Aporte de la técnica de PCR en el diagnóstico de Mansonella ozzardi en zonas endémicas de la Argentina / Contribution of the PCR assay to the diagnosis of Mansonella ozzardi in endemic areas of Argentina

Mansonella ozzardi es un nematode parásito tisular, agente etiológico de mansonellosis en casi la totalidad de los países latinoamericanos. En Argentina la mansonellosis ha sido descrita a lo largo de la región de las yungas. Su diagnóstico microscópico puede dar resultados falsos negativos en microfilaremias bajas. El objetivo del presente estudio fue optimizar su diagnóstico molecular y comparar los resultados con los obtenidos mediante las pruebas microscópicas de Knott, de gota gruesa y de extendido hemático fino, en 92 muestras de sangre de pacientes de zona endémica. La técnica de PCR seguida de la secuenciación del producto amplificado presentó una sensibilidad del 100 % frente al método de Knott, considerado como referencia, e incluso permitió identificar 7 casos más de la parasitosis.

Mansonella ozzardi is a tissue-dwelling parasitic nematode, the causative agent of mansonelliasis in almost all Latin American countries. It has been described along the Argentine Yungas region. The microscopic diagnosis can yield false-negative test results at low microfilaremia levels. The aim of this study was to optimize the molecular diagnostic technique and compare it with the Knott's method and standard blood smear procedures (thin blood films and thick smears) in 92 blood samples of individuals from an endemic area. The PCR technique followed by the sequencing of the amplified product yielded 100 % sensitivity compared to the Knott's test, which is considered a reference method. Seven more cases of this parasitosis could only be identified with the molecular technique.

Effect of a single dose of diethylcarbamazine, albendazole or both on the clearance of Wuchereria bancrofti microfilariae and antigenaemia among microfilaria carriers: A randomized trial.

Background. Lymphatic filariasis is a major vector-borne parasitic disease. The global programme to eliminate lymphatic filariasis was launched in 1997 and currently over 570 million people are covered under it in 48 countries. Mass annual single-dose drug administration of diethylcarbamazine (DEC), co-administrated with albendazole for 5–6 years and mass distribution of diethylcarbamazine-fortified salt are the two strategies for elimination of filariasis. Methods. Asymptomatic volunteers residing in Puducherry, India were screened for microfilaria (mf) by examining nocturnal thick blood smears. Those testing positive were randomly assigned to receive a single dose of DEC (6 mg/kg body weight) or albendazole 400 mg or both. Participants were hospitalized for 5 days. Membrane filtration count was used to assess microfilaraemia and ELISA (Og4C3) assay to measure circulating filarial antigens (CFA). Measurements were done before treatment and at 1, 2 and 3 years post-treatment. Viability of the adult worms was assessed by looking for the filarial dance sign (FDS) using ultrasound examination of the scrotum in men with hydrocele. Results. Fifty-four microfilaraemic individuals were studied. The mf prevalence started decreasing only by day 180 posttreatment in the DEC group but much earlier in the other two groups (day 30 in the albendazole and day 90 in the DEC with albendazole group). The decrease in mf was marginal (17.6%, 26.3% and 27.8%, respectively) by the end of year 1 posttreatment, but significant (96.7%, 78.6% and 93.3%, respectively) by the end of year 2 post-treatment (p<0.05). By the end of year 3, the level decreased to 80% in the DEC, 90% in the albendazole and to 100% in the DEC and albendazole groups. However, the mf intensity decreased © The National Medical Journal of India 2010 Vector Control Research Centre, Department of Health Research (ICMR), Indira Nagar, Puducherry 605006, India S. L. HOTI, S. P. PANI, P. VANAMAIL, K. ATHISAYA MARY, L. K. DAS, P. K. DAS Correspondence to S. L. HOTI; slhoti@yahoo.com significantly (by 39%; p<0.05) by day 7 post-treatment in both the DEC and DEC with albendazole groups, but only by day 30 in the albendazole group. In all the drug groups, the prevalence as well as intensity of CFA returned to pretreatment levels by the end of year 3 post-treatment. Conclusion. Annual single-dose administration of all the 3 drug regimens significantly reduced antigenaemia levels. There were no significant differences in the efficacy and overall pattern of CFA clearance between the 3 drug regimens.

In vitro effect of four herbal plants on the motility of Brugia malayi microfilariae.

BACKGROUND & OBJECTIVE: Disease burden due to lymphatic filariasis is disproportionately high despite mass drug administration with conventional drugs. Usage of herbal drugs in traditional medicine is quite well known but largely empirical. Hence the present study was designed to screen the in vitro antifilarial effect of four herbal plants on Brugia malayi. METHODS: Motility of microfilariae of B. malayi after incubation for 48 h with aqueous/methanol extracts of Vitex negundo L. (roots), Butea monosperma L. (roots and leaves), Ricinus communis L. (leaves), and Aegle marmelos Corr. (leaves) was explored in the concentration range of 20 to 100 ng/ml for possible antifilarial effect by comparing with suitable solvent control. RESULTS: Butea monosperma leaves and roots, Vitex negundo root and Aegle marmelo leaves showed significant inhibition of motility of microfilariae as compared to controls whereas inhibitory activity demonstrated by Ricinus communis L. leaves was not significant. Antifilarial effects imparted by all these extracts were found to be a function of their relative concentrations. Inhibitory concentrations (IC(50)) for the plant extracts with significant antifilarial activity against Brugia malayi microfilariae in in vitro system have been derived to be 82, 83 and 70 ng/ml for Vitex negundo L., Butea monosperma L. and Aegle marmelos Corr. respectively. INTERPRETATION & CONCLUSION: The present study recorded significant antifilarial effect of all plant extracts studied except for Ricinus communis L. leaves and contributes to the development of database for novel drug candidates for human lymphatic filariasis.

Population dynamics of onchocerca volvulus microfilariae in human host after six years of drug control.

BACKGROUND & OBJECTIVES: Mass administration of ivermectin drug was carried out annually between 1995 and 2001 in three villages that were endemic for onchocerciasis in the Lower Cross River Basin, Nigeria. The aim of this study was to evaluate the population dynamics (dispersion patterns, distribution, prevalence and intensity) of Onchocerca volvulus microfilariae in their human host after six years of ivermectin treatment. METHODS: A total of 1014 subjects from three rural areas in Etung Local Government Area of Cross River State, Nigeria were screened for skin microfilariae using standard parasitological method of diagnosis. RESULTS: Ivermectin drug intervention had significantly reduced the prevalence of skin microfilariae (PMF) from 69.3% pre-control to 39.3% and community microfilarial load (CMFL) from 7.11 to 2.31 microfilariae per skin snip. Males (45%) were significantly (p <0.05) more infected than females (34%). Both microfilarial prevalence and intensity increased with age. Pearson correlation test between intensity and age was not significant (r = 0.37; p >0.05). The correlation between age-dependent parasite prevalence and mean abundance was also not significant (r = 0.42; p >0.05). The degree of dispersion as measured by variance to mean ratio (VMR), coefficient of variation (CV) and exponent 'K' of the negative binomial model of distribution showed that the parasite aggregated, clumped and overdispersed in their human host. The relative index of potential infection of each age group showed that adults between the age of 21 and 50 yr accounted for 52.7% of microfilariae positive cases. INTERPRETATION & CONCLUSION: Aggregated and overdispersion of O. volvulus observed in this study showed that active transmission could still be going on, because the tendency of the vector, Simulium damnosum ingesting more microfilariae was high due to the aggregated and overdispersed nature of the parasite with its host.

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.

Independent assessment of Mass Drug Administration in filariasis affected Surat city.

The Mass Drug Administration (MDA) done in Surat city (Gujarat) during 2005, revealed good impact on infection and infectivity in mosquitoes and also on microfilaria rate & mean infection density. The overall impact seen was 23% on mf rate, 28% on mean mf density, 65% on infection rate and 50% on infectivity rate in vectors. Indigenous population contribution to microfilaria cases was 9.7%, whereas migratory population contributed 72.2%; predominant 51.9% from Orissa and 20.3% from U.P. Of the total 3640 persons interviewed for MDA compliance in seven zones of the Surat city revealed that actual drug consumption was 76.7% (2792/3640). Another 11.9% although took the drug but did not consume and 11.4% refused. Important reasons for consuming was fear to get the disease (40.7%) and for not consuming; 'will consume after meal' (6.9%), too many tablets (1.7%), seek consent from doctor (1.5%), lack of awareness (1.4%) etc. Refusal was mainly due to the reason as respondents felt apparently healthy. Assessment of IEC activities suggested that main awareness was created by media (local or national TV, banners or handbills, local news papers or mike announcement) alongwith some impact made through NGO's. These observations clearly indicated the utility of effective health education for optimum community participation and shown that it was crucial for successful community based elimination campaign. However some gray areas also suggest the scope for further improvements.

Short-term effects of treatment with 300 mg oral-dose diethylcarbamazine on nocturnally periodic Wuchereria bancrofti microfilaremia and antigenemia.

Seven microfilaremic Myanmar patients were treated with a single 300 mg dose of diethylcarbamazine (DEC) orally, as part of a case-finding survey in Ranong Province, Southern Thailand. This was conducted in order to evaluate the short-term effects of single-dose DEC on Wuchereria bancrofti microfilaremia and antigenemia during a 12-week course of treatment. Analysis of microfilarial periodicity on initial treatment revealed the microfilarial peak density (k) was at 52 minutes after midnight (0052). The periodicity index was then 103.26%. Single-dose DEC treatment did not affect the k values. A linear model of W. bancrofti microfilarial density reduction predicts a sharp decrease in the mean microfilarial density 2 weeks after DEC intake (Z = -2.197, p = 0.028). Over a longer period, a non-linear model predicts an increase in the mean microfilarial density to pre-treatment levels, having little or no macrofilaricidal effects. We reconfirmed the existence of nocturnally periodic W. bancrofti infection in Myanmar migrants in Ranong Province, and the short-term microfilaricidal activity of 300 mg single-dose DEC treatment used for biannual mass treatment and the DEC provocative test. Without an adequate DEC treatment dose, recrudescence can occur. A rational approach to the management of introduced nocturnally periodic W. bancrofti in Myanmar migrants, who came for short periods of stay in transmission-prone areas, is needed.

Potential antifilarial activity of fruit extracts of Ficus racemosa Linn. against Setaria cervi in vitro.

Effect of alcoholic and aqueous extracts of the fruits of F. racemosa Linn., on the spontaneous movements of both the whole worm and nerve muscle preparation of Setaria cervi and on the survival of microfilariae in vitro was studied. Alcoholic as well as aqueous extracts caused inhibition of spontaneous motility of whole worm and nerve muscle preparation of Setaria cervi characterized by increase in amplitude and tone of contractions. Initial stimulatory effect was not observed with aqueous extract on whole worm preparation, while effect of alcoholic extract on whole worm and nerve muscle preparation was characterized by an increase in amplitude and tone of contractions followed by paralysis. The concentrations required to inhibit the movement of the whole worm and nerve muscle preparation for alcoholic extract of fruits of F. racemosa were 250 and 50 microg/ml, respectively, whereas aqueous extract caused inhibition of the whole worm and nerve muscle preparation at 350 and 150 microg/ml, respectively, suggesting a cuticular barrier. Both alcoholic and aqueous extracts caused death of microfilariae in vitro. LC50 and LC90 were 21 and 35 ng/ml, respectively for alcoholic, which were 27 and 42 ng/ml for aqueous extracts.

Tolerability and efficacy of single dose diethylcarbamazine (DEC) alone or co-administration with Ivermectin in the clearance of Wuchereria bancrofti microfilaraemia in Pondicherry, South India.

The tolerability and efficacy of single dose DEC (12mg/kg body weight) or co-administration of DEC (6mg/kg body weight) with Ivermectin (200 or 400 mcg/kg of body weight) was studied in 60 asymptomatic W. bancrofti microfilariae (Mf) carriers following a double blind randomized design. The drugs were tolerated well. The incidence of adverse reactions of DEC (85.0%), DEC + Ivermectin 200mcg (95.0%) and DEC + Ivermectin 400mcg (100%) did not vary significantly (P>0.05). The mean score of adverse reaction intensity due to DEC + Ivermectin 200mcg (1.41) was significantly higher compared to DEC (0.61) (P<0.05). However, there was no significant difference between and DEC +Ivermectin 400mcg (0.89) and DEC + Ivermectin 200mcg (1.41) and DEC + Ivermectin 400mcg and DEC. The major adverse reactions were fever, headache and myalgia in all groups. The incidence and intensity of the adverse reactions were maximum between 24 to 48 hours of post therapy. The haematological and biochemical parameters did not vary significantly between pre and 7-day post therapy values in any of the study groups (P>0.05). Efficacy was measured in terms of proportion of cases clearing microfilaraemia completely and reduction in geometric mean parasite density in comparison to pre therapy levels. At the end of one year, DEC with Ivermectin 400mcg group showed significantly higher efficacy in complete clearance of Mf (94.4%) than that of DEC with Ivermectin 200mcg (60.0%) or DEC alone (52.6%) (P<0.05). However, no significant difference was observed in reduction of geometric mean Mf density (99.9%, 99.7%, 99.5% respectively). In all the groups, the tolerability and efficacy of the drugs were independent of host age and gender.

Treatment of microfilaraemics with DEC and its effect on vector infection and infectivity in tribal and non-tribal areas of Bankura district, West Bengal, India.

Single course DEC treatment (6 mg/kg body weight/day for 12 days) was administered to 66 tribal and 442 non-tribal microfilaria (mf) carriers detected through a Filariasis survey in Bankura district, West Bengal, India. All the mf carriers remained amicrofilaraemic on 22nd, 180th and 365th post-treatment day. As a result of DEC treatment to the mf carriers, vector (Culex quinquefasciatus) infection rate in tribal study areas reduced from 2.06% to 1.07%. Infectivity rate was "nil" both before and after treatment. In non-tribal study areas, vector infection rate reduced from 4.33% to 2.22% and infectivity rate from 0.51% to 0.29%.

Filariasis elimination in Egypt: impact of low microfilaraemics as sources of infection for mosquitoes

The elimination strategy for lymphatic filariasis aims at reducing blood microfilaraemia to levels at which vector transmission cannot be sustained. We aimed to determine whether patients with pre-treatment low or ultra-low microfilaria [MF] counts could be a reservoir of infection after mass drug administration [MDA] with a combined regimen. Laboratory-reared mosquitoes were fed on 30 volunteers after 2 rounds of MDA. Microfilaria uptake, infectivity rates and number of Wuchereria bancrofti L3 per mosquito were assessed. One year after MDA-1, 6 subjects transmitted MF, but up to 9 months after MDA-2 transmission failed. Six months after MDA-2 > 90% had clear MF smears and either failed to transmit MF or transmitted MF that did not develop to L3. We conclude that the transmission cycle is seriously weakened after MDA-2

Bancroftian filariasis: Clinical parasitologic and serologic evaluation after 4 years appling two antifilarial regimens

In August 1997, 124 individuals out of 1110 were selected as being seropositive for circulating filarial antigen OG4C3 [CFA]. Ten healthy children proven negative for CFA were used as controls. The patients were classified into: G1 [28 patients, 20 asymptomatic microfilaremic [MF] and eight symptomatic amicrofilaremic [AMF]], G2 [80 patients, 22 asymptomatic MF, 48 asymptomatic AMF and 10 symptomatic AMF] and G3 [16 asymptomatic AMF]. G1 was treated by a single annual dose of diethyl carbamazine [DEC] [6 mg/kg], G2 by a single annual dose of albendazole 400 mg and DEC [6 mg/kg] and G3 remained untreated. Four years later [2001], patients were reevaluated. Microfilaremia prevalence in MF patients was lowered to 20% [G1] and 9.1% [G2]. Antigenemia prevalence was lowered to 46.4%, 17.5% and 87.5% in the three groups, respectively. The disease became manifested among the asymptomatic in 5% [G1], 10% [G2] and 25% [G3]. The four years lowered the prevalence of microfilaremia, but it was not sufficient for its elimination from the blood

Circulating filarial antigens for monitoring the efficacy of ivermectin in treatment of filariasis

Seventy-three asymptomatic bancroftian filariasis patients with positive microfilaria in their blood films were included in this study. The patients were randomly divided into two groups: Ivermectin group given two doses [each of 100 ug/kg body weight three months apart] and 23 cases given two doses of placebo. The initial mean microfilaria [MF] count was 111/ml. At three months after ivermectin therapy, the mean MF became 7.8/ml and 24% of ivermectin treated cases had no detectable MF. At 6, 9 and 12 months; the mean MF count became 4.1, 6.5 and 11/ml with a microfilaria in 54%, 42% and 40% of treated cases, respectively. On the other hand, no statistically significant change in the mean MF count in placebo group was detected

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.

Effect of acetylcholine, L-glutamine and diethyl carbamazine citrate on the release of microfilariae from Setaria digitata.

Release of microfilariae from adult filarial parasite S. digitata was reversibly arrested in presence of 2 mM L-glutamine. Diethylcarbamazine also showed a similar effect, but at a lower concentration (0.26 mM). Both compounds inhibited activities of protease and leucine amino peptidase enzymes. The only difference being, different concentrations of the two materials were required for comparable effects. Levels of histamine and acetylcholine in parasite were lowered during incubation under in vitro conditions. Direct effect of L-glutamine on the release of mf was also confirmed.

Impact of surface modifications of Acanthocheilonema viteae microfilariae on cell adhesion.

Exposure of A. viteae microfilariae to various lectins reduced their capacity to react with the peritoneal exudate cells of the host, Mastomys natalensis. Sugars corresponding to these lectins with the exception of N-acetyl glucosamine, did not affect the adhesion per se. They however, protected the parasite against the adverse effect of lectins. Neuraminidase and chitinase also suppressed adhesion capacity of the microfilariae. Except sodium dodecylsulphate which enhanced cell attachment, other surfactants inhibited this reaction considerably. The results indicate that antibody dependent adhesion of the microfilariae with the macrophages involves surface moieties of the parasite, where N-acetylglucosamine acts as the principal sugar residue. Participation of -SH groups also is inferred from the observations that p-chloromercuribenzoate and dithiobis-(2-nitrobenzoic acid) inhibited cell attachment and dithiothreitol provided protection against these agents.

Influence of diethylcarbamazine on the patent period of infection in periodic Brugia malayi.

The patent period of infection which reflects the fecundic life span of the adult female worm was estimated for periodic B. malayi in south India by using immigration death model. Longitudinal data on natural loss of infection or that due to different degrees of DEC pressure in a cohort population of microfilaria carriers after a three years period, were used for the estimations. The patent period of infection was estimated to be 3.45 yr under natural conditions (without DEC therapy), which was reduced to 1.67 yr following one full course of selective therapy (6 mg/kg body wt/day for 12 days). When selective therapy was supplemented with four rounds of biannual single-dose mass DEC therapy (6 mg/kg body wt) the patent period was further reduced to 1.34 yr. The proportion loss of infection was relatively higher in microfilaria carriers who discontinued DEC selective therapy, when compared to natural loss. The percentage reduction in the mean microfilaria count was observed to decrease with increasing DEC pressure. Neither sex nor the age of the host was found to influence the fecundic life span or the survival of female adult worm.

A single diethylcarbamazine dose for treatment of Wuchereria bancrofti carriers in French Polynesia: efficacy and side effects.

In the fall of 1988, 14 Tahitian Wuchereria bancrofti carriers were treated by a single diethylcarbamazine (DEC) 3 mg/kg dose. Determination of blood microfilarial (mf) density was carried out on days 0, 7, 14, 30, 90 and 180 using the membrane filtration technique. Clinical signs and side effects were noted during the 3 days following treatment. Complete clearance of microfilaremia was observed in two carriers (negativation rate 14%). A decrease of mf density was noted in all of the 14 carriers, ranging from 35.2 to 99.2% (median 78.75%). The percentage decrease in mf density, determined for the whole group from the geometric mean of the 14 mf counts, was 86% by day 7 and reached 95% by day 180. Side effects were observed in 10 patients (71%) of whom 3 only were unable to perform usual activities for less than 24 hours. Though it induced an incomplete initial mf clearance, a single DEC 3 mg/kg dose was effective in reducing about 90% of the microfilaremia and in sustaining this reduction over a period of six months. Such long-term reduction (comparable to that observed in W. bancrofti carriers treated with a daily DEC 6 mg/kg dose during 12 days) is likely responsible for the consistent decrease of total mf counts observed in the Tahitian population which has been treated for years with single DEC doses given every six months.