A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (São Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 schoolchildren were submitted to ELISA and the data compared to the results of the parasitological method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5%, 43.0%, and 56.2% by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Improved Methods for the Diagnosis of African Trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.

Clinical, Parasitological and Immunological Aspects of Experimental Infection with Trypanosoma evansi in Dogs

This research investigated the pattern of antibody response by means of enzyme linked immunosorbent assay (Elisa) and indirect fluorescent antibody test (IFAT) through the course of experimental Trypanosoma evansi infection in dogs. Clinical and parasitological features were also studied. The average prepatent period was 11.2 days and parasitaemia showed an undulating course. Biometrical study of parasites revealed a mean total length of 21.68mm. The disease was characterized by intermittent fever closely related to the degree of parasitaemia and main clinical signs consisted of pallor of mucous membrane, edema, progressive emaciation and enlargement of palpable lymph nodes. Diagnostic antibody was detected within 12 to 15 days and 15 to 19 days of infection by IFAT and Elisa, respectively. High and persistent antibody levels were detected by both tests and appeared not to correlate with control of parasitaemia

The role of neutralizing antibodies for mouse mammary tumor virus transmission and mammary cancer development.

Mouse mammary tumor virus (MMTV) infection establishes chronic germinal centers and a lifelong neutralizing Ab response. We show that removal of the draining lymph node after establishment of the germinal center reaction led to complete loss of neutralizing Abs despite comparable infection levels in peripheral lymphocytes. Importantly, in the absence of neutralization, only the exocrine organs mammary gland, salivary gland, pancreas, and skin showed strikingly increased infection, resulting in accelerated mammary tumor development. Induction of stronger neutralization did not influence chronic infection levels of peripheral lymphoid organs but strongly inhibited mammary gland infection and virus transmission to the next generation. Taken together, we provide evidence that a tight equilibrium in virus neutralization allows limited infection of exocrine organs and controls cancer development in susceptible mouse strains. These experiments show that a strong neutralizing Ab response induced after infection is not able to control lymphoid MMTV infection. Strong neutralization, however, is capable of blocking amplification of mammary gland infection, tumor development, and virus transmission to the next generation. The results also indicate a role of neutralization in natural resistance to MMTV infection.

Hepatitis C virus prevalence among an immigrant community to the Southern Amazon, Brazil

A community-based random survey was conducted in a southern Brazilian Amazonian county aiming to investigate hepatitis C virus (HCV) infection prevalence and the association of demographic variables and lifestyle behaviours. Seven hundred eighty individuals were serologically screened with a third generation enzyme-linked immunosorbent assay to detect anti-HCV antibodies between 1994/1995. Positive samples were retested for confirmation with a line immunoassay (LIA, Inno-LIA HCV Ab III). Most of these subjects were low income and came from southern Brazilian states (65.8). Two point four percent (IC 95% 1.2%- 4.6%) of the subjects had LIA-confirmed anti-HCV antibodies reactivity. The age-specific prevalence of HCV antibodies slightly increased with age, with the highest prevalence after the age of 40 years. The results of multivariate analysis indicate a strong association between HCV antibodies and previous surgery and history of intravenous drug use. There were no apparent association with gender, hepatitis B virus markers, blood transfusion, and sexual activity. Mean time living in Amazon did not differ between confirmed and negative anti-HCV individuals. The present data point out an intermediate endemicity of HCV infection among this immigrant community to the Amazon region and that few HCV infected participants presented known risk factors.

Frequency of human toxocariasis in Jos, Plateau State, Nigeria

The enzyme-linked immunosorbent assay (Elisa) was used to examine sera of 104 children and adults in Jos, Plateau State, Nigeria for anti-toxocaral antibodies, out of which 31 (29.8%) were reactive. The seropositive rates were 30.4% for adults, 29.6% for children, 34% for females and 25.9% for males. However, the differences were not significant by age and sex. A highly significant association (p < 0.001) was observed between seropositivity and geophagia but none between seropositivity and dog ownership (p > 0.05).

Malaria vectors in the municipality of Serra do Navio, State of Amapá, Amazon Region, Brazil

We conducted a survey to determine the vectors of malaria in six localities of Serra do Navio municipality, State of Amapá, from 1990 to 1991. Malaria infection rates of 29.3%, 6.2% and 20.4% were detected by human blood smears in Colônia Água Branca, Porto Terezinha and Arrependido, respectively. There was no malaria infection detected in Serra do Navio. Fifteen species were identified among 3,053 anopheline mosquitoes collected by human bait and 64.4% were identified as Anopheles albitarsis s.l., 16.7% An. braziliensis, 9.5% An. nuneztovari and 5.8% An. triannulatus. An. darlingi, the main vector of malaria in the Amazon region of Brazil, was scare. Using enzyme-linked immunosorbent assay (ELISA), a total positive rate of 0.8% (23/2876) was found for six species: fifteen An. albitarsis s.l., four An. nuneztovari, and one of each: An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Nine of 23 positive mosquitoes were infected with Plasmodium malariae, eight with P. vivax VK210, three with P. vivax VK247 and three with P. falciparum. Since An. albitarsis s.l. was collected feeding on humans, was present in the highest density and was positive by ELISA for malaria sporozoites, it probably plays an important role in malaria transmission in this area.

High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

High prevalence anti-Trypanosoma cruzi antibodies, among blood donors in the State of Puebla, a non-endemic area of Mexico

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.

Measuring the inflammasome.

Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. The hallmarks of inflammasome activation are the secretion of the mature forms of Caspase-1 and IL-1β from cells of the innate immune system. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells (BMDCs) as a model system. The protocol includes the generation and handling of BMDCs, the stimulation of BMDCs with established Nlrp3 inflammasome activators, and the measurement of activation by both ELISA and western blot. These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types.

Prevalence of Toxocara infection in schoolchildren from the Butantã region, São Paulo, Brazil

Visceral larva migrans syndrome by Toxocara affects mainly children between 2 and 5 years of age, it is generally asymptomatic, and the seroprevalence varies from 3 to 86% in different countries. A total of 399 schoolchildren from 14 public schools of the Butantã region, São Paulo city, Brazil, were evaluated by Toxocara serology (enzyme-linked immunosorbent assay). Epidemiological data to the Toxocara infection obtained from a protocol were submitted to multiple logistic regression analysis for a risk profile definition. Blood was collected on filter paper by finger puncture, with all samples tested in duplicate. Considering titers > 1/160 as positive, the seroprevalence obtained was 38.8%. Among infected children, the mean age was 9.4 years, with a similar distribution between genders. A significant association was observed with the presence of onychophagia, residence with a dirty backyard, living in a slum, previous wheezing episodes, school attended, and family income (p < 0.05). All data, except "living in a slum", were considered to be determinant of a risk profile for the acquisition of Toxocara infection. A monthly income > 5 minimum salaries represented a protective factor, although of low relevance. Toxocara eggs were found in at least one of the soil samples obtained from five schools, with high prevalence of Toxocara infections, indicating the frequent soil contamination by this agent.

Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB) using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence antibody test (IFAT) results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals). S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.