Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Detection of a Giardia lamblia coproantigen by using a commercially available immunoenzymatic assay, in Belo Horizonte, Brazil

It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRÁS) to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0%) by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%). The assay identified 27 of the 30 positive patients (sensitivity = 90.0%) and was negative in 59 of the 60 negatives (specificity = 98.3%) in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Prevalence of human herpesvirus 8 antibodies in the population of Belém, Pará, Brazil

Serum samples from 497 children and adults inhabiting two neighbourhoods (Guamá and Terra Firme) in Belém, Pará, North Brazil were screened for the presence of human herpesvirus 8 (HHV-8) antibody using an enzyme-linked immunosorbent assay. An overall 16.3% prevalence was found for these urban communities. Taken both genders together, prevalence rates of HHV-8 antibody increase gradually, across age-groups, ranging from 12.0% to 33.3%. When seroprevalence is analysed by gender, similar rates are found for female (18.4%) and male (14.0%) individuals. In the former gender group, seroprevalence rates increased from 10.3%, in children £ 10 years of age, to 30.0% in adults 41-50 years of age. Conversely, among male subjects, the prevalence of HHV-8 antibodies decreased from 13.3% in children/young adults aged £ 10 to 20 years of age to 6.1% in adults aged 21-30 years. From the 31-40 year-old group male onwards, seropositivity rates increased gradually, ranging from 8.3% to 66.7%. A significant difference in seropositivity rates was noted when comparing 21-30 age groups for female and male subjects: 23.3% and 6.1%, respectively (P = 0.03). Geometric mean optical densities were found to increase slightly from the lower to the higher age-groups. Our data suggest that transmission of HHV-8 occurs frequently in the general urban population of Belém, and that prevalence of antibody seems to increase with age.

Distribution of phlebotomine sandflies (Diptera: Psychodidae) of medical importance in Mato Grosso State, Brazil

Intense environmental impacts, causing alterations of the natural habitats of fauna, including those of sandfly disease vectors are observed in Mato Grosso State, Central Brazil. Entomologic survey of phlebotomines was based on light trap and was carried out by entomological nucleus of the FUNASA and SES in the period between 1996 and 2001. Eighty eight species were identified, including the following sandflies with medical importance to leishmaniasis: Lutzomyia amazonensis, L. anduzei, L. antunesi, L. ayrozai, L. carrerai carrerai, L. complexa, L. cruzi, L. flaviscutellata, L. intermedia, L. longipalpis, L. migonei, L. paraensis, L. ubiquitalis, L. whitmani and L. yuilli yuilli. Most sandflies of medical importance occurred in the Amazon forest and savannah. L. longipalpis and L. cruzi had high densities in the savannah region. L. flaviscutellata is predominating in both the Amazon forest and the savannah region. L. whitmani and L. antunesi were sampled in the Amazon forest, savannah and marsh land.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

Sensitivity of Gomez-Nuñez boxes for the detection of household infestation with Panstrongylus megistus

Four methods for detecting household infestations with Panstrongylus megistus were compared: 1) manual collection; 2) collection after pyrethrum application; 3) search viable eggs; and 4) Gomez-Nuñez boxes. Manual collection was the most sensitive method (23% infested), followed by pyrethrum (21%), Gomez-Nuñez boxes (15%) and viable eggs (12%). About 10% of infested houses were positive exclusively on the Gomez-Nuñez box test. More over, 6 out of the 7 houses positive exclusively on the Gomez-Nuñez method were located in a recently sprayed area, where P. megistus density was low. Inspection of Gomez-Nuñez boxes at 12 weekspost-application was twice as effective as inspection at 6 weekspost-application. Triatomine feces was the most common evidencefor thepresence of P. megistus found within Gomez-Nuñez boxes. Gomez-Nuñez boxes area a useful adjunct to manual collection in detecting domestic infestations with P. megistus, especially in areas where bug densities are low. However, the utility of Gomez-Nuhez boxes must be weighed against the time and labor they require.

Nosocomial infections in a pediatric intensive care unit of a developing country: NHSN surveillance

INTRODUCTION: This study aimed to determine the epidemiology of the three most common nosocomial infections (NI), namely, sepsis, pneumonia, and urinary tract infection (UTI), in a pediatric intensive care unit (PICU) in a developing country and to define the risk factors associated with NI. METHODS: We performed a prospective study on the incidence of NI in a single PICU, between August 2009 and August 2010. Active surveillance by National Healthcare Safety Network (NHSN) was conducted in the unit and children with NI (cases) were compared with a group (matched controls) in a case-control fashion. RESULTS: We analyzed 172 patients; 22.1% had NI, 71.1% of whom acquired it in the unit. The incidence densities of sepsis, pneumonia, and UTI per 1,000 patients/day were 17.9, 11.4, and 4.3, respectively. The most common agents in sepsis were Enterococcus faecalis and Escherichia coli (18% each); Staphylococcus epidermidis was isolated in 13% of cases. In pneumonias Staphylococcus aureus was the most common cause (3.2%), and in UTI the most frequent agents were yeasts (33.3%). The presence of NI was associated with a long period of hospitalization, use of invasive devices (central venous catheter, nasogastric tube), and use of antibiotics. The last two were independent factors for NI. CONCLUSIONS: The incidence of NI acquired in this unit was high and was associated with extrinsic factors.

Spatial distribution of schistosomiasis and geohelminthiasis cases in the rural areas of Pernambuco, Brazil

INTRODUCTION: The prevalence and intensity of geohelminth infections and schistosomiasis remain high in the rural areas of Zona da Mata, Pernambuco (ZMP), Brazil, where these parasites still represent a significant public health problem. The present study aimed to spatially assess the occurrences of schistosomiasis and geohelminthiasis in the ZMP. METHODS: The ZMP has a population of 1,132,544 inhabitants, formed by 43 municipalities. An ecological study was conducted, using secondary data relating to positive human cases and parasite loads of schistosomiasis and positive human cases of geohelminthiasis that were worked up in Excel 2007. We used the coordinates of the municipal headquarters to represent the cities which served as the unit of analysis of this study. The Kernel estimator was used to spatially analyze the data and identify distribution patterns and case densities, with analysis done in ArcGIS software. RESULTS: Spatial analysis from the Kernel intensity estimator made it possible to construct density maps showing that the northern ZMP was the region with the greatest number of children infected with parasites and the populations most intensely infected by Schistosoma mansoni. In relation to geohelminths, there was higher spatial distribution of cases of Ascaris lumbricoides and Trichuris trichiura in the southern ZMP, and greater occurrence of hookworms in the northern/central ZMP. CONCLUSIONS: Despite several surveys and studies showing occurrences of schistosomiasis and geohelminthiasis in the ZMP, no preventive measures that are known to have been effective in decreasing these health hazards have yet been implemented in the endemic area.

Larval control of Anopheles (Nyssorhinchus) darlingiusing granular formulation of Bacillus sphaericus in abandoned gold-miners excavation pools in the Brazilian Amazon Rainforest

INTRODUCTION: Use of a Bacillus sphaericus based mosquito larvicide was evaluated as an intervention for malaria vector control at a mining site in Amapá, Brazil. Impacts on larval and adult densities of the primary vector Anopheles darlingi were measured over the course of a 52 week study period.METHODS: In Calçoene, State of Amapá, gold mining activity occurs in 19 mining sites in gold-miners of Lourenço. Large pools are formed in mining sites and naturally colonized by Anopheles darlingi. During one year, the impact of applications of VectoLex(r) CG to these larval sources was evaluated. Applications of 20kg/ha were made as needed, based on 10 immature (3rd, 4th instars and pupae) surveillance of health and established thresholds. RESULTS: One hundred percent initial control was observed 48h after each treatment. The pools received from 2-10 (5.3±1.6) treatments during the year. The average re-treatment interval in productive pools was 9.4±4.3 weeks. During weeks 3-52 of the study, mean density of late stage larvae was 78% and pupae were 93% lower in the treated pools than in untreated pools (p< 0.0001, n=51) while reduction of adult mosquitoes was 53% in comparison to the untreated area during the last five months of the study, which were the rainy season (p<0.001).CONCLUSIONS:VectoLex(r) CG reduced immature Anopheles darlingi infestation levels during the entire study period, and reduced adult mosquito populations during the rainy season.

Primate populations in continuous forest and forest fragments in Central Amazonia

Population densities of six primate species (Saguinus midas, Pithecia pithecia, Cebus apella, Chiropotes satanas, Alouatta seniculus and Ateles paniscus) were estimated in continuous forest and in isolated reserves (one of 100 ha and four of 10 ha). Saguinusdensities in the continuous forest were found to be low, probably due to the lack of edge habitat and second growth favoured by them; Pithecia, Cebus and Ateles populations are also low, possibly because of more widely distributed and/or less abundant food sources than is true for other Amazonian regions, although hunting in the past, particularly of Ateles may also be a contributing factor; and Chiropotes and Alouatta densities were found to be similar to those observed in other areas of Amazonas forests. Ateles and Chiropotes, which occupy ranges on the order of three km2 were excluded from the 100-ka reserve at the time of its isolation. Unfortunately populations were not known prior to isolation of this reserve but during isolation there remained four groups of Saguinus, two Pitheciagroups, one Cebus groups and five Alouatta groups. One Saguinus group disappeared two months later, and one year post-isolation the Cebus group also left the reserve. Single Alouatta groups survive in the isolated 10-ha reserves. Saguinus, present in the four 10-ha reserves following isolation, have disappeared from two of them. One 10-ha reserve retains a group of Pithecia.

Soil physical and Chemical properties of cultivated pasture on forest land, Roraima, Brazil .

Soil conditions under pasture were examined in a range of sites representing the sequence of conversion of forest to pasture at two locations in the vicinity of Ilha de Maracã, Roraima. Comparisons were made with adjacent savana. Soil bulk densities shown to increase after forest clearance and soil chemical data indicate that the initial beneficial effects on nutrient supply of burning forest debris are rather short-lived. Very low levels of available phosphorus prevail in areas of savanna and cultivated pasture of all ages. Variations in the status of older cultivated pastures are mainly attributable to different grazing levelt.

PRELIMINARY RESULTS OF A LARGE-SCALE TREE INVENTORY OF UPLAND RAIN FOREST IN THE CENTRAL AMAZON

A large-scale inventory of trees > 10cm DBH was conducted in the upland "terra firme" rain forest of the Distrito Agropecuário da SUFRAMA (Manaus Free Zone Authority Agricultural District) approximately 65Km north of the city of Manaus (AM), Srasil. Thegeneral appearance and structure of the forest is described together with local topography and soil texture. Thepreliminary results of the Inventory provide a minimum estimate of 698 tree species in 53 families in the 40Km radius sampled, including 17 undescribed species. Themost numerically abundant families, Lecythidaceae, Leguminosae, 5apotaceae and Burseraceae as also among the most species rich families. One aspect of this diverse assemblage is the proliferation of species within certain genera, Including 26 genera In 17 families with 6 or more species or morphospecies. Most species have very low abundances of less than 1 tree per hectare. While more abundant species do exist at densities ranging up to a mean of 12 trees per ha, many have clumped distributions leading to great variation in local species abundance. The degree of similarity between hectare samples based int the Coefficient of Community similarity Index varies widely over different sample hectares for five ecologically different families. Soil texture apparently plays a significant role In determining species composition in the different one hectare plots examined while results for other variable were less consistent. Greater differences in similarity indices are found for comparisons with a one hectare sample within the same formation approximately 40Km to the south. It is concluded that homogeneity of tree community composition within this single large and diverse yet continuous upland forest formation can not be assumed.

Heliconia acuminata reproductive success is independent of local floral density

Reproductive plants in tropical forests are patchily distributed, with some in large aggregations of reproductive consepecifics while others are relatively isolated. This variation in floral density is hypothesized to have a major effect on plant reproductive success, since individuals in higher density neighborhoods can attract more or higher quality pollinators. We experimentally tested this hypothesis with populations of the understory herb Heliconia acuminata in central Amazonia. We created replicated plots in which reproductive plant density spanned the range of naturally occurring floral neighborhood size, then measured three surrogates of plant fitness in focal plants in each array. There was no significant difference between any of the three floral neighborhood treatments in total seed production, fruit set, or the number of seeds produced per fruit. Pollinator visitation rates to plants in all treatments were extremely low, with many plants not visited at all during the observation period. This could be because H. acuminata's hummingbird pollinators are unable to find the widely scattered reproductive plants, however this hypothesis appears unlikely. Instead, natural flowering plant densities may simply be below the threshold value at which neighborhood effects become important, even in "high-density" aggregations. Nutrient limitation, selective fruit abortion, and reproduction via male rather than female function may also be playing a role. We argue the absence of neighborhood effects may be a general phenomenon in central Amazonian forests, though additional experiments with other plant-pollinator systems are needed to determine the extent to which this hypothesis is supported.

Outcrossing rate of Couratari multiflora (J.Smith) Eyma (Lecythidaceae), a low-density tropical tree species from a central Amazonian rainforest

A multilocus mixed-mating model was used to evaluate the mating system of a population of Couratari multiflora, an emergent tree species found in low densities (1 individual/10 ha) in lowland forests of central Amazonia. We surveyed and observed phenologically 41 trees in an area of 400 ha. From these, only four mother trees were analyzed here because few of them set fruits, which also suffered high predation. No difference was observed between the population multilocus outcrossing rate (t mp = 0.953 ± 0.040) and the average single locus rate (t sp = 0.968 ± 0.132). The four mother trees were highly outcrossed (t m ~ 1). Two out of five loci showed departures from the Hardy-Weinberg Equilibrium (HWE) expectations, and the same results occurred with the mixed-mating model. Besides the low number of trees analyzed, the proportion of loci in HWE suggests random mating in the population. However, the pollen pool was heterogeneous among families, probably due to both the small sample number and the flowering of trees at different times of the flowering season. Reproductive phenology of the population and the results presented here suggest, at least for part of the population, a long-distance pollen movement, around 1,000 m.