Mf Counts Research Articles

An automated, high-resolution phenotypic assay for adult Brugia malayi and microfilaria

Brugia malayi are thread-like parasitic worms and one of the etiological agents of Lymphatic filariasis (LF). Existing anthelmintic drugs to treat LF are effective in reducing the larval microfilaria (mf) counts in human bloodstream but are less effective on adult parasites. To test potential drug candidates, we report a multi-parameter phenotypic assay based on tracking the motility of adult B. malayi and mf in vitro. For adult B. malayi, motility is characterized by the centroid velocity, path curvature, angular velocity, eccentricity, extent, and Euler Number. These parameters are evaluated in experiments with three anthelmintic drugs. For B. malayi mf, motility is extracted from the evolving body skeleton to yield positional data and bending angles at 74 key point. We achieved high-fidelity tracking of complex worm postures (self-occlusions, omega turns, body bending, and reversals) while providing a visual representation of pose estimates and behavioral attributes in both space and time scales.

Long-term evaluation of viability of microfilariae and intravenously transplanted adult Dirofilaria immitis in microfilaremic dogs treated with low-dose, short- and long-treatment regimens of doxycycline and ivermectin

BackgroundMicrofilarial (mf) counts were monitored over 21.3 months for any rebound that might occur in counts, and adulticidal efficacy was assessed following administration of low dosage with short- and long-treatment regimens of doxycycline and ivermectin to heartworm-microfilaremic dogs.MethodsTwelve heartworm-naïve beagles infected with 10 pairs of adult Dirofilaria immitis by intravenous transplantation were randomly allocated to three groups of four dogs. All treatments started on day 0. On day 0, Group 1 (short-treatment regimen) received doxycycline orally at 10 mg/kg once daily for 30 days plus ivermectin orally (minimum, 6 mcg/kg) on days 0 and 30. Group 2 (long-treatment regimen) received doxycycline orally at 10 mg/kg once daily until individual dogs became mf-negative (72–98 days) and ivermectin every other week until individual dogs became mf-negative (6–7 doses). Group 3 was the untreated control. Mf counts and antigen (Ag) tests were conducted. Dogs were necropsied for recovery and enumeration of heartworms on day 647.ResultsDay −1 mean mf counts were 15,613, 23,950, and 15,513 mf/ml for groups 1, 2, and 3, respectively. Mean counts for Groups 1 and 2 declined until days 239 and 97, respectively, when all were negative. Group 3 had high mf counts throughout the study. There was not a rebound in mf counts in any of the treated dogs after they became amicrofilaremic. All dogs in group 1 and group 3 were Ag-positive throughout the study and had at least one live female worm at necropsy. All dogs in treated Group 2 were positive for Ag through day 154, but were antigen-negative on days 644 and 647, as all had only male worms. Mean live adult worm recoveries for Groups 1, 2, and 3 were 6.8 (range, 5–8), 3.3 (range, 1–6), and 16.0 (range, 14–17), respectively, with a percent reduction in adult worm counts of 57.5% for Group 1 and 79.3% for Group 2.ConclusionsThese data lend support to the use of the American Heartworm Society Canine Guidelines for adulticide therapy recommending the initiation of doxycycline plus a macrocyclic lactone (ML) at the time of the heartworm-positive diagnosis.Graphical

Characterization of a novel microfilarial antigen for diagnosis of Wuchereria bancrofti infections.

BackgroundLymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori. The Global Program to Eliminate LF uses mass drug administration (MDA) of anti-filarial drugs that clear microfilariae (Mf) from blood to interrupt transmission by mosquitos. New diagnostic tools are needed to assess the impact of MDA on bancroftian filariasis, because available serologic tests can remain positive after successful treatment.Methodology/Principal findingsWe identified Wb-bhp-1, which encodes a W. bancrofti homologue of BmR1, the B. malayi protein used in the Brugia Rapid antibody test for brugian filariasis. Wb-bhp-1 has a single exon that encodes a 16.3 kD protein (Wb-Bhp-1) with 45% amino acid identity to BmR1. Immunohistology shows that anti-Wb-Bhp-1 antibodies primarily bind to Mf. Plasma from 124 of 224 (55%) microfilaremic individuals had IgG4 antibodies to Wb-Bhp-1 by ELISA. Serologic reactivity to Wb-Bhp-1 varied widely with samples from different regions (sensitivity range 32–92%), with 77% sensitivity for 116 samples collected from microfilaremic individuals outside of sub-Saharan Africa. This variable sensitivity highlights the importance of validating new diagnostic tests for parasitic diseases with samples from different geographical regions. Individuals with higher Mf counts were more likely to have anti-Wb-Bhp-1 antibodies. Cross-reactivity was observed with a minority of plasma samples from people with onchocerciasis (17%) or loiasis (10%). We also identified, cloned and characterized BmR1 homologues from O. volvulus and L. loa that have 41% and 38% identity to BmR1, respectively. However, antibody assays with these antigens were not sensitive for onchocerciasis or loiasis.ConclusionsWb-Bhp-1 is a novel antigen that is useful for serologic diagnosis of bancroftian filariasis. Additional studies are needed to assess the value of this antigen for monitoring the success of filariasis elimination programs.

Relationship between skin snip and Ov16 ELISA: Two diagnostic tools for onchocerciasis in a focus in Cameroon after two decades of ivermectin-based preventive chemotherapy.

BackgroundOnchocerciasis elimination currently relies on repeated ivermectin-based preventive chemotherapy. Current World Health Organization’s guidelines strongly recommend, though with low evidence of certainty, the use of Ov16 serology testing in children younger than 10 years old to assess whether mass drugs administration can be safely stopped. Therefore, more evidences are needed to support the use of this marker as sero-evaluation tool. This study aimed at determining the relationship between microfilaridermia and anti-Ov16 IgG4, and their variation according to age, gender and ivermectin intake history.MethodologyA cross-sectional survey was conducted in an area where ivermectin-based MDA has been implemented since more than 20 years. A questionnaire was used to record ivermectin intake history for the last 5 years. All volunteers aged ≥2 years were tested for microfilaridermia. IgG4 antibodies against Ov16 antigen were determined using the Standard Diagnostic Ov16 IgG4 ELISA kits and the recombinant anti-Ov16 AbD19432 antibodies. Prevalences, microfilaridermia counts and IgG4 concentrations were compared with regards to age, gender and history of ivermectin intake.Principal findingsThe prevalence of skin microfilariae was 23.4% (95% CI: 23.4–30.8), whereas Ov16 seroprevalence was 53.2% (95% CI: 47.9–58.4). A moderate positive percentage agreement (50.4%) and a high negative percentage agreement (69.2%) was found between skin snip and Ov16 serology in the whole population, while in children aged <10 years, the agreements were higher (positive percentage agreement: 62.6%; negative percentage agreement: 83.5%). In addition, no associations were found between ivermectin intake, Mf counts and estimated IgG4 concentration of participants. Anti-Ov16 IgG4 were higher in individuals harboring microfilariae than their negative counterparts (p<0.0001), though a negative correlation was found between skin microfilarial counts and anti-Ov16 IgG4 levels (r = -0.2400; p = 0.03). No variation in microfilarial counts according to age and gender was observed. Though positively correlated with age (r = 0.4020; p<0.0001), IgG4 was significantly different between the different age classes (p<0.0001).Conclusion/SignificanceOur results revealed moderate positive and negative agreements between parasitological and immunological parameters of onchocerciasis infection after several rounds MDA. Anti-Ov16 IgG4 levels increased with age but decreased with microfilarial counts, suggesting a variation of anti-Ov16 IgG4 as a result of constant exposure and accumulation of infection. This brings evidence sustaining the use of Ov16 serology in children as evaluation tool. However, additional investigations are needed to further reshape the appropriate age range among children aged <10 years old.

Semiannual Treatment of Albendazole Alone is Efficacious for Treatment of Lymphatic Filariasis: A Randomized Open-label Trial in Cote d'Ivoire.

Ivermectin (IVM) plus albendazole (ALB), or IA, is widely used in mass drug administration (MDA) programs that aim to eliminate lymphatic filariasis (LF) in Africa. However, IVM can cause severe adverse events in persons with heavy Loa loa infections that are common in Central Africa. ALB is safe in loiasis, but more information is needed on its efficacy for LF. This study compared the efficacy and safety of 3 years of semiannual treatment with ALB to annual IA in persons with bancroftian filariasis. Adults with Wuchereria bancrofti microfilaremia (Mf) were randomized to receive either 3 annual doses of IA (N = 52), 6 semiannual doses of ALB 400 mg (N = 45), or 6 semiannual doses of ALB 800 mg (N = 47). The primary outcome is amicrofilaremia at 36 months. IA was more effective for completely clearing Mf than ALB 400mg or ALB 800mg (79%, 95% confidence interval [CI]: 67-91; vs 48%, 95% CI: 32-66 and 57%, 95% CI: 41-73, respectively). Mean percentage reductions in Mf counts at 36 months relative to baseline tended to be greater after IA (98%, 95% CI: 88-100) than after ALB 400 mg (88%, 95% CI: 78-98) and ALB 800 mg (89%, 95% CI: 79-99) (P = .07 and P = .06, respectively). Adult worm nest numbers (assessed by ultrasound) were reduced in all treatment groups. Treatments were well tolerated. Repeated semiannual treatment with ALB is macrofilaricidal for W. bancrofti and leads to sustained reductions in Mf counts. This is a safe and effective regimen that could be used as MDA to eliminate LF in areas where ivermectin cannot be used. NCT02974049.

A Reevaluation of the Tolerability and Effects of Single-Dose Ivermectin Treatment on Onchocerca volvulus Microfilariae in the Skin and Eyes in Eastern Ghana.

ABSTRACT.Mass administration of ivermectin (IVM) has significantly reduced onchocerciasis prevalence, intensity, and morbidity in most endemic areas. Most IVM clinical trials were performed long ago in persons with high-intensity infections that are uncommon in West Africa today. This cohort treatment study recruited participants from a hypoendemic area in eastern Ghana to reevaluate the efficacy and tolerability of IVM with a special focus on the kinetics of microfilaria (Mf) clearance. Mf in the skin and anterior chambers (AC) were assessed by skin snip and slit lamp examinations at baseline and at 3 and 6 months after treatment with IVM 150 μg/kg. Most participants (184–231, 79.7%) enrolled were treatment-naïve. The baseline geometric mean skin Mf count was 12.67/mg (range 3–86). Although persons with MfAC at baseline (64/231, 27%) had significantly higher skin Mf counts than people without MfAC, 7 of 39 (15%) of persons with skin Mf counts in the range of 3–5 Mf/mg had MfAC. Skin Mf were detected in 14% (31/218) and 45% (96/216) of participants 3 and 6 months after IVM treatment, respectively. MfAC were detected in 12 of 212 (5.7%) study participants at 6 months. 81% (187 of 231) of participants experienced 439 adverse events within 7 days after treatment; all adverse events were mild (96.1%) or moderate. This study has provided new data on the kinetics of Mf in the skin and eyes after IVM treatment of persons with light to moderate intensity Onchocerca volvulus infections that are common in Africa at this time.

An open label, block randomized, community study of the safety and efficacy of co-administered ivermectin, diethylcarbamazine plus albendazole vs. diethylcarbamazine plus albendazole for lymphatic filariasis in India.

Better drug regimens for mass drug administration (MDA) could accelerate the Global Programme to Eliminate Lymphatic Filariasis (LF). This community study was designed to compare the safety and efficacy of MDA with IDA (ivermectin, diethylcarbamazine and albendazole) or DA (diethylcarbamazine and albendazole) in India. This two-armed, open-labelled, block randomised, community study was conducted in LF endemic villages in Yadgir district, Karnataka, India. Consenting participants ≥5 years of age were tested for circulating filarial antigenemia (CFA) and microfilaremia (Mf) before treatment with a single oral dose of IDA or DA. Adverse events (AEs) were monitored actively for two days and passively for five more days. Persons with positive CFA or Mf tests at baseline were retested 12-months post-treatment to assess treatment efficacy. Baseline CFA and Mf-rates were 26.4% and 6.9% in IDA and 24.5% and 6.4% in DA villages respectively. 4758 and 4160 participants received IDA and DA. Most AEs were mild after both treatments; fewer than 0.1% of participants experienced AEs with severity > grade 1. No serious AEs were observed. Fever, headache and dizziness were the most common AEs. AE rates were slightly higher after IDA than DA (8.3% vs. 6.4%, P<0.01). AEs were more frequent in females and Mf-positives after either treatment, but significantly more frequent after IDA (40.5% vs 20.2%, P < 0.001). IDA was more effective for clearing Mf than DA (84% vs. 61.8%, P < 0.001). Geometric mean Mf counts per 60μl in retested Mf-positives decreased by 96.4% from 11.8 after IDA and by 90.0% from 9.5 after DA. Neither treatment was effective for clearing CFA. IDA had an acceptable safety profile and was more effective for clearing Mf than DA. With adequate compliance and medical support to manage AEs, IDA has the potential to accelerate LF elimination in India. Clinical Trial Registry of India (CTRI No/2016/10/007399).

Heterogeneous response of Wuchereria bancrofti-infected persons to diethylcarbamazine (DEC) and its implications for the Global Programme to Eliminate Lymphatic Filariasis (GPELF).

DEC or ivermectin (IVM) in combination with albendazole (ALB) has been the recommended strategy of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) since 2000. Despite effective population coverage (> 65%) with several rounds of MDA with DEC or combination of DEC plus ALB, microfilariae persist in few individuals and they continue to be the source of infection for transmitting LF. We report an individual's variability in response to DEC by defining the response as complete absence of microfilaria (mf) (post-treatment mf count = 0) and non-response as presence of mf (post-treatment mf count ≥ 1). We analyzed follow-up data on individual's response to treatment from two randomized clinical trials in which 46 microfilaremic individuals were treated with single-dose DEC (6 mg/kg body weight). They were classified into low, medium, and high mf density categories based on their pre-treatment mf counts. Of the 46 individuals, 65.2% have not responded throughout the 12-month post-treatment period. Application of a logistic regression model with fixed (age, gender, mf density, post-treatment time, and their interactions) and random (individual's response over time) effects indicated that treatment response is independent of age, gender, and time. The overall treatment response increases in low and decreases in high mf density categories. Furthermore, the estimates for the random coefficients model showed that there is a greater variability in response between individuals over post-treatment time. The results substantiate that individual variation in response to DEC exists which indicate the importance of studying the parasite as well as host genetic factors associated with DEC action.

Fecundity of adult female worms were affected when Brugia malayi infected Mongolian gerbils were immunized with a multivalent vaccine (rBmHAXT) against human lymphatic filarial parasite

A multivalent recombinant fusion protein prophylactic vaccine, rBmHAXT developed against lymphatic filariasis (LF) demonstrated over 57% protection against challenge infection in rhesus macaque model. Currently, we do not know if the rBmHAXT vaccination has any effect on adult worms and/or on the fecundity of adult female worms. Thus, the major focus of this study was to determine the effect of rBmHAXT vaccination on Brugia malayi infected mongolian gerbils. We performed two sets of experiments. In the first set of experiment, gerbils were infected with 100 B. malayi L3. After confirming the establishment of infection, four rounds of DEC treatment and rBmHAXT vaccination was given. Results showed that following vaccination with rBmHAXT, the microfilaria (Mf) count was significantly decreased in all vaccinated animals compared to controls. At the end of these experiments, we collected and counted the established adult worms. There was a 36% reduction in the recovery of adult female worms, which might account for the low Mf load in vaccinated animals. In the second set of experiments, animals were vaccinated first with rBmHAXT followed by surgically implanting adult male or female B. malayi parasites into the peritoneal cavity to determine the effect of vaccination on each sex of the parasite. Our results show that the rBmHAXT vaccination has no effect on male adult worms compared to controls. However, there was 40% reduction in the Mf load in vaccinated animals that were transplanted with adult female worms. These findings suggested that the rBmHAXT vaccination has potential damaging effect on the fecundity of adult female worms. Scanning electron microscopy studies showed cuticular damage on the surface of adult female worms. These studies thus show that the rBmHAXT vaccination in infected gerbils has partial microfilaricidal effect and potentially affect the fecundity of adult female worms.

In Southern Nigeria Loa loa Blood Microfilaria Density is Very Low Even in Areas with High Prevalence of Loiasis: Results of a Survey Using the New LoaScope Technology.

Ivermectin treatment can cause central nervous system adverse events (CNS-AEs) in persons with very high-density Loa loa microfilaremia (≥ 30,000 mf/mL blood). Hypoendemic onchocerciasis areas where L. loa is endemic have been excluded from ivermectin mass drug administration programs (MDA) because of the concern for CNS AEs. The rapid assessment procedure for L. loa (RAPLOA) is a questionnaire survey to assess history of eye worm. If ≥ 40% of respondents report eye worm, this correlates with ≥ 2% prevalence of very high-density loiasis microfilaremia, posing an unacceptable risk of CNS-AEs after MDA. In 2016, we conducted a L. loa study in 110 ivermectin-naïve, suspected onchocerciasis hypoendemic villages in southern Nigeria. In previous RAPLOA surveys these villages had prevalences between 10% and 67%. We examined 10,605 residents using the LoaScope, a cell phone-based imaging device for rapidly determining the microfilaria (mf) density of L. loa infections. The mean L. loa village mf prevalence was 6.3% (range 0-29%) and the mean individual mf count among positives was 326 mf/mL. The maximum individual mf count was only 11,429 mf/mL, and among 2,748 persons sampled from the 28 villages with ≥ 40% RAPLOA, the ≥ 2% threshold of very high Loa mf density could be excluded with high statistical confidence (P < 0.01). These findings indicate that ivermectin MDA can be delivered in this area with extremely low risk of L. loa-related CNS-AEs. We also concluded that in Nigeria the RAPLOA survey methodology is not predictive of ≥ 2% prevalence of very high-density L. loa microfilaremia.

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

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. 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. 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). 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.

Efficacy and tolerability of treatment with single doses of diethylcarbamazine (DEC) and DEC plus albendazole (ABZ) for three consecutive years in lymphatic filariasis: a field study in India.

Lymphatic filariasis (LF) affects 73 countries, causes morbidity and impedes socioeconomic development. We had found no difference in safety and micro (Mf) and macro filarial action of single-dose diethylcarbamazine (DEC) and DEC + albendazole (ABZ) in an F01 study done in India (year 2000). There was a programmatic need to evaluate safety and efficacy of multiple annual treatments (F02). Subjects (155) from the F01 study, meeting inclusion-exclusion criteria, were enrolled in F02 and treated with further two annual doses of DEC or DEC + ABZ. Efficacy was evaluated for Mf positivity by peripheral smear (PS) and nucleopore (NP) filter, circulating filarial antigen (CFA) and filarial dance sign (FDS) positivity and Mf count at yearly follow-up. Safety was assessed for 5days after drug administration. Total of 139 subjects evaluated for efficacy (69 DEC and 70 DEC + ABZ group). Mf positivity prevalence declined progressively by 95% (PS), 66% (NP), and 95% (PS) and 86% (NP); CFA positivity prevalence declined by 15% and 9%; FDS by 100% each; Mf count declined by 75.5 and 76.9% with three annual treatment of DEC and DEC + ABZ, respectively. Addition of ABZ did not show any advantage over DEC given as three annual rounds for LF. DEC and DEC + ABZ were well tolerated. There was no correlation between result of CFA and FDS, (both claimed to be indicative of adult worm). Analysis of published studies and our data indicate that macrofilaricidal effect of DEC/DEC + ABZ may be seen in children and not adults, with three or more annual dosing.

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.

Canine dirofilariosis endemic in Central Europe-10years of epidemiological study in Slovakia.

The study presents the comprehensive results of a detailed epidemiological study on canine dirofilariosis in Slovakia, Central Europe. More than 4000 dogs were investigated, and several epidemiological factors were considered. The mean prevalence in individual regions ranged from 2.0% in northern Slovakia to more than 25.0% in the south-western part of the country, with the nematode Dirofilaria repens confirmed as the dominant causative agent. Canine dirofilariosis occurred more often in animals more than 3years old and in dogs of large and giant breed sizes. Short-haired animals were infected more often than dogs with a long coat. Also, the infection was significantly more prevalent in animals kept in rural areas in comparison with urban environments.Counts of microfilariae (mf) in peripheral blood reached their highest levels in May and August and corresponded to activity peaks and population maximums of potential vectors, the mosquito species Aedes vexans and Culex pipiens, the two most prevalent species in Slovakia. Moreover, two dogs naturally infested with D. repens were included in the experiment in order to monitor daily microfilarial periodicity. This fluctuation showed the same tendency in both animals, with a peak of circulating mf recorded at 4 a.m. and minimal mf counts at 4 p.m.

The impact of two semiannual treatments with albendazole alone on lymphatic filariasis and soil-transmitted helminth infections: a community-based study in the Republic of Congo.

Implementation of mass drug administration (MDA) with ivermectin plus albendazole (ALB) for lymphatic filariasis (LF) has been delayed in central Africa because of the risk of serious adverse events in subjects with high Loa loa microfilaremia. We conducted a community trial to assess the impact of semiannual MDA with ALB (400 mg) alone on LF and soil-transmitted helminth (STH) infections in the Republic of Congo. Evaluation at 12 months showed that ALB MDA had not significantly reduced Wuchereria bancrofti antigenemia or microfilaria (mf) rates in the community (from 17.3% to 16.6% and from 5.3% to 4.2%, respectively). However, the geometric mean mf count in mf-positive subjects was reduced from 202.2 to 80.9 mf/mL (60% reduction, P = 0.01). The effect of ALB was impressive in 38 subjects who were mf-positive at baseline and retested at 12 months: 37% had total mf clearance, and individual mf densities were reduced by 73.0%. MDA also dramatically reduced the hookworm infection rate in the community from 6.5% to 0.6% (91% reduction), with less impressive effects on Ascaris and Trichuris. These preliminary results suggest that semiannual community MDA with ALB is a promising strategy for controlling LF and STH in areas with coendemic loiasis.

Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs.

BackgroundStrains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods.MethodsTo validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal ‘hotspots’ of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce L3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 μg/kg) monthly for 6 months at twice the label dose (6 μg/kg), and one untreated control.ResultsBoth treated and untreated dogs from Group I and Group II developed patent D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott’s test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg, given monthly for 3 months.ConclusionsThese experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.

Filarial antigenemia and Loa loa night blood microfilaremia in an area without bancroftian filariasis in the Democratic Republic of Congo.

Implementation of mass drug administration for lymphatic filariasis (LF) has been delayed in central Africa because of incomplete mapping and coendemic loiasis. We mapped two regions in eastern Democratic Republic of Congo that were suspected to have LF. Night blood samples were collected from 2,724 subjects in 30 villages. Filarial antigenemia rates by card test exceeded 1% in 28 villages (range = 0–14%). Prevalence rates for large sheathed microfilariae (Mf) ranged from 4% to 40%; Mansonella perstans rates ranged from 22% to 98%. Large Mf were exclusively Loa loa by microscopy, and only 1 of 337 samples tested by quantitative polymerase chain reaction (qPCR) was positive for Wuchereria bancrofti DNA. Filarial antigen positivity was strongly associated with high L. loa Mf counts. Periodicity studies revealed atypical patterns, with no significant diurnal periodicity in some individuals. Thus, methods routinely used for LF mapping may not be reliable in areas in central Africa that are highly endemic for loiasis.

Detection of circulating parasite-derived microRNAs in filarial infections.

Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.

Semi-Quantitative Scoring of an Immunochromatographic Test for Circulating Filarial Antigen

Abstract. The value of a semi-quantitative scoring of the filarial antigen test (Binax Now Filariasis card test, ICT) results was evaluated during a field survey in the Republic of Congo. One hundred and thirty-four (134) of 774 tests (17.3%) were clearly positive and were scored 1, 2, or 3; and 11 (1.4%) had questionable results. Wuchereria bancrofti microfilariae (mf) were detected in 41 of those 133 individuals with an ICT test score ≥ 1 who also had a night blood smear; none of the 11 individuals with questionable ICT results harbored night mf. Cuzick's test showed a significant trend for higher microfilarial densities in groups with higher ICT scores (P < 0.001). The ICT scores were also significantly correlated with blood mf counts. Because filarial antigen levels provide an indication of adult worm infection intensity, our results suggest that semi-quantitative reading of the ICT may be useful for grading the intensity of filarial infections in individuals and populations.

Evaluation of the Efficacy of Imidacloprid 10 % / Moxidectin 2.5 % (Advocate®, Advantage® Multi, Bayer) for the Prevention of Dirofilaria repens Infection in Dogs

The efficacy of imidacloprid 10 %/moxidectin 2.5 % (Advocate®, Advantage® Multi, Bayer) against experimental Dirofilaria (D.) repens infection in dogs was evaluated in a blinded, negative controlled randomised laboratory efficacy study. On SD (study day) 0, eight dogs received a spot-on treatment at a dose of 10 mg imidacloprid and 2.5 mg moxidectin per kg body weight. Another 8 dogs were left untreated. On SD 28 each dog was infected with approximately 75 infective D. repens larvae. Blood samples were collected every 4 weeks after treatment. A modified Knott test was conducted to detect mf (microfilaria). PCR analysis was performed with mf-positive blood samples. On SDs 245 and 246, all dogs were euthanised for detection of D. repens worms. Blood samples of all treated dogs were negative for mf at all sampling days. Blood samples of control dogs were positive for mf in 5 out of 8 control dogs. Individual mf counts ranged from 7 to 2800 mf/ml. In mf-positive blood samples, only D. repens was identified by PCR analysis. During necropsy D. repens worms could be detected in eight untreated control dogs (range: 3–21 worms per dog), whereas no worm could be detected in any of the treated dogs. These results indicate a 100 % preventive efficiency of a single spot-on treatment of imidacloprid 10 %/moxidectin 2.5 % in dogs against experimental infection with D. repens (L3 larvae). The product was well tolerated in all study animals, no treatment related adverse reactions were observed throughout the study.