Treatment and seroconversion in a cohort of children suffering from recent chronic Chagas infection in Yoro, Honduras

Between 1999-2002, Médécins Sans Frontières-Spain implemented a project seeking to determine the efficacy and safety of benznidazole in the treatment of recent chronic Chagas disease in a cohort of seropositive children in the Yoro Department, Honduras. A total of 24,471 children were screened for Trypanosoma cruzi IgG antibodies through conventional enzyme-linked immunosorbent assays (ELISA) on filter paper. Recombinant ELISA (0.93% seroprevalence) showed 256 initially reactive cases, including 232 confirmed positive cases. Of these, 231 individuals were treated with benznidazole (7.5 mg/kg/day) for 60 days and were followed with a strict weekly medical control and follow-up protocol. At the end of the project, 229 patients were examined by the Honduras Secretariat of Health for post-treatment serological assessments; 88.2% seroconverted after 18 months and 93.9% seroconverted after three years. No differences were found in the seroconversion rates according to age or sex. Most of the side effects of the treatment were minor. These results support the argument that in areas where T. cruzi I is predominant and in areas affected by T. cruzi II, when vector transmission has been interrupted, Chagas disease diagnosis and treatment are feasible, necessary and ethically indisputable.

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite's immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2-carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation.

Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.

Evaluation of local immune response to Fasciola hepatica experimental infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine antigen

Protection against Fasciola hepatica in goats immunized with a synthetic recombinant antigen from Schistosoma mansoni fatty acid-binding protein 14 (rSm14) was investigated by assessing worm burdens, serum levels of hepatic enzymes, faecal egg count and hepatic damage, which was evaluated using gross and microscopic morphometric observation. The nature of the local immune response was assessed by examining the distribution of CD2+, CD4+, CD8+ and γ´+ T lymphocytes along with IgG+, IL-4+ and IFN-γ+ cells in the liver and hepatic lymph nodes (HLN). The goats used consisted of group 1 (unimmunized and uninfected), group 2 [infected control - immunized with Quillaia A (Quil A)] and group 3 (immunized with rSm14 in Quil A and infected), each containing seven animals. Immunization with rSm14 in Quil A adjuvant induced a reduction in gross hepatic lesions of 56.6% (p < 0.001) and reduced hepatic and HLN infiltration of CD2+, CD4+, CD8+ and γ´+ T lymphocytes as well as IL-4+ and IFN-γ+ cells (p < 0.05). This is the first report of caprine immunization against F. hepatica using a complete rSm14 molecule derived from S. mansoni. Immunization reduced hepatic damage and local inflammatory infiltration into the liver and HLN. However, considering that Quil A is not the preferential/first choice adjuvant for Sm14 immunization, further studies will be undertaken using the monophosphoryl lipid A-based family of adjuvants during clinical trials to facilitate anti-Fasciolavaccine development.

Parvovirus B19 antibodies and correlates of infection in pregnant women attending an antenatal clinic in central Nigeria

Human parvovirus B19 infection is associated with spontaneous abortion, hydrops foetalis, intrauterine foetal death, erythema infectiosum (5th disease), aplastic crisis and acute symmetric polyarthropathy. However, data concerning Nigerian patients with B19 infection have not been published yet. The purpose of this study was to establish the prevalence of B19 IgG and IgM antibodies, including correlates of infection, among pregnant women attending an antenatal clinic in Nigeria. Subsequent to clearance from an ethical committee, blood samples were collected between August-November 2008 from 273 pregnant women between the ages of 15-40 years who have given their informed consent and completed self-administered questionnaires. Recombinant IgG and IgM enzyme linked immunosorbent assay kits (Demeditec Diagnostics, Germany) were used for the assays. Out of the 273 participants, 111 (40.7%) had either IgG or IgM antibodies. Out of these, 75 (27.5%) had IgG antibodies whereas 36 (13.2%) had IgM antibodies, and those aged 36-40 years had the highest prevalence of IgG antibodies. Significant determinants of infection (p < 0.05) included the receipt of a blood transfusion, occupation and the presence of a large number of children in the household. Our findings have important implications for transfusion and foeto-maternal health policy in Nigeria. Routine screening for B19 IgM antibodies and accompanying clinical management of positive cases should be made mandatory for all Nigerian blood donors and women of childbearing age.

Serological evidence of hantavirus infection in rural and urban regions in the state of Amazonas, Brazil

Hantavirus disease is caused by the hantavirus, which is an RNA virus belonging to the family Bunyaviridae. Hantavirus disease is an anthropozoonotic infection transmitted through the inhalation of aerosols from the excreta of hantavirus-infected rodents. In the county of Itacoatiara in the state of Amazonas (AM), Brazil, the first human cases of hantavirus pulmonary and cardiovascular syndrome were described in July 2004. These first cases were followed by two fatal cases, one in the municipality of Maués in 2005 and another in Itacoatiara in 2007. In this study, we investigated the antibody levels to hantavirus in a population of 1,731 individuals from four different counties of AM. Sera were tested by IgG/IgM- enzyme-linked immune-sorbent assay using a recombinant nucleocapsid protein of the Araraquara hantavirus as an antigen. Ten sera were IgG positive to hantavirus (0.6%). Among the positive sera, 0.8% (1/122), 0.4% (1/256), 0.2% (1/556) and 0.9% (7/797) were from Atalaia do Norte, Careiro Castanho, Itacoatiara and Lábrea, respectively. None of the sera in this survey were IgM-positive. Because these counties are distributed in different areas of AM, we can assume that infected individuals are found throughout the entire state, which suggests that hantavirus disease could be a local emerging health problem.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

Immunological diagnostic methods for Trypanosoma cruzi depend specifically on the presence of antibodies and parasitological methods lack sensitivity during the chronic and “indeterminate” stages of the disease. This study performed a serological survey of 1,033 subjects from 52 rural communities in 12 of the 18 municipalities in the state of Querétaro, Mexico. We detected anti-T. cruzi antibodies using the following tests: indirect haemagglutination assay (IHA), indirect immunofluorescence assay (IFA), ELISA and recombinant ELISA (rELISA). We also performed Western blot (WB) analysis using iron superoxide dismutase (FeSOD), a detoxifying enzyme excreted by the parasite, as the antigen. Positive test results were distributed as follows: ELISA 8%, rELISA 6.2%, IFA and IHA 5.4% in both cases and FeSOD 8%. A comparative study of the five tests was undertaken. Sensitivity levels, specificity, positive and negative predictive values, concordance percentage and kappa index were considered. Living with animals, trips to other communities, gender, age, type of housing and symptomatology at the time of the survey were statistically analysed using SPSS software v.11.5. Detection of the FeSOD enzyme that was secreted by the parasite and used as an antigenic fraction in WBs showed a 100% correlation with traditional ELISA tests.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Evidence of hantavirus infection in wild rodents captured in a rural area of the state of São Paulo, Brazil

Hantaviruses are the etiological agents of the Hantavirus Cardio-Pulmonary Syndrome, a serious rodent-borne disease in Brazil. In order to investigate the occurrence of hantavirus infection in wild rodents, a survey was conducted in three different suburban areas of the municipality of Pedreira, State of São Paulo, Brazil. Of the 145 wild animals captured belonging to 12 different species identified by morphology and karyological analysis, 107 were rodents of the following species: Akodon montensis, Bolomys lasiurus, Calomys tener, Oligoryzomys nigripes, Oligoryzomys flavescens, and Myocastor coypus. Blood samples from these rodents were assayed for the presence of antibodies against hantavirus by IgG ELISA using Andes recombinant nucleocapsid antigen. Antibody reactive to Andes virus was found in two different species, O. nigripes and O. flavescens. These results indicate a potential risk for hantavirus transmission to humans in this area, where reservoir rodents are present in peridomestic settings.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 10(6.9)TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 10(7.6)TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV56/90, 10(7.1)TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 10(7.5)TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (10(7.5)TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 10(7.3)TCID50). All vaccinated animals developed VN titers from 2 to 16 at day 30 pv. A boost vaccination performed at day 240 pv resulted in a rapid and strong anamnestic antibody response, with VN titers reaching from 16 to 256 at day 14 post-booster. Again, serum samples remained negative for gE antibodies. Selected serum samples from vaccinated animals showed a broad VN activity against nine BoHV-5 and eight BoHV-1 field isolates. These results show that the recombinant virus is attenuated, immunogenic for calves and induces an antibody response differentiable from that induced by natural infection. Thus, the recombinant BoHV-5gE/TKΔ is an adequate candidate strain for a modified live vaccine.

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5) recombinant deleted in the gene encoding the enzyme thymidine kinase (TK) in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99) (n=18) at a low titer (10(5.5)TCID50) shed virus in nasal secretions in titers up to 10(4.5)TCID50 for up to 12 days (average: 9.8 days [5-12]) and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1)TCID50) also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3)TCID50) and for a shorter period (average: 6.6 days [2-11]) and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi) revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi), DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx) administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9]) and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability to invade the brain and to establish latent infection. Additional studies are underway to determine the biological and molecular mechanisms underlying the inability of BoHV-5TKΔ to reactivate from latency.