An allele specific PCR assay for screening for drug resistance among Wuchereria bancrofti populations in India.

Background & objectives: Albendazole, a commonly used anthelminthic drug that targets the polymerization of α- and β-tubulin dimer is currently co-administered with the antifilarial drug, diethylcarbamazine citrate (DEC) in the ongoing Global Programme for Elimination of Lymphatic Filariasis (GPELF). The experience in veterinary field has shown that there can be a rapid development of resistance to this drug, which therefore, needs to be monitored regularly in GPELF. Hence, we investigated the nucleotide polymorphism in the albendazole-binding domain of the isotype 1 β-tubulin gene from several populations of Wuchereria bancrofti and developed an AS-PCR assay useful in screening for sensitive/resistance alleles among parasite populations and also evaluated its utility. Methods: For studying the polymorphism of isotype 1 β-tubulin gene, a 475 bp fragment spanning exon 5 and 6 of the gene was amplified and sequenced from the genomic DNA of W. bancrofti collected from six geographic regions of India. An allele specific (AS) PCR for screening albendazole sensitivity/resistance was developed and a total of 55 mf samples from blood smears on slides collected from Thiruvannamalai, Thanjavur and Puducherry were screened. Selective therapy with DEC was in place in three areas, mass drug administration (MDA) with DEC alone was implemented in four areas, while DEC plus albendazole was administered in one district. Results: The analysis of the nucleotide sequence of the fragment from 20 W. bancrofti populations showed the domain to be highly conserved. An allele-specific PCR assay developed was used to detect sensitive/ resistance alleles among 55 isolates of W. bancrofti and no albendazole resistance alleles were detected among the populations tested. Interpretation & conclusion: The drug-binding domain of isotype 1 β-tubulin gene of W. bancrofti from different geographical locations was highly conserved. The AS-PCR developed showed potential application as a tool for monitoring albendazole sensitivity/resistance alleles among W. bancrofti populations, in areas where combination therapy of DEC-albendazole is being mass administered in the LF elimination programme.

Anti-microfilarial activity of methanolic extract of Vitex negundo and Aegle marmelos and their phytochemical analysis.

In the present study, methanolic extracts of roots of Vitex negundo L. and extracts of leaves of Vitex negundo L., Ricinus communis L. and Aegle marmelos Corr. were explored for possible antifilarial effect against Brugia malayi microfilariae. It was observed that among the herbal extracts, root extract of Vitex negundo L. and leaves extract of Aegle marmelos Corr. at 100 ng/ml concentration showed complete loss of motility of microfilariae after 48 hr of incubation. Thin layer chromatography of the extracts revealed the presence of alkaloids, saponin and flavonoids in the roots of Vitex negundo L. and coumarin in the leaves of Aegle marmelos Corr.

Caracterização do efeito da dietilcarbazina (DEC) sobre a espermatogênese de camundongos / Characterization of the effect of the diethylcarbazine (DEC) on spermatogenesis of mice

A dietilcarbamazina tem sido altamente efetiva contra filariose linfática, mas seu efeito sobre células de vertebrados permanece controverso. Apesar de ter sido descrita sua ação sobre microtúbulos, a hipótese de que esta droga atue sobre a espermatogênese não havia sido testada. Após o tratamento de camundongos Swiss com 200mg de DEC/kg durante doze dias, as análises ultraestruturais mostraram vacuolização das células de Sertoli e espermatogônias com características morfológicas de apoptose, como condensação do citoplasma e aumento da densidade cromossomal. Algumas espermátides apresentaram mitocôndrias vacuoladas, as quais estavam desorganizadas em relação ao eixo microtubular do flagelo. Além disso, as células de Leydig mostraram numerosas gotículas de lipídio distribuídas no citoplasma, corpos multivesiculares e círculos concêntricos gigantes de retículo endoplasmático liso, alguns dos quais englobando gotículas de lipídio. Algumas células de Leydig não apresentaram núcleo indicando um processo de esteatose. Baixas dosagens de DEC produziram alterações similares nas células de Leydig, entretanto seus efeitos foram diretamente proporcionais à concentração da droga. Os níveis de testosterona no soro foram significativamente menores no grupo tratado com 200mg de DEC/kg quando comparados com o controle. Entretanto, nenhuma mudança significante foi observada nas taxas de gravidez e no número de filhotes quando machos tratados com DEC acasalaram com fêmeas não tratadas, nas doses estudadas. Os resultados do presente estudo indicam que a DEC apresenta um importante efeito sobre a morfologia testicular e esteroidogênese de camundongos provavelmente por afetar tubulina e/ou complexo de microtúbulos. Porém, os presentes dados não excluem a possibilidade desta droga atuar diretamente sobre as vias enzimáticas hormonais.

Macrofilaricidal effects of chloroquine on adult Onchocerca volvulus by local infiltration of palpable onchocercal nodules

O efeito macrofilaricida da infiltração local, com uma alta concentração de cloroquina, dentro da cápsula do nódulo oncocercótico sobre o verme adulto de Onchocerca volvulus foi determinado. Seis semanas depois da infiltração, estudos histopatológicos de nódulos simple demonstraram todos os vermes adultos mortos. Em conglomerados de nódulos a ação da cloroquina foi só sobre os vermes adultos do nódulo infiltrado, não acontecendo a difusão da droga aos nódulos adjacentes. A infiltração de cloroquina a nódulos novos ou de recente formação reduz a carga de vermes adultos dos indivíduos parasitados e pode ser uma alternativa para os altos custos das nodulectomias.

The macrofilaricidal effects of local infiltration of high concentrations of chloroquine into the capsule of onchocercal nodules on adult worms of Onchocerca volvulus was determined. Six weeks post infiltration, histological examination of single nodules showed all adult worms to be dead. With nodule conglomerates, there was localized action of chloroquine only on the adult worms in the infiltrated nodule, with no diffusion of the drug to adjacent nodules. Chloroquine infiltration of young, recently formed nodules to reduce the adult worm load of infected individuals may be an alternative method to costly nodulectomy.

Dietilcarbamazina no tratamento da filariose bancroftiana / Diethylcarbamazine in the treatment of Bancroft's filariasis

Os autores realizaram uma ampla revisão sobre o tratamento da filariose bancroftiana com a droga dietilcarbamazina. Os aspectos interessantes sobre o histórico de sua descoberta e os conceitos básicos de sua farmacologia foram relatados de forma resumida. Ênfase especial, por outro lado, foi dada às especulações feitas pelos diversos autores sobre os achados intrigantes descritos na literatura. Foram trazidos os novos avanços sobre o conhecimento da doença, como por exemplo, a visualização pela ultra-sonografia do verme vivo de Wuchereria bancrofti, no seu hospedeiro natural, o homem. Isso possibilitou a compreensão de muitos dos achados aparentemente paradoxais encontrados na literatura sobre o tratamento da infeção com a DEC. Assim, devido à inexistência de uma droga sucessora que reunisse efeitos micro e macrofilaricidas ideais e aos novos conhecimentos sobre a bancroftose e sobre a própria dietilcarbamazina, foi-lhe conferido um novo realce. Esses aspectos a colocaram numa posição de destaque no cenário da infecção, à época do seu quase cinqüentenário de existência.

The authors presented a detailed review about the treatment of bancroftian filariasis with diethylcarbamazine. The interesting aspects about the drug discovery and the basic concepts about its pharmacology were reported in a summarised form. On the other hand, emphasis was made about the speculation done by several authors about the intriguing findings regarding its efficacy reported in the literature. Latter, it was brought the new advances about the disease, as for example, the visualization by ultrasound of living Wuchereria bancrofti adult worm on its natural host--the human being. This made possible the comprehension of several paradoxical issues reported, focusing the treatment of infection using diethylcarbamazine. So far, because of the lack of ideal drug with micro and macrofilaricidal properties, together with the new understand about the disease and the new parameters for monitoring the efficacy of the drug, diethylcarbamazine has back its importance conquered at the begin of its discovery, almost fifth years ago.

In vitro antifilarial activity of Sencio nudicaulis Buch. Ham. Effect on Setaria cervi (Nematoda Filarioidea).

The effect on aqueous and alcoholic extracts of the leaves of Sencio nudicaulis Buch. Ham. was studied on the spontaneous movements of the whole worm and nerve-muscle preparation of Setaria cervi and on the survival of micro-filariae in vitro. Aqueous as well as alcoholic extracts caused inhibition of spontaneous motility of the whole worm and nerve-muscle preparation of S. cervi characterized by decreased amplitude, rate and tone of contractions. The concentration required to inhibit the movements of n.m. preparation was 1/3rd for aqueous and 1/20th for alcoholic extract suggesting a cuticular permeability barrier. The effect of S nudicaulis extracts was different than that produced by calcium channel blocker nifedipine on the whole worm and n.m. preparation. While nifedipine blocks the stimulant effect of Ach the extracts of S. nudicaulis fails to do so. While the response bears similarity with DEC which also does not block Ach response. Both aqueous and alcoholic extracts exhibited microfilaricidal action in vitro LC50 and LC90 being 10 and 15 ng/ml for aqueous extract, 5 and 12 ng/ml for alcoholic extract.

Antifilarial effect of two triterpenoid saponins isolated from Acacia auriculiformis.

The active principle, isolated from the funicles of A. auriculiformis, consisted of two triterpenoid saponins, acaciaside A and acaciaside B which killed in vitro 97% microfilaria of Setaria cervi in 100 min at 4mg/ml concentration and 100% of adults in 35 min. The drug, when administered orally at 100 mg/kg on rats, in which S. cervi adults were implanted intra-peritoneally, increased the blood mf count by 1.5-fold after the first phase of treatment for 10 days. Following the third phase of treatment and thereafter, the mf density was reduced by more than 80%. No toxic effect of the saponins was observed in rats. The rise in mf count indicated that the drug induced a very high physiological stress on the adult worms which increased the rate of discharge of the mf before impending death. The treated rats on autopsy did not show any adult worms.

Hydrophobicity and antifilarial activities of 4-aminoquinolines.

The apparent partition coefficients (Papp.) of eight 4-aminoquinolines in 1-octanol/pH 7.4 buffered solutions have been determined and correlated with their reported antifilarial activities. Antifilarial activity appears to be present only in those 4-aminoquinolines which have log Papp. values falling within a narrow range of 2.8 to 3.2.