The use of dacron plates for DOT enzyme-linked immunosorbent assay (DOT-ELISA)

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Triration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparasion with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4ºC, 28ºC and -20ºC for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4ºC. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.

Genotoxicity of plant extracts

Aqueous extracts of seven species used in Brazilian popular medicine (Achyrocline satureoides, Iodina rhombifolia, Desmodium incanum, Baccharis anomala, Tibouchina asperior, Luehea divaricata, Maytenus ilicifolia) were screened to the presence of mutagenic activity in the Ames test (Salmonella/microsome). Positive results were obtained for A. satureoides, B anomala and L. divaricata with microsomal activation. As shown elsewhere (Vargas et al., 1990) the metabolites of A. satureoides extract also show the capacity to induce prophage and/or SOS response in microscreen phage induction assay and SOS spot chromotest.

Milky spots reactions to schistosomal mansoni infection

Milky spots (MS), considered by the authors as a Coelomatic Lympho-myelopoietic Organ (CLMO), present a strong reactivity during experimental schistosomal mansoni infection, characterized by an increase of lymphocytes, macrophages, plasmocytes, mast cells, neutrophils and expression of eosinophil metaplasia. Intraperitoneal injection of purified Schistosoma mansoni (Sm) eggs provoked a rise in the number and size of MS, which developed the sessile marginal and pedunculated types. The authors conclude that egg antigens are, at least partially, responsible for MS reactivity during Sm infection.

The identification of Anopheles (Nyssorhynchus) rondoni (Diptera: Culicidae) in Mato Grosso, Brazil: an analysis of key character variability

A morphological study was made of a population of Anopheles (Nyssorhynchus) rondoni (Neiva and Pinto) from northern Mato Grosso, Brazil. This population usually lacked the primary key character of a dark basal band on hindtarsomere 3, i.e., hindtarsomere 3 was all white as in most other members of the subgenus. It was determined that this species can be recognized instead by the presence of a dark spot on the thorax made up of a large dark prescutellar space that is contiguous with a concolorous central area on the scutellum. A secondary character of a dark area on the costa created by the fusion of the humeral dark, presector dark and sector dark proximal spots is also usually reliable. Regression analyses comparing the lengths and ratios of the dark bands on hindtarsomeres 2 to those on 3 describe a straight line relationship. This suggests that the "atypical" population is at one end of a character gradient. We propose that in the subgenus Nyssorhynchus individuals that have a long basal band on hindtarsomere 2 are more likely to also have a basal band on hindtarsomere 3. The pupal stage of this species has not been previously described. Reared-associated specimens from this study show that the pupa can be easily differentiated from all other Nyssorhynchus by the relatively stout, usually 2 or 3 branched (1-5), setae 1 and 5 on segments IV-VII.

Malaria control in an agro-industrial settlement of Rondônia (Western Amazon Region, Brazil)

A malaria control pilot project was developed in the Urupá agro-industrial farm that is situated in the State of Rondônia (Western Amazon Region, Brazil). Around 180 inhabitants had been surveyed for the past five years. The control measures were based on (1) training a community agent to perform on the spot microscopical diagnosis of malaria and to treat the uncomplicated cases of malaria; (2) limiting the use of insecticides to a short period before the high transmission season. This resulted in a significant reduction in the time between the onset of clinical symptoms and specific chemotherapy which fell from 3.5 to 1.3 days. In relation to the previous three reference years the total number of malaria cases was reduced to 50% in the first year and to 25% in the second year. The introduction of these measures coincided with pronounced reduction in the frequency of Plasmodium falciparum infections but this was less marked for P. vivax infections. In the second year of the pilot experiment there was no P. falciparum transmission on the farm. During the last decade there was a general decrease in the endemicity of malaria in the State of Rondônia. The linear regression coefficient values indicate that the decline was more pronounced in Urupá than in the general municipality and that the falciparum malaria API in Urupá farm is significantly lower than in the general municipality of Candeias were the farm is situated.

Wing geometry as a tool for studying the Lutzomyia longipalpis (Diptera: Psychodidae) complex

Toro Toro (T) and Yungas (Y) have been described as genetically well differentiated populations of the Lutzomyia longipalpis (Lutz & Neiva, 1912) complex in Bolivia. Here we use geometric morphometrics to compare samples from these populations and new populations (Bolivia and Nicaragua), representing distant geographical origins, qualitative morphological variation ("one-spot" or "two-spots" phenotypes), ecologically distinct traits (peridomestic and silvatic populations), and possibly different epidemiological roles (transmitting or nor transmitting Leishmania chagasi). The Nicaragua (N) (Somotillo) sample was "one-spot" phenotype and a possible peridomestic vector. The Bolivian sample of the Y was also "one-spot" phenotype and a demonstrated peridomestic vector of visceral leishmaniasis (VL). The three remaining samples were silvatic, "two-spots" phenotypes. Two of them (Uyuni and T) were collected in the highlands of Bolivian where VL never has been reported. The last one (Robore, R) came from the lowlands of Bolivia, where human cases of VL are sporadically reported. The decomposition of metric variation into size and shape by geometric morphometric techniques suggests the existence of two groups (N/Y/R, and U/T). Several arguments indicate that such subdivision of Lu. longipalpis could correspond to different evolutionary units.

Eleutherinone, a novel fungitoxic naphthoquinone from Eleutherine bulbosa (Iridaceae)

The dichloromethane extract prepared from the underground parts of Eleutherine bulbosa (Miller) Urban (Iridaceae) showed strong activity in the direct bioautography assay with the phytopathogenic fungus Cladosporium sphaerospermum. This assay was used to guide the fractionation of this extract and allowed the isolation of four compounds: the new naphthoquinone eleutherinone[8-methoxy-1-methyl-1,3-dihydro-naphtho(2,3-c)furan-4,9 -dione] and the known compounds, previously isolated from this species, eleutherin [9-methoxy-1(R),3(S)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], isoeleutherin [9-methoxy-1(R),3(R)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], and eleutherol [4-hydroxy-5-methoxy-3(R)-methyl-3H-naphtho(2,3-c)furan-1 -one]. All quinonoid compounds showed strong antifungal activity in the bioautography assay at 100 µg/spot, while eleutherol was inactive.

Intradermal vaccination of adults with three low doses (2 µg) of recombinant hepatitis B vaccine. I. Seroconversion rate and adverse effects

A total of 250 dentists (53.6% men and 46.4% women), with a mean age of 35.1 ± 9.8 years, were submitted to serological tests for the diagnosis of hepatitis B (HB) - HBsAg, anti-HBs, anti-HBc, HBeAg, and anti-HBe - using a radioimmunoassay. One or more of these markers were detected in 78 individuals (31.2%) who were excluded from the group to be vaccinated. Of the 172 HB-susceptible individuals, 135 (78.5%) responded to the call and were intradermally injected with three 2 µg doses of the Belgian HB recombinant vaccine, applied at an interval of one month between the 1st and 2nd dose and of five months between the 2nd and 3rd dose. A new determination of HB markers carried out 50 days after the 3rd dose showed that 110 (81.5%) individuals had become anti-HBs positive (65.5% good responders and 34.5% poor responders). Mean serum anti-HBs titer of these 110 dentists was 42.4 U S/N, similar in both sexes. The adverse effects analyzed in 106 dentists were: (a) local: pain (12.3%), burning sensation (14.1%), pruritus (25.5%), erythema (28.3%), local heat (18.9%), and a hypochromic spot (32.1%); (b) systemic (4.7%): discomfort in two patients, and fever, anorexia, and asthenia in one patient each. Intradermal administration of a fourth 2 µg vaccine dose to 39 dentists (poor or non-responders) increased the total number of anti-HBs-positive individuals from 110 (81.5%) to 114 (84.4%), with the number of good responders increasing from 72 (65.5%) to 85 (74.6%). We conclude that the Belgian recombinant vaccine applied in the scheme used here induces a high rate of seroconversion and causes only mild and transitory adverse effects.

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional Löwenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.

Morphological analysis of three populations of Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae) from Colombia

Based on the results of comparative analyses of 1,039 specimens of several progenies of Anopheles nuneztovarifrom three localities in Colombia, eight costal wing spot patterns were observed. Patterns I and III were the most frequent: 77.96% and 11.36%, respectively. Using the diagnostic characters ratio of the length of the basal dark area of hind tarsomere II/length of hind tarsomere II, ratio of the length of the humeral pale spot/length of the pre-humeral dark spot, and the ratio of the length of the subcostal pale spot/length of the distal sector dark spot (DS-III2/Ta-III2, HP/PHD, SCP/DSD) approximately 5% of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5% of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34% shared a HP/PHD ratio less than 1.50. Consequently, 1.52% of An. nuneztovari individuals can be misidentified as Anopheles oswaldoi. In those specimens with the DS-III2/Ta-III2 ratios higher than 0.25, 34.45% displayed SCP/DSD values greater than 0.50 and of these, 3.65% displayed HP/PHD values greater than 1.8. This combination of characters could lead one to misidentify samples of An. nuneztovari as Anopheles rangeli. Similarly, 2.43% of the females could be identified erroneously as either Anopheles aquasalis or Anopheles benarrochi. Individuals with a HP/PHD ratio greater than 2.0, could be misidentified as Anopheles trinkae, Anopheles strodei or Anopheles evansae. A distinct combination of diagnostic characters for An. nuneztovari from Colombia is proposed.

Congenital toxoplasmosis and DALYs in the Netherlands

The calculation of disability-adjusted life years (DALYs) enables public health policy makers to compare the burden of disease of a specific disease with that of other (infectious) diseases. The incidence of a disease is important for the calculation of DALYs. To estimate the incidence of congenital toxoplasmosis (CT), a random sample of 10,008 dried blood spot filter paper cards from babies born in 2006 in the Netherlands were tested for Toxoplasma gondii-specific IgM antibodies. Eighteen samples were confirmed as positive for IgM, resulting in an observed birth incidence of CT of 1.8 cases per 1,000 live-born children in 2006 and an adjusted incidence of 2.0 cases per 1,000. This means that 388 infected children were born in 2006. The most likely burden of disease is estimated to be 2,300 DALYs (range 820-6,710 DALYs). In the previous calculations, using data from a regional study from 1987, this estimate was 620 DALYs (range 220-1,900 DALYs). The incidence of CT in the Netherlands is much higher than previously reported; it is 10 times higher than in Denmark and 20 times higher than in Ireland, based on estimates obtained using the same methods. There is no screening program in the Netherlands; most children will be born asymptomatic and therefore will not be detected or treated.

Omental and pleural milky spots: different reactivity patterns in mice infected with Schistosoma mansoni reveals coelomic compartmentalisation

In vertebrate animals, pleural and peritoneal cavities are repositories of milky spots (MS), which constitute an organised coelom-associated lymphomyeloid tissue that is intensively activated by Schistosoma mansoni infection. This study compared the reactive patterns of peritoneal MS to pleural MS and concluded from histological analysis that they represent independent responsive compartments. Whole omentum, lungs and the entire mediastinum of 54 S. mansoni-infected mice were studied morphologically. The omental MS of infected animals were highly activated, modulating from myeloid-lymphocytic (60 days of infection) to lymphomyeloid (90 days of infection) and lymphocytic or lymphoplasmacytic (160 days of infection) types. The non-lymphoid component predominated in the acute phase of infection and was expressed by monocytopoietic, eosinopoietic and neutropoietic foci, with isolated megakaryocytes and small foci of late normoblasts and mast cells. Nevertheless, pleural or thoracic MS of infected mice were monotonous, consisting of small and medium lymphocytes with few mast and plasma cells and no myeloid component. Our data indicate that compartmentalisation of the MS response is dependent on the lymphatic vascularisation of each coelomic cavity, limiting the effects or consequences of any stimulating or aggressive agents, as is the case with S. mansoni infection.

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardiand Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region.

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

Declining prevalence of hepatitis A virus antibodies among children from low socioeconomic groups reinforces the need for the implementation of hepatitis A vaccination in Brazil

Age-related seroprevalence studies that have been conducted in Brazil have indicated a transition from a high to a medium endemicity of hepatitis A virus (HAV) infection in the population. However, most of these studies have focused on urban populations that experience lower incidence rates of HAV infection. In the current study, the prevalence of anti-HAV antibodies was investigated in children with a low socioeconomic status (SES) that live on the periphery of three capital cities in Brazil. A total of 1,162 dried blood spot samples were collected from individuals whose ages ranged from one-18 years and tested for anti-HAV antibodies. A large number of children under five years old (74.1-90%) were identified to be susceptible to HAV infection. The anti-HAV antibody prevalence reached ≥ 50% among those that were 10-14 years of age or older. The anti-HAV prevalence rates observed were characteristics of regions with intermediate level of hepatitis A endemicity. These data indicated that a large proportion of children with a low SES that live at the periphery of urban cities might be at risk of contracting an HAV infection. The hepatitis A vaccine that is currently offered in Brazil is only available for high-risk groups or at private clinics and is unaffordable for individuals with a lower SES. The results from this study suggest that the hepatitis A vaccine should be included in the Brazilian National Program for Immunisation.