudinal parasitological assessment of lymphatic filariasis-positive individuals: impact of a biannual treatment with ivermectin and albendazole.

OBJECTIVE: Mass drug administration (MDA) for the control of lymphatic filariasis (LF), in Ghana, started in the year 2000. While this had great success in many implementation units, there remain areas with persistent transmission, after more than 10 years of treatment. A closer examination of the parasite populations could help understand the reasons for persistent infections and formulate appropriate strategies to control LF in these areas of persistent transmission. MATERIALS AND METHODS: In a longitudinal study, we assessed the prevalence of microfilaraemia (mf) in two communities with 12 years of MDA in Ghana. In baseline surveys 6 months after the National MDA in 2014, 370 consenting individuals were tested for antigenaemia using immunochromatographic test (ICT) cards and had their mf count determined through night blood surveys. 48 ICT positives, of whom, 17 were positive for mf, were treated with 400 µg/kg ivermectin + 400 mg albendazole and subsequently followed for parasitological assessment at 3-month intervals for 1 year. This overlapped with the National MDA in 2015. RESULTS: There was a 68% parasite clearance 3 months after treatment. The pre-treatment mf count differed significantly from the post-treatment mf counts at 3 months (P = 0.0023), 6 months (P = 0.0051), 9 months (P = 0.0113) and 12 months (P = 0.0008). CONCLUSION: In these settings with persistent LF transmission, twice-yearly treatment may help accelerate LF elimination. Further large-scale evaluations are required to ascertain these findings.

Comparison and functional characterisation of peripheral blood mononuclear cells isolated from filarial lymphoedema and endemic normals of a South Indian population.

OBJECTIVE: The underlying problem in lymphatic filariasis is irreversible swelling of the limbs (lymphoedema), which is a unique feature of lymphatic insufficiency. It is still unclear whether the natural ability of lymphatics to form functional lymphatic vasculature is achieved or attenuated in the lymphoedemal pathology. Clinical studies have clearly shown that circulating lymphatic progenitors (CLPs), a subset of bone marrow-derived mononuclear cells (PBMCs), contribute to post-natal lymph vasculogenesis. CLP-based revascularisation could be a promising strategy to bypass the endothelial disruption and damage incurred by the filarial parasites. Thus our aim was to compare and characterise the functional prowess of PBMCs in physiological and lymphoedemal pathology. METHODS: PBMCs were isolated from venous blood sample from drug-naive endemic normals (EN) and drug-deprived filarial lymphoedema (FL) individuals using density gradient centrifugation. Adhesion, transwell migration and in vitro matrigel assays were employed to characterise the lymphvasculogenic potential of PBMCs. CLPs were phenotypically characterised using flow cytometry; expression levels of lymphatic markers and inflammatory cytokines were quantified using qRT-PCR and ELISA, respectively. RESULTS: PBMCs from FL group display poor adherence to fibronectin (P = 0.040), reduced migration towards SDF-1α (P = 0.035), impaired tubular network (P = 0.004) and branching point (P = 0.048) formation. The PBMC mRNA expression of VEGFR3 (P = 0.039) and podoplanin (P = 0.050) was elevated, whereas integrin α9 (P = 0.046) was inhibited in FL individuals; additionally, the surface expression of CD34 (P = 0.048) was significantly reduced in the FL group compared to the EN group. CONCLUSION: PBMCs from filarial lymphoedema show defective and dysregulated lymphvasculogenic function compared to endemic normals.

Molecular identification and phylogenetic analysis of Wuchereria bancrofti from human blood samples in Egypt.

Lymphatic filariasis (LF) is a serious vector-borne health problem, and Wuchereria bancrofti (W.b) is the major cause of LF worldwide and is focally endemic in Egypt. Identification of filarial infection using traditional morphologic and immunological criteria can be difficult and lead to misdiagnosis. The aim of the present study was molecular detection of W.b in residents in endemic areas in Egypt, sequence variance analysis, and phylogenetic analysis of W.b DNA. Collected blood samples from residents in filariasis endemic areas in five governorates were subjected to semi-nested PCR targeting repeated DNA sequence, for detection of W.b DNA. PCR products were sequenced; subsequently, a phylogenetic analysis of the obtained sequences was performed. Out of 300 blood samples, W.b DNA was identified in 48 (16%). Sequencing analysis confirmed PCR results identifying only W.b species. Sequence alignment and phylogenetic analysis indicated genetically distinct clusters of W.b among the study population. Study results demonstrated that the semi-nested PCR proved to be an effective diagnostic tool for accurate and rapid detection of W.b infections in nano-epidemics and is applicable for samples collected in the daytime as well as the night time. PCR products sequencing and phylogenitic analysis revealed three different nucleotide sequences variants. Further genetic studies of W.b in Egypt and other endemic areas are needed to distinguish related strains and the various ecological as well as drug effects exerted on them to support W.b elimination.

Evaluation of Lymphatic Filariasis and Onchocerciasis in Three Senegalese Districts Treated for Onchocerciasis with Ivermectin.

In Africa, onchocerciasis and lymphatic filariasis (LF) are co-endemic in many areas. Current efforts to eliminate both diseases are through ivermectin-based mass drug administration (MDA). Years of ivermectin distribution for onchocerciasis may have interrupted LF transmission in certain areas. The Kédougou region, Senegal, is co-endemic for LF and onchocerciasis. Though MDA for onchocerciasis started in 1988, in 2014 albendazole had not yet been added for LF. The objective of this study was to assess in an integrated manner the LF and onchocerciasis status in the three districts of the Kédougou region after ≥10 years of ivermectin-based MDA. The study employed an African Programme for Onchocerciasis Control (APOC) onchocerciasis-related methodology. In the three districts, 14 villages close to three rivers that have Simulium damnosum breeding sites were surveyed. Convenience sampling of residents ≥5 years old was performed. Assessment for LF antigenemia by immunochromatographic testing (ICT) was added to skin snip microscopy for onchocerciasis. Participants were also tested for antibodies against Wb123 (LF) and Ov16 (onchocerciasis) antigens. In two districts, no participants were ICT or skin snip positive. In the third district, 3.5% were ICT positive and 0.7% were skin snip positive. In all the three districts, Wb123 prevalence was 0.6%. Overall, Ov16 prevalence was 6.9%. Ov16 prevalence among children 5-9 years old in the study was 2.5%. LF antigenemia prevalence was still above treatment threshold in one district despite ≥10 years of ivermectin-based MDA. The presence of Ov16 positive children suggested recent transmission of Onchocerca volvulus. This study showed the feasibility of integrated evaluation of onchocerciasis and LF but development of integrated robust methods for assessing transmission of both LF and onchocerciasis are needed to determine where MDA can be stopped safely in co-endemic areas.

Burden of lymphatic filariasis morbidity in an area of low endemicity in Brazil.

The Global Programme to Eliminate Lymphatic Filariasis has two main components: interrupting transmission of lymphatic filariasis (LF) and managing morbidity and preventing disability. However, interventions to prevent and manage LF-related disabilities in endemic communities have been of limited extent. The aim of this study was to describe the prevalence of morbidity and its correlation with filarial infection, thereby filling a gap that existed regarding the data on morbidity in Brazil. Presence of Wuchereria bancrofti microfilaria was investigated using the thick smear technique. Information on parasitosis-related clinical manifestations was obtained using a questionnaire applied by community health agents with previous training and capacitation to know about and identify the disease. To analyze correlations, Pearson's correlation coefficient was used with the corresponding statistical significance test. 23,673 individuals were investigated: 323 presented microfilaremia (1.36%) and 741 (3.13%) had clinical complaints that were attributable to LF. Acute dermatolymphangioadenitis (ADLA) was the most prevalent condition (2.2%). Lymphedema, ADLA and chyluria were more commonly reported among female patients. There were positive associations between all the clinical complaints reported and filarial infection. Hydrocele presented the most strongly positive association (r=0.699; p<0.001). The present study showed that there is an association between clinical condition reported and the rate of infection among people living in an area of low endemicity for LF. It contributes data that might provide support for healthcare systems and thus optimize disease management, through incorporating surveillance measures directed towards preventing disability and reducing the psychosocial and economic impact of the disease on poor populations living in areas endemic for LF.

Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.