Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64% of all infections, P. vivax for 28%, and P. malariae for 4%. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4% of total cases. Children under 10 years accounted for 58% of the cases; the risk for malaria in this age group was 77% higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42%), P. malariae (0.25%), and P. vivax-247 (0.1%). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.

Chagas disease: what is known and what is needed - A background article

Chagas disease began millions of years ago as an enzootic disease of wild animals and started to be transmitted to man accidentally in the form of an anthropozoonosis when man invaded wild ecotopes. Endemic Chagas disease became established as a zoonosis over the last 200-300 years through forest clearance for agriculture and livestock rearing and adaptation of triatomines to domestic environments and to man and domestic animals as a food source. It is estimated that 15 to 16 million people are infected with Trypanosoma cruzi in Latin America and 75 to 90 million people are exposed to infection. When T. cruzi is transmitted to man through the feces of triatomines, at bite sites or in mucosa, through blood transfusion or orally through contaminated food, it invades the bloodstream and lymphatic system and becomes established in the muscle and cardiac tissue, the digestive system and phagocytic cells. This causes inflammatory lesions and immune responses, particularly mediated by CD4+, CD8+, interleukin-2 (IL) and IL-4, with cell and neuron destruction and fibrosis, and leads to blockage of the cardiac conduction system, arrhythmia, cardiac insufficiency, aperistalsis, and dilatation of hollow viscera, particularly the esophagus and colon. T. cruzi may also be transmitted from mother to child across the placenta and through the birth canal, thus causing abortion, prematurity, and organic lesions in the fetus. In immunosuppressed individuals, T. cruzi infection may become reactivated such that it spreads as a severe disease causing diffuse myocarditis and lesions of the central nervous system. Chagas disease is characterized by an acute phase with or without symptoms, and with entry point signs (inoculation chagoma or Romaña's sign), fever, adenomegaly, hepatosplenomegaly, and evident parasitemia, and an indeterminate chronic phase (asymptomatic, with normal results from electrocardiogram and x-ray of the heart, esophagus, and colon) or with a cardiac, digestive or cardiac-digestive form. There is great regional variation in the morbidity due to Chagas disease, and severe cardiac or digestive forms may occur in 10 to 50% of the cases, or the indeterminate form in the other asymptomatic cases, but with positive serology. Several acute cases have been reported from Amazon region most of them by T. cruzi I, Z3, and a hybrid ZI/Z3. We conclude this article presenting the ten top Chagas disease needs for the near future.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

Anopheles darlingi bionomics and transmission of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae in Amerindian villages of the Upper-Maroni Amazonian forest, French Guiana

French Guiana is one of the areas in South America most affected by malaria and where the disease has become a serious public health problem. In spite of this situation, little recent entomological data are available from the main localities where the disease occurs, even though they are crucial for development of an effective vector control strategy. A longitudinal entomological survey was carried out from March 2000-February 2002 in three Amerindian villages, namely Twenké, Taluène and Cayodé, located in the Amazonian forest of the Upper-Maroni area, to assess anopheline mosquitoes and malaria transmission dynamics. Anopheles darlingi (Diptera: Culicidae) was the most abundant mosquito species caught during the study. This efficient American malaria vector was active the entire year, but showed an evident peak of abundance during the main rainfall season, from April-June, with an average human biting rate of 255.5 bites per person per night. Parity rates were homogeneous all year, indicating no significant seasonal variability in female survival rates. Estimated vectorial capacity indices were higher during the rainy season, even though the risk of transmission was present throughout the year (VCI > 1). A total of 14 An. darlingi were found infected with Plasmodium falciparum, Plasmodium vivax or Plasmodium malariae. The annual circumsporozoite indices were 0.15, 0.14 and 0.05, and the entomological inoculation rates were 22.8, 27.4 and 14.4 infected bites per person per year in Twenké, Taluène and Cayodé, respectively. An. darlingiwas endo-exophagic and rather exophilic in these localities. The species was collected throughout the night but was more aggressive between 21:30-03:30 h and after 05:30 h. Parity rates were homogeneous during the entire night. Impregnated hammock and/or bed nets, coupled with the use of mosquito repellents, as well as the early treatment of malarial cases, appear to be the most suitable tools for fighting malaria in these Amerindian villages since the spraying of residual insecticides is inefficient because of vector biology and the housing structure.

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France

Water is a vehicle for disseminating human and veterinary toxoplasmosis due to oocyst contamination. Several outbreaks of toxoplasmosis throughout the world have been related to contaminated drinking water. We have developed a method for the detection of Toxoplasma gondii oocysts in water and we propose a strategy for the detection of multiple waterborne parasites, including Cryptosporidium spp. and Giardia. Water samples were filtered to recover Toxoplasma oocysts and, after the detection of Cryptosporidium oocysts and Giardia cysts by immunofluorescence, as recommended by French norm procedure NF T 90-455, the samples were purified on a sucrose density gradient. Detection of Toxoplasma was based on PCR amplification and mouse inoculation to determine the presence and infectivity of recovered oocysts. After experimental seeding assays, we determined that the PCR assay was more sensitive than the bioassay. This strategy was then applied to 482 environmental water samples collected since 2001. We detected Toxoplasma DNA in 37 environmental samples (7.7%), including public drinking water; however, none of them were positive by bioassay. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method. Alternative methods can be used in conjunction with this one to determine the infectivity of parasites that were detected by molecular methods.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24%) were infected. The transmission rates of Toxoplasma gondii were 7%, 24% and 59% in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91% and 99.5%, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52% and 99%, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53% and 64%, respectively, and specificity values were 91% and 92%. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Growth, cysts and kinetics of Borrelia garinii (Spirochaetales: Spirochaetacea) in different culture media

The aim of the present paper was to evaluate cyst formation and growth parameters of Borrelia garinii in a range of media differing in formulation and cost. A qualitative assessment of morphology and motility of B. garinii was conducted. All media were prepared aseptically and used in test tubes or Petri dishes. For each medium, the initial spirochete concentration was standardized to 10³ spirochets/mL. The following culture media were suitable to grow B. garinii: Barbour-Stoenner-Kelly, brain heart infusion and PMR. Growth was minimal at six weeks post-inoculation and maximum spirochete density was observed between 9-12 weeks. Often, the cultures developed cysts of different sizes, isolated or in groups, with a spiraled portion of variable sizes, mainly in unfavorable culture media. Brazilian Lyme disease-like illness, also known as Baggio-Yoshinari syndrome (BYS), is a new and interesting emerging tick-borne disease, caused by Borrelia burgdorferi sensu lato spirochetes, only during its cystic forms. It has been assumed that the peculiar clinical and laboratory features of BYS are consequential to the absence of a human sucker Ixodes ricinus complex tick at risk areas in Brazil, supporting the concept that the borrelia phenotypic expression pattern is modified as it is transmitted through the host.

The benefits of using selenium in the treatment of Chagas disease: prevention of right ventricle chamber dilatation and reversion of Trypanosoma cruzi-induced acute and chronic cardiomyopathy in mice

Cardiac damage is a frequent manifestation of Chagas disease, which is caused by the parasite Trypanosoma cruzi. Selenium (Se) is an essential micronutrient, the deficiency of which has been implicated in the development of cardiomyopathy. Our group has previously demonstrated that Se supplementation prevents myocardial damage during acute T. cruzi infection in mice. In this study, we analyzed the effect of Se treatment in cases of T. cruzi infection using prevention and reversion schemes. In the Se prevention scheme, mice were given Se supplements (2 ppm) starting two weeks prior to inoculation with T. cruzi(Brazil strain) and continuing until 120 days post-infection (dpi). In the Se reversion scheme, mice were treated with Se (4 ppm) for 100 days, starting at 160 dpi. Dilatation of the right ventricle was observed in the infected control group at both phases of T. cruzi infection, but it was not observed in the infected group that received Se treatment. Surviving infected mice that were submitted to the Se reversion scheme presented normal P wave values and reduced inflammation of the pericardium. These data indicate that Se treatment prevents right ventricular chamber increase and thus can be proposed as an adjuvant therapy for cardiac alterations already established by T. cruziinfection.

Biomphalaria alexandrina snails as immunogens against Schistosoma mansoni infection in mice

Despite effective chemotherapy, schistosomiasis remains the second largest public health problem in the developing world. Currently, vaccination is the new strategy for schistosomiasis control. The presence of common antigenic fractions between Schistosoma mansoni and its intermediate host provides a source for the preparation of a proper vaccine. The objective of this paper is to evaluate the nucleoprotein extracted from either susceptible or resistant snails to protect against schistosomiasis. The vaccination schedule consisted of a subcutaneous injection of 50 µg protein of each antigen followed by another inoculation 15 days later. Analyses of marker enzymes for different cell organelles [succinate dehydrogenase, lactate dehydrogenase (LDH), glucose-6-phosphatase, acid phosphatase and 5'-nucleotidase] were carried out. Energetic parameters (ATP, ADP, AMP, phosphate potentials, inorganic phosphate, amino acids and LDH isoenzymes) were also investigated. The work was extended to record worm and ova counts, oogram determination in the liver and intestine and the histopathological pattern of the liver. The nucleoprotein of susceptible snails showed reduction in worm and ova counts by 70.96% and 51.31%, respectively, whereas the nucleoprotein of resistant snails showed reductions of 9.67% and 16.77%, respectively. In conclusion, we found that the nucleoprotein of susceptible snails was more effective in protecting against schistosomiasis.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite

The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Malaria entomological risk factors in relation to land cover in the Lower Caura River Basin, Venezuela

To explore the effects of deforestation and resulting differences in vegetation and land cover on entomological parameters, such as anopheline species composition, abundance, biting rate, parity and entomological inoculation rate (EIR), three villages were selected in the Lower Caura River Basin, state of Bolívar, Venezuela. All-night mosquito collections were conducted between March 2008-January 2009 using CDC light traps and Mosquito Magnet(r) Liberty Plus. Human landing catches were performed between 06:00 pm-10:00 pm, when anophelines were most active. Four types of vegetation were identified. The Annual Parasite Index was not correlated with the type of vegetation. The least abundantly forested village had the highest anopheline abundance, biting rate and species diversity. Anopheles darlingi and Anopheles nuneztovari were the most abundant species and were collected in all three villages. Both species showed unique biting cycles. The more abundantly forested village of El Palmar reported the highest EIR. The results confirmed previous observations that the impacts of deforestation and resulting changes in vegetation cover on malaria transmission are complex and vary locally.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.