Doxycycline for the treatment of nodding syndrome: a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Nodding syndrome is a poorly understood neurological disorder that predominantly occurs in Africa. We hypothesised that nodding syndrome is a neuroinflammatory disorder, induced by antibodies to Onchocerca volvulus or its Wolbachia symbiont, cross-reacting with host neuronal proteins (HNPs), and that doxycycline can be used as treatment. METHODS: In this randomised, double-blind, placebo-controlled, phase 2 trial, we recruited participants from districts affected by nodding syndrome in northern Uganda. We included children and adolescents aged 8-18 years with nodding syndrome, as defined by WHO consensus criteria. Participants were randomly assigned (1:1) to receive either 100 mg doxycycline daily or placebo for 6 weeks via a computer-generated schedule stratified by skin microscopy results, and all parties were masked to group assignment. Diagnoses of O volvulus and antibodies to HNPs were made using luciferase immunoprecipitation system assays and immunohistochemistry. The primary outcome was change in the proportion with antibodies to HNPs, assessed at 24 months. All participants were included in safety analyses, and surviving participants (those with samples at 24 months) were included in primary analyses. Secondary outcomes were: change in concentrations of antibodies to HNPs at 24 months compared with baseline; proportion of participants testing positive for antibodies to O volvulus-specific proteins and concentrations of Ov16 or OVOC3261 antibodies at 24 months compared with baseline; change in seizure burden, proportion achieving seizure freedom, and the proportions with interictal epileptiform discharges on the diagnostic EEG; overall quality of life; disease severity at 24 months; and incidence of all-cause adverse events, serious adverse events, and seizure-related mortality by 24 months. This trial is registered with ClinicalTrials.gov, NCT02850913. FINDINGS: Between Sept 1, 2016, and Aug 31, 2018, 329 children and adolescents were screened, of whom 240 were included in the study. 140 (58%) participants were boys and 100 (42%) were girls. 120 (50%) participants were allocated to receive doxycycline and 120 (50%) to receive placebo. At recruitment, the median duration of symptoms was 9 years (IQR 6-10); 232 (97%) participants had O volvulus-specific antibodies and 157 (65%) had autoantibodies to HNPs. The most common plasma autoantibodies were to human protein deglycase DJ-1 (85 [35%] participants) and leiomodin-1 (77 [32%] participants) and, in cerebrospinal fluid (CSF), to human DJ-1 (27 [11%] participants) and leiomodin-1 (14 [6%] participants). On immunohistochemistry, 46 (19%) participants had CSF autoantibodies to HNPs, including leiomodin-1 (26 [11%]), γ-aminobutyric acid B receptors (two [<1%]), CASPR2 (one [<1%]), or unknown targets (28 [12%]). At 24 months, 161 (72%) of 225 participants had antibodies to HNPs compared with 157 (65%) of 240 at baseline. 6 weeks of doxycycline did not affect the concentration of autoantibodies to HNPs, seizure control, disease severity, or quality of life at the 24-month follow-up but substantially decreased Ov16 antibody concentrations; the median plasma signal-to-noise Ov16 ratio was 16·4 (95% CI 6·4-38·4), compared with 27·9 (8·2-65·8; p=0·033) for placebo. 14 (6%) participants died and, other than one traffic death, all deaths were seizure-related. Acute seizure-related hospitalisations (rate ratio [RR] 0·43 [95% CI 0·20-0·94], p=0·028) and deaths (RR 0·46 [0·24-0·89], p=0·028) were significantly lower in the doxycycline group. At 24 months, 96 (84%) of 114 participants who received doxycycline tested positive for antibodies to Ov16, compared with 97 (87%) of 111 on placebo (p=0·50), and 74 (65%) participants on doxycycline tested positive for antibodies to OVOC3261, compared with 57 (51%) on placebo (p=0·039). Doxycycline was safe; there was no difference in the incidence of grade 3-5 adverse events across the two groups. INTERPRETATION: Nodding syndrome is strongly associated with O volvulus and the pathogenesis is probably mediated through an O volvulus induced autoantibody response to multiple proteins. Although it did not reverse disease symptoms, doxycycline or another prophylactic antibiotic could be considered as adjunct therapy to antiseizure medication, as it might reduce fatal complications from acute seizures and status epilepticus induced by febrile infections. FUNDING: Medical Research Council (UK). TRANSLATION: For the Luo translation of the abstract see Supplementary Materials section.

Comparison of Structural Changes in Nodding Syndrome and Other Epilepsies Associated With Onchocerca volvulus.

BACKGROUND AND OBJECTIVE: Nodding syndrome (NS) is a unique childhood-onset epileptic disorder that occurs predominantly in several regions of sub-Saharan Africa. The disease has been associated with Onchocerca volvulus (Ov)-induced immune responses and possible cross-reactivity with host proteins. The aim of this study was to compare structural changes in the brain on MRI between NS and other forms of onchocerciasis-associated epilepsies (OAEs) and to relate structural changes to the Ov-induced immune responses and level of disability. METHODS: Thirty-nine children with NS and 14 age-matched participants with other forms of OAE from an endemic region in Uganda underwent detailed clinical examination, serologic evaluation (including Ov-associated antibodies to Ov-16 and Hu-leiomodin-1) and quantitative volumetric analysis of brain MRIs (1.5 T scanner) using Neuroreader, a cloud-based software. RESULTS: Cerebral and cerebellar atrophy were the predominant features in both NS and OAE. On quantitative volumetric analysis, participants with NS had larger ventricular volumes compared with participants with OAE, indicative of increased global cortical atrophy (pcorr = 0.036). Among children with NS, severe disability correlated with higher degree of atrophy in the gray matter volume (pcorr = 0.009) and cerebellar volume (pcorr = 0.009). NS cases had lower anti-Ov-16 IgG signal-to-noise ratios than the OAE cases (p < 0.01), but no difference in the levels of the Hu-leiomodin-1 antibodies (p = 0.64). The levels of Ov-associated antibodies did not relate to the degree of cerebral or cerebellar atrophy in either NS or OAE cases. DISCUSSION: This is the first study to show that cerebral and cerebellar atrophy correlated with the severity of NS disability, providing an imaging marker for these endemic epileptic disorders that until now have remained poorly characterized. Both NS and OAE have cerebral and cerebellar atrophy, and the levels of Ov-associated antibodies do not seem to be related to the structural changes on MRI.

Zoonotic Ancylostoma ceylanicum Hookworm Infections, Ecuador.

Ancylostoma ceylanicum hookworms are zoonotic parasites that can infect humans. To detect autochthonous transmission, we analyzed human fecal samples collected in 2000. Multiparallel quantitative PCR detected infection in persons who had never traveled outside Ecuador. These data indicate human transmission of A. ceylanicum in the Americas, although endemicity remains unknown.

Loa loa Microfilariae in Skin Snips: Consequences for Onchocerciasis Monitoring and Evaluation in L. loa-Endemic Areas.

The specificity of skin snips for onchocerciasis diagnoses is considered to be almost 100%. Our molecular methods revealed that microfilariae emerging from skin snips collected from highly microfilaremic Loa loa-infected individuals were largely misidentified as Onchocerca volvulus. This has important implications for onchocerciasis diagnostic testing in Loa-endemic areas.

Differences in the Clinical and Laboratory Features of Imported Onchocerciasis in Endemic Individuals and Temporary Residents.

Many parasitic infections have different presenting features in endemic individuals (ENDs) and immunologically naive temporary residents (TRs). Temporary residents with loiasis often display acute symptoms and hypereosinophilia, in contrast to a parasite-induced subclinical state in chronically infected ENDs. Few studies have examined differences in ENDs and TRs infected with the related filarial parasite Onchocerca volvulus. We identified 40 TRs and 36 ENDs with imported onchocerciasis at the National Institutes of Health between 1976 and 2016. All study subjects received an extensive pretreatment medical history, physical examination, and laboratory investigations. We performed additional parasite-specific serologic testing on stored patient sera. Asymptomatic infection occurred in 12.5% of TRs and no ENDs (P = 0.06). Papular dermatitis was more common in TRs (47.5% versus 2.7%, P < 0.001), whereas more pigmentation changes occurred in ENDs (41.7% versus 15%, P = 0.01). Only endemic patients reported visual disturbance (13% versus 0%, P = 0.03). One TR (3.3%) had onchocercal eye disease, compared with 22.6% of ENDs (P = 0.053). Absolute eosinophil counts (AECs) were similar in ENDs and TRs (P = 0.5), and one-third of subjects had a normal AEC. Endemic individuals had higher filarial-specific IgG4 and were more likely to be positive for IgG4 antibodies to Ov-16. Temporary residents and ENDs with imported O. volvulus infection presented with different dermatologic manifestations; ocular involvement occurred almost exclusively in ENDs. Unlike Loa loa, clinical differences appear not to be eosinophil-mediated and may reflect chronicity, intensity of infection, or the presence of Wolbachia in O. volvulus.

A Serological Survey of Human Onchocerciasis in Yemen.

Yemen is a country that has been treating severe cases of oncho-dermatitis since 1992 and is now moving to a program aimed at the elimination of the transmission of Onchocerca volvulus. It is important to ensure that the currently acceptable tools used in epidemiological assessment of onchocerciasis in Africa and Latin America also apply to Yemen. Five hundred and ten blood samples from three known O. volvulus-endemic areas, locations that have never been under a mass treatment program, were tested for the presence of antibodies against a panel of O. volvulus-specific antigens using enzyme-linked immunosorbent assay (Ov16) and luciferase immunoprecipitation system (Ov-FAR-1 and Ov-MSA-1) assays. Overall, 31.4% of the samples tested were positive, with positivity increasing with age. Positivity was seen in 76.5% of those presenting with clinical onchocerciasis but importantly also in more than 28.5% of those defined as free of oncho-dermatitis; these latter individuals are likely to be serving as a source for persistent reinfection. This study supports the use of the current O. volvulus-specific serologic methodology in Yemen.

Comparison of antigen and antibody responses in repeat lymphatic filariasis transmission assessment surveys in American Samoa.

BACKGROUND: Current WHO recommendations for lymphatic filariasis (LF) surveillance advise programs to implement activities to monitor for new foci of transmission after stopping mass drug administration (MDA). A current need in the global effort to eliminate LF is to standardize diagnostic tools and surveillance activities beyond the recommended transmission assessment survey (TAS). METHODOLOGY: TAS was first conducted in American Samoa in 2011 (TAS 1) and a repeat TAS was carried out in 2015 (TAS 2). Circulating filarial antigen (CFA) and serologic results from both surveys were analyzed to determine whether interruption of LF transmission has been achieved in American Samoa. PRINCIPAL FINDINGS: A total of 1,134 and 864 children (5-10 years old) were enrolled in TAS 1 and TAS 2, respectively. Two CFA-positive children were identified in TAS 1, and one CFA-positive child was identified in TAS 2. Results of both surveys were below the threshold for which MDA was warranted. Additionally, 1,112 and 836 dried blood spots from TAS 1 and TAS 2, respectively were tested for antibodies to Wb123, Bm14 and Bm33 by luciferase immunoprecipitation system (LIPS) assay and multiplex bead assay. In 2011, overall prevalence of responses to Wb123, Bm14, and Bm33 was 1.0%, 6.8% and 12.0%, respectively. In 2015, overall prevalence of positive Bm14 and Bm33 responses declined significantly to 3.0% (p<0.001) and 7.8% (p = 0.013), respectively. CONCLUSIONS/SIGNIFICANCE: Although passing TAS 1 and TAS 2 and an overall decline in the prevalence of antibodies to Bm14 and Bm33 between these surveys suggests decreased exposure and infection among young children, there were persistent responses in some schools. Clustering and persistence of positive antibody responses in schools may be an indication of ongoing transmission. There is a need to better understand the limitations of current antibody tests, but our results suggest that serologic tools can have a role in guiding programmatic decision making.

Measuring changes in transmission of neglected tropical diseases, malaria, and enteric pathogens from quantitative antibody levels.

BACKGROUND: Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. METHODS/PRINCIPAL FINDINGS: We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman's rho = 0.75). In both high- and low transmission settings, mean antibody curves revealed changes in population mean antibody levels that were masked by seroprevalence measures because changes took place above or below the seropositivity cutoff. CONCLUSIONS/SIGNIFICANCE: Age-dependent antibody curves and summary means provided a robust and sensitive measure of changes in transmission, with greatest sensitivity among young children. The method generalizes to pathogens that can be measured in high-throughput, multiplex serological assays, and scales to surveillance activities that require high spatiotemporal resolution. Our results suggest quantitative antibody levels will be particularly useful to measure differences in exposure for pathogens that elicit a transient antibody response or for monitoring populations with very high- or very low transmission, when seroprevalence is less informative. The approach represents a new opportunity to conduct integrated serological surveillance for neglected tropical diseases, malaria, and other infectious diseases with well-defined antigen targets.

Nodding syndrome may be an autoimmune reaction to the parasitic worm Onchocerca volvulus.

Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is an epidemiological association with Onchocerca volvulus, the parasitic worm that causes onchocerciasis (river blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoantibodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cortical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and suggests that patients may benefit from immunomodulatory therapies.

Molecular Epidemiology of Blood-Borne Human Parasites in a Loa loa-, Mansonella perstans-, and Plasmodium falciparum-Endemic Region of Cameroon.

The study of the interactions among parasites within their hosts is crucial to the understanding of epidemiology of disease and for the design of effective control strategies. We have conducted an assessment of infections with Loa loa, Mansonella perstans, Wuchereria bancrofti, and Plasmodium falciparum in eastern Cameroon using a highly sensitive and specific quantitative polymerase chain reaction assay using archived dried whole blood spots. The resident population (N = 1,085) was parasitized with M. perstans (76%), L. loa (39%), and P. falciparum (33%), but not with W. bancrofti Compared with single infections (40.1%), coinfection was more common (48.8%): 21.0% had L. loa-M. perstans (Ll(+)/Mp(+)/Pf(-)), 2.7% had L. loa-P. falciparum (Ll(+)/Pf(+)/Mp(-)), 15.1% had M. perstans-P. falciparum (Mp(+)/Pf(+)/Ll(-)), and 10.0% had L. loa-M. perstans-P. falciparum (Ll(+)/Mp(+)/Pf(+)). Interestingly, those with all three infections (Ll(+)/Mp(+)/Pf(+)) had significantly higher L. loa microfilaria (mf) counts than either single Ll(+) (P = 0.004) or double Ll(+)/Mp(+) (P = 0.024) infected individuals. Of those infected with L. loa, the mean estimated counts of L. loa mf varied based on location and were positively correlated with estimated intensities of M. perstans mf. Finally, at a community level, heavy L. loa infections were concentrated in a few individuals whereby they were likely the major reservoir for infection.

Isolation of Onchocerca lupi in Dogs and Black Flies, California, USA.

In southern California, ocular infections caused by Onchocerca lupi were diagnosed in 3 dogs (1 in 2006, 2 in 2012). The infectious agent was confirmed through morphologic analysis of fixed parasites in tissues and by PCR and sequencing of amplicons derived from 2 mitochondrially encoded genes and 1 nuclear-encoded gene. A nested PCR based on the sequence of the cytochrome oxidase subunit 1 gene of the parasite was developed and used to screen Simulium black flies collected from southern California for O. lupi DNA. Six (2.8%; 95% CI 0.6%-5.0%) of 213 black flies contained O. lupi DNA. Partial mitochondrial16S rRNA gene sequences from the infected flies matched sequences derived from black fly larvae cytotaxonomically identified as Simulium tribulatum. These data implicate S. tribulatum flies as a putative vector for O. lupi in southern California.

Comparison of three immunoassays for detection of antibodies to Strongyloides stercoralis.

Due to the limited sensitivities of stool-based microscopy and/or culture techniques for Strongyloides stercoralis, the detection of antibodies to this intestinal nematode is relied upon as a surrogate for determining exposure status or making a diagnosis of S. stercoralis infection. Here, we evaluated three immunoassays, including the recently released InBios Strongy Detect IgG enzyme-linked immunosorbent assay (ELISA) (InBios International, Inc., Seattle, WA), the SciMedx Strongyloides serology microwell ELISA (SciMedx Corporation, Denville, NJ), and the luciferase immunoprecipitation system (LIPS) assay performed at the National Institutes of Health (NIH), for their detection of IgG antibodies to S. stercoralis. A total of 101 retrospective serum samples, previously submitted for routine S. stercoralis antibody detection using the SciMedx assay, were also evaluated by the InBios and LIPS assays. The qualitative results from each assay were compared using a Venn diagram analysis, to the consensus result among the three assays, and each ELISA was also evaluated using the LIPS assay as the reference standard. By Venn diagram analysis, 65% (66/101) of the samples demonstrated perfect agreement by all three assays. Also, the numbers of samples considered positive or negative by a single method were similar. Compared to the consensus result, the overall percent agreement of the InBios, SciMedx, and LIPS assays were comparable at 87.1%, 84.2%, and 89.1%, respectively. Finally, the two ELISAs performed analogously but demonstrated only moderate agreement (kappa coefficient for the two assays, 0.53) with the LIPS assay. Collectively, while the two commercially available ELISAs perform equivalently, neither should be used independently of clinical evaluation to diagnose strongyloidiasis.

Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools.

Onchocerciasis is a neglected tropical disease caused by infection with the parasite Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% (95% confidence interval: 86.2%-92.0%), and specificity of 97% (95% confidence interval: 95.4%-98.6%). Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions (37°C, 80% relative humidity) and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test.

Rapid Wuchereria bancrofti-specific antigen Wb123-based IgG4 immunoassays as tools for surveillance following mass drug administration programs on lymphatic filariasis.

The Global Programme to Eliminate Lymphatic Filariasis has an urgent need for rapid assays to detect ongoing transmission of lymphatic filariasis (LF) following multiple rounds of mass drug administration (MDA). Current WHO guidelines support using the antigen card immunochromatographic test (ICT), which detects active filarial infection but does not detect early exposure to LF. Recent studies found that antibody-based assays better serve this function. In the present study, two tests, a rapid IgG4 enzyme-linked immunosorbent assay (ELISA) and a lateral-flow strip immunoassay, were developed based on the highly sensitive and specific Wuchereria bancrofti antigen Wb123. A comparison of W. bancrofti-infected and -uninfected patients (with or without other helminth infections) demonstrated that both tests had high sensitivities and specificities (93 and 97% [ELISA] and 92 and 96% [strips], respectively). When the W. bancrofti-uninfected group was separated into those with other filarial/helminth infections (i.e., onchocerciasis, loiasis, and strongyloidiasis) and those who were parasite uninfected, the specificities of the assays varied between 91 and 100%. In addition, the geometric mean response by ELISA of W. bancrofti-infected patients was significantly higher than the response of those without W. bancrofti infection (P < 0.0001). Furthermore, the Wb123 ELISA and the lateral-flow strips had high positive and negative predictive values, giving valuable information on the size of survey population needed to be reasonably certain whether or not transmission is ongoing. These highly sensitive and specific IgG4 tests to the W. bancrofti Wb123 protein give every indication that they will serve as useful tools for post-MDA monitoring.

Identification of Wb123 as an early and specific marker of Wuchereria bancrofti infection.

BACKGROUND: The current antibody tests used for monitoring in lymphatic filariasis (LF) elimination programs suffer from poor specificity because of the considerable geographical overlap with other filarial infections such as Loa loa (Ll), Onchocerca volvulus (Ov), and Mansonella perstans (Mp). METHODS: Using bioinformatics to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of W. bancrofti (Wb), these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of B. malayi (Bm - n = 5068), Ov (n = 4166), and Ll (n = 3315). Nineteen potential L3- and Wb- and/or Bm-specific antigens were identified. Sixteen of the 19 antigens could be expressed as fusion proteins with Renilla luciferase (Ruc); these were used in a rapid Luciferase Immunopreciptation System (LIPS) assay. RESULTS: One of the 16 expressed antigens (Wb123) was both highly immunogenic and specific for Wb. Using Wb123-based IgG and IgG4 LIPS assays on well-defined sera from normal North Americans and those infected exclusively with intestinal helminths, we could detect all of the Wb-infected individuals (from diverse geographic regions) with 100% sensitivity and 100% specificity. Using sera from exclusively Ll-infected, Ov-infected Mp-infected or Bm-infected subjects as the negative comparator, the sensitivities were between 98-100% and the specificities ranged between 84-100% (for IgG anti-Wb123) and between 98-100% (for IgG4 anti-Wb123). Blinded assessments using panels of sera from various Wb-, Bm- or non-Wb helminth-infected subjects demonstrated equally high degrees of sensitivity and specificity. SIGNIFICANCE: We have identified a Wb-encoded antigen that can be used both as a rapid, high throughput tool to diagnose individual Wb infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.

Antibody to the filarial antigen Wb123 reflects reduced transmission and decreased exposure in children born following single mass drug administration (MDA).

BACKGROUND: Antibody (Ab) to the Wuchereria bancrofti (Wb) infective larval (L3) antigen Wb123, using a Luciferase Immunoprecipitation System (LIPS) assay, has been shown to be a species-specific, early marker of infection developed for potential use as a surveillance tool following transmission interruption post mass drug administration. To examine its usefulness in a single filarial-endemic island assessed at two time points with markedly different levels of transmission, Ab to Wb123 was measured in sera collected from subjects from Mauke, Cook Islands in 1975 (no previous treatment) and 1992 (5 years after a one time island-wide treatment with diethylcarbamazine [DEC]). FINDINGS: Between 1975 and 1992, Wb transmission decreased dramatically as evidenced by reduced prevalences of microfilariae (31% vs. 5%) and circulating Ag (CAg, 49% vs. 16%). Age specific prevalence analysis showed a dramatic reduction in Wb123 Ab positivity from 54% (25/46) in 1975 to 8% (3/38) in 1992 in children 1-5 years (p<0.0001), reflecting the effects of single-dose treatment five years earlier. By 1992, Wb123 Ab prevalence in children 6-10 years had fallen from 75% (42/56) in 1975 to 42% (33/79) consistent with a lower cumulative transmission potential. In the whole population, Wb123 seropositivity decreased from 86% to 60% between 1975 and 1992. In CAg+ subjects the levels of Wb123 Ab were indistinguishable between the 2 time points but differed in those who were CAg- (p<0.0001). In paired sample analysis, individuals who were CAg+ in 1975 but became CAg- in 1992 had significantly lower Ab levels in 1992 (p<0.0001), with 9/40 (23%) becoming seronegative for Wb123. CONCLUSIONS: The relationship between reduction in Wb123 Ab prevalence and the reduction of transmission, seen most clearly in young children, strongly advocates for the continuing assessment and rapid development of Wb123 as a surveillance tool to detect potential transmission of bancroftian filariasis in treated endemic areas.

Longitudinal monitoring of the development of antifilarial antibodies and acquisition of Wuchereria bancrofti in a highly endemic area of Haiti.

Antifilarial antibody testing has been established as a sensitive and specific method of diagnosing lymphatic filariasis. However, the development of serological responses to specific filarial antigens and their relationship to acquisition of infection is poorly understood. In order to evaluate whether the development of antigen specific antifilarial antibodies precedes microfilaremia and antigenemia, we compared the antibody responses of serum samples collected between 1990 and 1999 from a cohort of 142 Haitian children followed longitudinally. Antigen status was determined using the Og4C3 ELISA and the presence of microfilaremia was detected using microscopy. Antibody responses to Wb123, a Wuchereria bancrofti L3 antigen, were measured using a Luciferase Immunoprecipitation System (LIPS) assay. Antibody responses to Bm14 and Bm33, Brugia malayi antigens and to a major surface protein (WSP) from Wolbachia were analyzed using a multiplex bead assay. Over follow-up, 80 (56%) of the children became antigen-positive and 30 (21%) developed microfilaremia. Detectable antibody responses to Bm14, Bm33, Wb123, and WSP developed in 95%, 100%, 92%, and 29% of children, respectively. With the exception of WSP, the development of antibody responses generally preceded detection of filarial antigen. Our results show that antifilarial antibody responses can serve as an important epidemiological indicator in a sentinel population of young children and thus, may be valuable as tool for surveillance in the context of lymphatic filariasis elimination programs.

A randomized trial of doxycycline for Mansonella perstans infection.

BACKGROUND: Mansonella perstans infection is common in areas of Africa where Wuchereria bancrofti, a causative agent of lymphatic filariasis, is endemic. M. perstans is refractory to standard antifilarial therapies. The recent discovery of bacterial endosymbionts (e.g., wolbachia) in most filarial species, including M. perstans, provides new therapeutic options for reducing microfilaremia. METHODS: In an open-label, randomized trial, we recruited subjects with M. perstans microfilaremia, with or without concomitant W. bancrofti infection, from four villages in Mali and randomly assigned them to receive doxycycline, at a dose of 200 mg daily for 6 weeks (106 subjects), or no treatment (110). At 6 months, subjects who were coinfected with W. bancrofti underwent a second random assignment, to treatment with a single dose of albendazole (400 mg) and ivermectin (150 microg per kilogram of body weight) or no treatment. Subjects were monitored daily during the first 6-week study period for adverse events. M. perstans and W. bancrofti microfilarial levels were assessed at 6, 12, and 36 months. RESULTS: At 12 months, 67 of 69 subjects who had received treatment with doxycycline only (97%) had no detectable M. perstans microfilariae per 60 microl of blood, as compared with 10 of 63 subjects who had received no treatment (16%) (relative risk, 6.18; 95% confidence interval, 3.63 to 11.89; P<0.001). At 36 months, M. perstans microfilaremia remained suppressed in 48 of 64 subjects who had received treatment with doxycycline only (75%), a finding that was consistent with a macrofilaricidal effect of doxycycline. Vomiting was more frequent in the doxycycline-treated group than in the untreated group (17% vs. 4%). CONCLUSIONS: These results are consistent with previous findings that M. perstans harbors the intracellular endosymbiont, wolbachia, and suggest that doxycycline is an effective therapy for M. perstans infection. (ClinicalTrials.gov number, NCT00340691.)

Molecular identification of Wolbachia from the filarial nematode Mansonella perstans.

Wolbachiae are bacterial endosymbionts of insects and many filarial nematodes whose products trigger inflammation in filarial infections. The dependence of the parasites on their endosymbionts has also led to the use of antibiotics directed against the Wolbachiae, therapy that has been demonstrated to have a profound salutary effect on filarial infections. The identification of Wolbachiae in Mansonella species has been conclusively shown for Mansonella ozzardi (Mo), but not for Mansonella perstans (Mp). Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found in all samples tested. Sequence analysis of these samples demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes. When aligned with the only other Mansonella Wolbachia sequence (Mo) there were only 8 nucleotide differences in the 1369bp overlapping sequence. Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia.

Seroconversion to filarial antigens in Australian defence force personnel in Timor-Leste.

To investigate whether Australian soldiers were exposed to filarial parasites that cause lymphatic filariasis during a 6-month deployment to Timor-Leste, antifilarial antibody levels were measured in 907 soldiers using an enzyme linked immunosorbent assay (ELISA). Initial testing using Dirofilaria immitis antigen demonstrated that 49 of 907 (5.4%) soldiers developed antifilarial antibodies of the IgG1 subclass after deployment, whereas 1 of 944 (0.1%) seroconverted to the IgG4 subclass. When a sub sample of 88 D. immitis-reactive sera was subject to testing with an antifilarial antibody test using Brugia malayi antigen, 46 had elevated IgG antibodies, whereas 5 had elevated antibodies of the IgG4 subclass. A total of 24 soldiers seroconverted to B. malayi, as measured by parasite-specific IgG, whereas 1 seroconverted to IgG4. The relatively low number of seroconversions indicates a low but measurable risk of exposure to human filarial parasites among Australian soldiers deployed to Timor-Leste. However, to reduce the risk of exposure to these parasites, soldiers deploying to endemic areas should practice strict adherence to personal protective measures against mosquito bites.