RESUMEN
BACKGROUND: Preventive chemotherapy with ivermectin and albendazole (IA) in mass drug administration (MDA) programs for all at-risk populations is the core public health intervention to eliminate lymphatic filariasis (LF). Achieving this goal depends on drug effectiveness in reducing parasite reservoirs in the community to halt transmission. We assessed the efficacy of ivermectin and albendazole in clearing microfilariae and circulating filarial antigens (CFA) following MDA. METHODS: This community-based prospective study was conducted in Mkinga district, Tanga region, Tanzania, from November 2018 to June 2019. A total of 4115 MDA-eligible individuals were screened for CFA using Filarial test strips. CFA positives were re-examined for microfilariae by microscopy. CFA and microfilariae positive individuals were enrolled and received IA through MDA campaign. The status of microfilariae and CFA was monitored before MDA, and on day 7 and six-month following MDA. The primary efficacy outcomes were the clearance rates of microfilariae on day 7 and six-months, and CFA at 6 months of post-MDA. The McNemar test assessed the proportions of microfilariae positive pre- and post-MDA, while Chi-square tests were utilized to examine factors associated with CFA status six months post-MDA. RESULTS: Out of 4115 individuals screened, 239 (5.8%) tested positive for CFA, of whom 11 (4.6%) were also positive for microfilariae. Out of the ten microfilariae-positive individuals available for follow-up on day 7, nine tested negative, yielding a microfilariae clearance rate of 90% [95% confidence interval (CI): 59.6-98.2%]. Participants who tested negative for microfilariae on day 7 remained free of microfilariae six months after MDA. However, those who did not clear microfilariae on day-7 remained positive six-months post-MDA. The McNemar test revealed a significant improvement in microfilariae clearance on day 7 following MDA (P = 0.02). Out of 183 CFA-positive individuals who were available at 6-month follow-up, 160 (87.4%) remained CFA positive, while 23 became CFA negative. The CFA clearance rate at 6 months post-MDA was 12.6% (95% CI: 8.5-8.5%). There was no significant association of variability in ivermectin plasma exposure, measured by maximum concentration or area under the curve, and the clearance status of microfilariae or CFA post-MDA. CONCLUSIONS: Preventive chemotherapy with IA effectively clears microfilariae within a week. However, it is less effective in clearing CFA at six months of post-MDA. The low clearance rate for filarial antigenemia underscores the need for alternative drug combinations and additional preventive measures to achieve LF elimination by 2030.
Asunto(s)
Albendazol , Filariasis Linfática , Filaricidas , Ivermectina , Administración Masiva de Medicamentos , Ivermectina/uso terapéutico , Ivermectina/administración & dosificación , Albendazol/uso terapéutico , Albendazol/administración & dosificación , Tanzanía/epidemiología , Humanos , Filariasis Linfática/prevención & control , Filariasis Linfática/tratamiento farmacológico , Filariasis Linfática/transmisión , Estudios Prospectivos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Adolescente , Adulto Joven , Animales , Niño , Filaricidas/uso terapéutico , Filaricidas/administración & dosificación , Quimioterapia Combinada , Microfilarias/efectos de los fármacos , Anciano , Preescolar , Antígenos Helmínticos/sangre , Resultado del TratamientoRESUMEN
Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.
Asunto(s)
Dirofilaria immitis , Dirofilariasis , Enfermedades de los Perros , Resistencia a Medicamentos , Animales , Dirofilaria immitis/efectos de los fármacos , Dirofilaria immitis/genética , Perros , Dirofilariasis/parasitología , Dirofilariasis/tratamiento farmacológico , Enfermedades de los Perros/parasitología , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/epidemiología , Europa (Continente) , Italia/epidemiología , Macrólidos/farmacología , Macrólidos/uso terapéutico , Lactonas/farmacología , Microfilarias/efectos de los fármacos , Polimorfismo de Nucleótido Simple , Filaricidas/farmacología , Filaricidas/uso terapéuticoRESUMEN
Mass drug administration (MDA) of antifilarial drugs is the main strategy for the elimination of lymphatic filariasis (LF). Recent clinical trials indicated that the triple-drug therapy with ivermectin, diethylcarbamazine, and albendazole (IDA) is much more effective against LF than the widely used two-drug combinations (albendazole plus either ivermectin or diethylcarbamazine). For IDA-based MDA, the stop-MDA decision is made based on microfilariae (mf) prevalence in adults. In this study, we assess how the probability of eventually reaching elimination of transmission depends on the critical threshold used in transmission assessment surveys (TAS-es) to define whether transmission was successfully suppressed and triple-drug MDA can be stopped. This analysis focuses on treatment-naive Indian settings. We do this for a range of epidemiological and programmatic contexts, using the established LYMFASIM model for transmission and control of LF. Based on our simulations, a single TAS, one year after the last MDA round, provides limited predictive value of having achieved suppressed transmission, while a higher MDA coverage increases elimination probability, thus leading to a higher predictive value. Every additional TAS, conditional on previous TAS-es being passed with the same threshold, further improves the predictive value for low values of stop-MDA thresholds. An mf prevalence threshold of 0.5% corresponding to TAS-3 results in ≥95% predictive value even when the MDA coverage is relatively low.
Asunto(s)
Albendazol , Dietilcarbamazina , Quimioterapia Combinada , Filariasis Linfática , Filaricidas , Ivermectina , Administración Masiva de Medicamentos , Microfilarias , Filariasis Linfática/tratamiento farmacológico , Filariasis Linfática/epidemiología , Filariasis Linfática/prevención & control , Humanos , Albendazol/uso terapéutico , Albendazol/administración & dosificación , Filaricidas/uso terapéutico , Dietilcarbamazina/uso terapéutico , Dietilcarbamazina/administración & dosificación , Ivermectina/uso terapéutico , Ivermectina/administración & dosificación , Animales , India/epidemiología , Microfilarias/efectos de los fármacos , Adulto , PrevalenciaRESUMEN
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.
Asunto(s)
Antihelmínticos/uso terapéutico , Ivermectina/uso terapéutico , Microfilarias/efectos de los fármacos , Onchocerca volvulus/efectos de los fármacos , Oncocercosis/tratamiento farmacológico , Adulto , Animales , Estudios de Cohortes , Ojo/efectos de los fármacos , Ojo/parasitología , Femenino , Ghana/epidemiología , Humanos , Masculino , Administración Masiva de Medicamentos , Persona de Mediana Edad , Onchocerca volvulus/crecimiento & desarrollo , Onchocerca volvulus/parasitología , Oncocercosis/epidemiología , Oncocercosis/parasitología , Oncocercosis/patología , Piel/efectos de los fármacos , Piel/parasitologíaRESUMEN
Control and treatment of onchocerciasis, a devastating tropical filarial disease caused by Onchocerca volvulus, rely solely on the community directed treatment with ivermectin. However, ivermectin is only microfilaricidal with evidence of resistance of the parasite among other limitations, which necessitate the search for new efficacious and safe filaricides. Ten synthetic thienylazoryl dyes were screened in vitro against adult and microfilariae worm stages of Onchocerca ochengi based on worm motility and MTT formazan assay. Cytotoxicity of active compounds was assessed on monkey kidney epithelial cells (LLC-MK2) using the MTT formazan assay. Seven (7) compounds showed both macrofilaricidal activity against adult male worms and microfilaricidal activity among which three 4a, 4c and 4e recorded the highest activity (IC50 = 4.2 to 8.8µM) against adult male worms, comparable to some standard anthelmintics. Five compounds showed rapid activity against microfilariae with 100% inhibition after 24-h incubation. The active compounds were nontoxic on monkey kidney cells (CC50> 4µg/mL), but their selectivity index values were relatively low (≤ 3). The thienylazoaryls with both macrofilaricidal and microfilaricidal activities may yield molecules which could be used for eradication of onchocerciasis following further medicinal chemistry modification of their structures to enhance their selectivity.
Asunto(s)
Compuestos Azo/farmacología , Filaricidas/farmacología , Microfilarias/efectos de los fármacos , Onchocerca/efectos de los fármacos , Tiofenos/farmacología , Animales , Compuestos Azo/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Concentración 50 Inhibidora , Masculino , Tiofenos/químicaRESUMEN
BACKGROUND: Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are two human neglected tropical diseases that cause major disabilities. Mass administration of drugs targeting the microfilarial stage has reduced transmission and eliminated these diseases in several countries but a macrofilaricidal drug that kills or sterilizes the adult worms is critically needed to eradicate the diseases. The causative agents of onchocerciasis and lymphatic filariasis are filarial worms that harbor the endosymbiotic bacterium Wolbachia. Because filarial worms depend on Wolbachia for reproduction and survival, drugs targeting Wolbachia hold great promise as a means to eliminate these diseases. METHODS: To better understand the relationship between Wolbachia and its worm host, adult Brugia pahangi were exposed to varying concentrations of doxycycline, minocycline, tetracycline and rifampicin in vitro and assessed for Wolbachia numbers and worm motility. Worm motility was monitored using the Worminator system, and Wolbachia titers were assessed by qPCR of the single copy gene wsp from Wolbachia and gst from Brugia to calculate IC50s and in time course experiments. Confocal microscopy was also used to quantify Wolbachia located at the distal tip region of worm ovaries to assess the effects of antibiotic treatment in this region of the worm where Wolbachia are transmitted vertically to the microfilarial stage. RESULTS: Worms treated with higher concentrations of antibiotics had higher Wolbachia titers, i.e. as antibiotic concentrations increased there was a corresponding increase in Wolbachia titers. As the concentration of antibiotic increased, worms stopped moving and never recovered despite maintaining Wolbachia titers comparable to controls. Thus, worms were rendered moribund by the higher concentrations of antibiotics but Wolbachia persisted suggesting that these antibiotics may act directly on the worms at high concentration. Surprisingly, in contrast to these results, antibiotics given at low concentrations reduced Wolbachia titers. CONCLUSION: Wolbachia in B. pahangi display a counterintuitive dose response known as the "Eagle effect." This effect in Wolbachia suggests a common underlying mechanism that allows diverse bacterial and fungal species to persist despite exposure to high concentrations of antimicrobial compounds. To our knowledge this is the first report of this phenomenon occurring in an intracellular endosymbiont, Wolbachia, in its filarial host.
Asunto(s)
Brugia Malayi/fisiología , Microfilarias/microbiología , Onchocerca/fisiología , Simbiosis , Wolbachia/fisiología , Animales , Antibacterianos/farmacología , Brugia Malayi/efectos de los fármacos , Brugia Malayi/microbiología , Doxiciclina/farmacología , Femenino , Masculino , Microfilarias/efectos de los fármacos , Microfilarias/fisiología , Onchocerca/efectos de los fármacos , Onchocerca/microbiología , Simbiosis/efectos de los fármacos , Wolbachia/efectos de los fármacosRESUMEN
Inhibition of an imperative antioxidant enzyme with subsequent death is a victorious and widely accepted strategy to combat various infectious diseases. Among different antioxidant enzymes, thioredoxin reductase (TrxR) is an exclusive one. Studies have revealed that direct inhibition of TrxR by different classes of chemical moieties promptly results in the death of an organism. Especially the structural as well as biochemical modifications of the enzyme upon inhibition project serious threat towards the subject organism. Herein, an attempt was made to inhibit TrxR of filarial species by administering Auranofin, 1 chloro 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo dithioperoxo(thioate) derivative (4a). Our study has revealed that inhibition of TrxR resulted in the induction of the classical CED pathway of apoptosis along with the intrinsic and extrinsic pathways of apoptosis (Caspase mediated) routed through the ASK-1/p38 axis. Druggability analysis of filarial TrxR for the selected compounds was performed in silico through molecular docking studies. Therefore, this study attempts to decipher the mechanism of apoptosis induction following TrxR inhibition. The safety of those four compounds in terms of dose and toxicity was taken under consideration. Thitherto, the mechanism of TrxR mediated initiation of cell death in filarial parasite has remained undercover, and therefore, it is a maiden report on the characterization of apoptosis induction upon TrxR inhibition which will eventually help in generating effective antifilarial drugs in the future.
Asunto(s)
Antihelmínticos/farmacología , Auranofina/farmacología , Caspasas/genética , Curcumina/farmacología , Dinitroclorobenceno/farmacología , Setaria (Nematodo)/efectos de los fármacos , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Animales , Antihelmínticos/química , Apoptosis/efectos de los fármacos , Apoptosis/genética , Auranofina/química , Sitios de Unión , Caspasas/metabolismo , Bovinos , Curcumina/química , Dinitroclorobenceno/química , Regulación de la Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Microfilarias/efectos de los fármacos , Microfilarias/enzimología , Microfilarias/crecimiento & desarrollo , Modelos Moleculares , Estrés Oxidativo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismo , Setaria (Nematodo)/enzimología , Setaria (Nematodo)/crecimiento & desarrollo , Transducción de Señal , Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Current measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation. METHODS: Dirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR). RESULTS: In vitro conditions that best promote early development of D. immitis microfilariae to the "late sausage stage" have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro. CONCLUSIONS: The arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae.
Asunto(s)
Dirofilaria immitis/efectos de los fármacos , Ecdisona/farmacología , Microfilarias/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Transducción de Señal/efectos de los fármacos , Animales , Anopheles/efectos de los fármacos , Dirofilaria immitis/genética , Dirofilariasis/tratamiento farmacológico , Enfermedades de los Perros/tratamiento farmacológico , Perros , Ecdisterona/farmacología , Larva/efectos de los fármacos , Microfilarias/fisiologíaRESUMEN
Lymphatic filariasis (LF) is a disease caused by parasitic filarial nematodes that is endemic in 49 countries of the world and affects or threatens over 890 million people. Strategies to control LF rely heavily on mass administration of anthelmintic drugs including ivermectin (IVM), a macrocyclic lactone drug considered an Essential Medicine by the WHO. However, despite its widespread use the therapeutic mode of action of IVM against filarial nematodes is not clear. We have previously reported that filarial nematodes secrete extracellular vesicles (EVs) and that their cargo has immunomodulatory properties. Here we investigate the effects of IVM and other anti-filarial drugs on parasitic nematode EV secretion, motility, and protein secretion. We show that inhibition of EV secretion was a specific property of IVM, which had consistent and significant inhibitory effects across nematode life stages and species, with the exception of male parasites. IVM inhibited EV secretion, but not parasite motility, at therapeutically relevant concentrations. Protein secretion was inhibited by IVM in the microfilariae stage, but not in any other stage tested. Our data provides evidence that inhibiting the secretion of immunomodulatory EVs by parasitic nematodes could explain, at least in part, IVM mode of action and provides a phenotype for novel drug discovery.
Asunto(s)
Brugia Malayi/efectos de los fármacos , Filariasis Linfática/tratamiento farmacológico , Vesículas Extracelulares/metabolismo , Tracto Gastrointestinal/efectos de los fármacos , Proteínas del Helminto/metabolismo , Ivermectina/farmacología , Microfilarias/efectos de los fármacos , Animales , Antiparasitarios/farmacología , Brugia Malayi/fisiología , Filariasis Linfática/metabolismo , Filariasis Linfática/parasitología , Vesículas Extracelulares/efectos de los fármacos , Femenino , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/parasitología , Locomoción , Masculino , Microfilarias/parasitología , FagocitosisRESUMEN
Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816-831 µm as compared to 767 µm of 2-D cultured larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite's biology.
Asunto(s)
Antiparasitarios/uso terapéutico , Órganos Bioartificiales/parasitología , Desarrollo de Medicamentos/métodos , Onchocerca volvulus/crecimiento & desarrollo , Oncocercosis Ocular/tratamiento farmacológico , Piel/parasitología , África , Animales , Humanos , Ivermectina/uso terapéutico , Microfilarias/efectos de los fármacos , Onchocerca volvulus/efectos de los fármacos , Oncocercosis Ocular/patología , Técnicas de Cultivo de Órganos , Prueba de Estudio ConceptualRESUMEN
BACKGROUND: Dirofilaria immitis is a life-threatening nematode spreading globally. Arsenical treatment is currently recommended for removal of adult worms. However, arsenical treatment is not available in some countries, and there are dogs that cannot tolerate the rapid kill of adult worms; therefore, alternative adulticide slow-kill treatments are needed. Criticisms against the use of these alternative protocols include the potential for allowing disease to progress and for the development of ML-resistant worms. METHODS: The efficacy of a protocol that includes semi-annual doses (i.e. every 6 months) of commercially available extended-release injectable moxidectin suspension (ProHeart® SR-12) with 30-day oral administration of doxycycline was studied in 20 dogs with naturally occurring D. immitis infections. Each dog received treatment with ProHeart® SR-12 (0.5 mg moxidectin/kg) by subcutaneous injection and oral doxycycline (10 mg/kg/bid × 30 days) every 6 months until two consecutive negative antigen test results were obtained. Pulmonary and cardiac evaluations were performed by radiographic and echocardiographic parameters. Physical examinations, complete blood counts, clinical chemistry profiles, microfilariae and antigen tests were performed periodically. RESULTS: At enrollment, all dogs were positive for D. immitis antigen and 18 were microfilaremic. On day 30, microfilaremia counts decreased, and all dogs became amicrofilaremic by day 150. On day 180, 11 dogs were antigen-negative, and 7 more became negative by day 360. The two remaining antigen-positive dogs converted to negative by day 540 or 810. All antigen tests performed 180 days after the first negative test were negative. There was no decline in cardiac performance of the dogs throughout the study. Overall, pulmonary clinical conditions, presence of worms by echocardiography, and enlargement of caudal and main pulmonary arteries improved after treatment. Physical examinations, complete blood count results, and clinical chemistry profiles were within normal reference values. Respiratory conditions were improved, no damage to the heart was observed, and the treatment protocol was well tolerated by the animals. CONCLUSIONS: This alternative adulticide treatment was efficacious and well tolerated in naturally infected dogs. The injectable formulation provides the advantage of having veterinarians able to administer, monitor, and assess the efficacy and condition of the dog throughout the treatment and post-treatment periods.
Asunto(s)
Dirofilaria immitis/efectos de los fármacos , Dirofilariasis/tratamiento farmacológico , Enfermedades de los Perros/tratamiento farmacológico , Filaricidas , Administración Oral , Animales , Antihelmínticos/administración & dosificación , Antihelmínticos/farmacología , Antígenos Helmínticos/sangre , Perros , Doxiciclina/administración & dosificación , Doxiciclina/farmacología , Quimioterapia Combinada/veterinaria , Filaricidas/administración & dosificación , Filaricidas/farmacología , Inyecciones Subcutáneas/veterinaria , Macrólidos/administración & dosificación , Macrólidos/farmacología , Microfilarias/efectos de los fármacosRESUMEN
A major impediment to eliminate lymphatic filariasis and onchocerciasis is the lack of effective short-course macrofilaricidal drugs or regimens that are proven to be safe for both infections. In this study we tested oxfendazole, an anthelmintic shown to be well tolerated in phase 1 clinical trials. In vitro, oxfendazole exhibited modest to marginal motility inhibition of adult worms of Onchocerca gutturosa, pre-adult worms of Onchocerca volvulus and Onchocerca lienalis microfilariae. In vivo, five days of oral treatments provided sterile cure with up to 100% macrofilaricidal efficacy in the murine Litomosoides sigmodontis model of filariasis. In addition, 10 days of oral treatments with oxfendazole inhibited filarial embryogenesis in patent L. sigmodontis-infected jirds and subsequently led to a protracted but complete clearance of microfilaremia. The macrofilaricidal effect observed in vivo was selective, as treatment with oxfendazole of microfilariae-injected naïve mice was ineffective. Based on pharmacokinetic analysis, the driver of efficacy is the maintenance of a minimal efficacious concentration of approximately 100 ng/ml (based on subcutaneous treatment at 25 mg/kg in mice). From animal models, the human efficacious dose is predicted to range from 1.5 to 4.1 mg/kg. Such a dose has already been proven to be safe in phase 1 clinical trials. Oxfendazole therefore has potential to be efficacious for treatment of human filariasis without causing adverse reactions due to drug-induced microfilariae killing.
Asunto(s)
Bencimidazoles/farmacología , Filariasis Linfática/tratamiento farmacológico , Filarioidea/efectos de los fármacos , Animales , Antihelmínticos/uso terapéutico , Modelos Animales de Enfermedad , Filariasis Linfática/parasitología , Femenino , Filarioidea/embriología , Gerbillinae/parasitología , Ratones , Ratones Endogámicos BALB C , Microfilarias/efectos de los fármacos , Onchocerca/efectos de los fármacos , Onchocerca volvulus/efectos de los fármacos , Oncocercosis/tratamiento farmacológicoRESUMEN
Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
Asunto(s)
Dirofilaria immitis/genética , Resistencia a Medicamentos/genética , Filaricidas/farmacología , Lactonas/farmacología , Repeticiones de Microsatélite , Animales , Dirofilaria immitis/efectos de los fármacos , Dirofilaria immitis/crecimiento & desarrollo , Marcadores Genéticos , Geografía , Compuestos Macrocíclicos/farmacología , Microfilarias/efectos de los fármacos , Microfilarias/genética , Microfilarias/crecimiento & desarrollo , Estados UnidosRESUMEN
The availability of a safe macrofilaricidal drug would help to accelerate onchocerciasis elimination. A trial was conducted in Cameroon to evaluate the effects of a subcutaneous injectable long-acting formulation of ivermectin (LAFI) on the microfilariae (mf) and adult stages of Onchocerca ochengi. Ten zebu cattle naturally infected with the parasite were injected subcutaneously with either 500 mg (group A, N = 4), or 1000 mg long-acting ivermectin (group B, N = 4) or the vehicle (group C, N = 2). Skin samples were collected from each animal before, and 6, 12, and 24 months after treatment to measure microfilarial densities (MFDs). Nodules excised before, and 6 and 12 months after treatment were examined histologically to assess the adult worms' viability and reproductive status. Blood samples were collected at pre-determined time-points to obtain pharmacokinetic data. Before treatment, the average O. ochengi MFDs were similar in the three groups. Six months after treatment, all animals in groups A and B were free of skin mf, whereas those in group C still showed high MFDs (mean = 324.5 mf/g). Only one ivermectin-treated animal (belonging to group A) had skin mf 12 months after treatment (0.9 mf/g). At 24 months, another animal in group A showed skin mf (10.0 mf/g). The histologic examination of nodules at 6 and 12 months showed that LAFI was not macrofilaricidal but had a strong effect on embryogenesis. The new LAFI regimen might be an additional tool to accelerate the elimination of human onchocerciasis in specific settings.
TITLE: Effets d'une formulation injectable d'ivermectine à activité prolongée sur Onchocerca ochengi chez les bovins zébu. ABSTRACT: La disponibilité d'un médicament macrofilaricide et sans danger permettrait d'accélérer l'élimination de l'onchocercose. Un essai a été mené au Cameroun pour évaluer les effets d'une formulation injectable en sous-cutané d'ivermectine à activité prolongée (FIAP) sur les microfilaires (mf) et les stades adultes d'Onchocerca ochengi. Dix vaches zébu infectées naturellement par le parasite ont reçu une injection sous-cutanée de 500 mg (groupe A, N = 4) ou de 1000 mg d'ivermectine à activité prolongée (groupe B, N = 4) ou le véhicule (groupe C, N = 2). Des échantillons de peau ont été collectés de chaque animal avant, puis 6, 12 et 24 mois après traitement pour mesurer les densités microfilariennes (DMF). Des nodules prélevés avant et 6 et 12 mois après traitement ont été examinés histologiquement pour évaluer la viabilité et le statut reproductif des vers adultes. Des échantillons de sang ont été prélevés pour obtenir des données de pharmacocinétique. Avant traitement, les DMF à O. ochengi étaient similaires dans les 3 groupes. Six mois après traitement, aucun des animaux des groupes A et B ne présentait de mf dermiques, alors que ceux du groupe C présentaient encore des DMF élevées (moyenne : 324,5 mf/g). Parmi les animaux traités par ivermectine, un seul (du groupe A) avait des mf dermiques 12 mois après traitement (0,9 mf/g). A 24 mois, un autre animal du groupe A avait des mf (10,0 mf/g). L'examen histologique des nodules collectés à 6 et 12 mois montrait que la FIAP n'était pas macrofilaricide mais avait un effet marqué sur l'embryogénèse. La nouvelle FIAP pourrait représenter un outil pour accélérer l'élimination de l'onchocercose dans certaines circonstances spécifiques.
Asunto(s)
Antiparasitarios/uso terapéutico , Enfermedades de los Bovinos/tratamiento farmacológico , Ivermectina/uso terapéutico , Onchocerca/efectos de los fármacos , Oncocercosis/veterinaria , Animales , Camerún , Bovinos/parasitología , Enfermedades de los Bovinos/parasitología , Preparaciones de Acción Retardada/uso terapéutico , Femenino , Inyecciones , Microfilarias/efectos de los fármacos , Oncocercosis/tratamiento farmacológico , Piel/parasitología , Resultado del TratamientoRESUMEN
The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16-18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10-14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days.
Asunto(s)
Antibacterianos/uso terapéutico , Filariasis/tratamiento farmacológico , Oncocercosis/tratamiento farmacológico , Quinazolinas/uso terapéutico , Wolbachia/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Brugia pahangi/efectos de los fármacos , Femenino , Filariasis/microbiología , Filarioidea/efectos de los fármacos , Gerbillinae/microbiología , Gerbillinae/parasitología , Microfilarias/efectos de los fármacos , Quinazolinas/administración & dosificación , Simbiosis/efectos de los fármacosRESUMEN
A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.
Asunto(s)
Brugia Malayi/efectos de los fármacos , Filariasis Linfática/tratamiento farmacológico , Filaricidas/farmacología , Piperidinas/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Animales , Chalcona/farmacología , Replicación del ADN/efectos de los fármacos , Filariasis Linfática/parasitología , Antagonistas del Ácido Fólico/farmacología , Humanos , Microfilarias/efectos de los fármacos , Simulación del Acoplamiento Molecular , Tetrahidrofolato Deshidrogenasa/efectos de los fármacos , Sales de Tetrazolio , Timidina/farmacologíaRESUMEN
BACKGROUND: Mansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth. METHODS: The ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility. RESULTS: FBS supplement and additional LLC-MK2 cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK2 cells sustained the maintenance of mf for at least 20 days (100.00⯱â¯0.00% survival). In co-cultures with LLC-MK2 cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate. CONCLUSION: Both RPMI and DMEM in the presence of LLC-MK2 cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans mf was documented for the first time in this study and can therefore be considered as reference for further screening of agents against this parasite stage.
Asunto(s)
Artesunato/farmacología , Filaricidas/farmacología , Mansonella/efectos de los fármacos , Mansonella/crecimiento & desarrollo , Mefloquina/farmacología , Amodiaquina/farmacología , Animales , Antimaláricos/farmacología , Antinematodos/farmacología , Área Bajo la Curva , Bovinos , Línea Celular , Medios de Cultivo/química , Haplorrinos , Ivermectina/farmacología , Mansonella/fisiología , Microfilarias/efectos de los fármacos , Microfilarias/crecimiento & desarrollo , Microfilarias/fisiología , Movimiento/efectos de los fármacos , Rifampin/farmacologíaRESUMEN
Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans.
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Brugia Malayi/efectos de los fármacos , Brugia Malayi/enzimología , Glucuronosiltransferasa/metabolismo , Albendazol/farmacología , Animales , Antígenos Helmínticos/sangre , Brugia Malayi/inmunología , Brugia Malayi/metabolismo , Quimioterapia Combinada , Filariasis Linfática/tratamiento farmacológico , Filariasis Linfática/prevención & control , Femenino , Filaricidas/farmacología , Glucuronosiltransferasa/antagonistas & inhibidores , Glucuronosiltransferasa/genética , Humanos , Inmunoglobulina E/sangre , Intestinos/enzimología , Microfilarias/efectos de los fármacos , Movimiento , Probenecid/farmacología , ARN Interferente Pequeño , Sulfinpirazona/farmacologíaRESUMEN
Depletion of Wolbachia endosymbionts of human pathogenic filariae using 4-6 weeks of doxycycline treatment can lead to permanent sterilization and adult filarial death. We investigated the anti-Wolbachia drug candidate ABBV-4083 in the Litomosoides sigmodontis rodent model to determine Wolbachia depletion kinetics with different regimens. Wolbachia reduction occurred in mice as early as 3 days after the initiation of ABBV-4083 treatment and continued throughout a 10-day treatment period. Importantly, Wolbachia levels continued to decline after a 5-day-treatment from 91.5% to 99.9% during a 3-week washout period. In jirds, two weeks of ABBV-4083 treatment (100mg/kg once-per-day) caused a >99.9% Wolbachia depletion in female adult worms, and the kinetics of Wolbachia depletion were recapitulated in peripheral blood microfilariae. Similar to Wolbachia depletion, inhibition of embryogenesis was time-dependent in ABBV-4083-treated jirds, leading to a complete lack of late embryonic stages (stretched microfilariae) and lack of peripheral microfilariae in 5/6 ABBV-4083-treated jirds by 14 weeks after treatment. Twice daily treatment in comparison to once daily treatment with ABBV-4083 did not significantly improve Wolbachia depletion. Moreover, up to 4 nonconsecutive daily treatments within a 14-dose regimen did not significantly erode Wolbachia depletion. Within the limitations of an animal model that does not fully recapitulate human filarial disease, our studies suggest that Wolbachia depletion should be assessed clinically no earlier than 3-4 weeks after the end of treatment, and that Wolbachia depletion in microfilariae may be a viable surrogate marker for the depletion within adult worms. Furthermore, strict daily adherence to the dosing regimen with anti-Wolbachia candidates may not be required, provided that the full regimen is subsequently completed.
Asunto(s)
Antibacterianos/farmacología , Filarioidea/microbiología , Microfilarias/microbiología , Wolbachia/efectos de los fármacos , Wolbachia/fisiología , Animales , Doxiciclina/farmacología , Femenino , Filariasis , Filarioidea/efectos de los fármacos , Gerbillinae , Cinética , Ratones , Ratones Endogámicos BALB C , Microfilarias/efectos de los fármacos , Microfilarias/embriología , Modelos AnimalesRESUMEN
The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10â¯nM), but much higher concentrations of ivermectin (>100â¯nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10â¯nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.