Toxoplasma gondii: Comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 × 107 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as gold standard and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA × IFAT (κ = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs. © 2005 Elsevier Inc. All rights reserved.

Detection of HTLV-IIa in blood donors in an urban area of the Amazon Region of Brazil (Belém, PA)

Os vírus linfotrópicos de células T humanas tipo I (HTLV-I) e tipo II (HTLV-II) são membros de um grupo de retrovírus de mamíferos com propriedades biológicas similares que apresentam como uma das principais rotas de transmissão a transfusão sangüínea. O HTLV-I é endêmico em diferentes áreas geográficas e está associado a vários distúrbios clínicos. O HTLV-II é endêmico em vários grupos indígenas das Américas e em usuários de drogas intravenosas na América do Norte e do Sul, Europa e Sudeste da Ásia. Durante o ano de 1995, todos os doadores de sangue positivos para HTLV-I/II no Banco de Sangue do Estado (HEMOPA), foram direcionados a um médico e ao Laboratório de Virologia na Universidade Federal do Pará, para consulta, aconselhamento e confirmação do diagnóstico laboratorial. Trinta e cinco soros foram testados por um ensaio imunoenzimático e confirmados por um Western blot que discrimina as infecções por HTLV-I e HTLV-II. Amostras soropositivas para HTLV-II foram submetidas à reação em cadeia da polimerase (PCR) para as regiões genômicas env e pX e confirmaram ser do subtipo IIa. Esta é a primeira detecção, em Belém, da presença da infecção pelo HTLV-IIa em doadores de sangue. Estes resultados enfatizam que o HTLV-II está presente em áreas urbanas da região Amazônica e a necessidade de incluir testes de triagem capazes de detectar anticorpos para ambos os tipos de HTLV.

De novo transcriptome assembly for a non-model species, the blood-sucking bug Triatoma brasiliensis, a vector of Chagas disease

High throughput sequencing (HTS) provides new research opportunities for work on non-model organisms, such as differential expression studies between populations exposed to different environmental conditions. However, such transcriptomic studies first require the production of a reference assembly. The choice of sampling procedure, sequencing strategy and assembly workflow is crucial. To develop a reliable reference transcriptome for Triatoma brasiliensis, the major Chagas disease vector in Northeastern Brazil, different de novo assembly protocols were generated using various datasets and software. Both 454 and Illumina sequencing technologies were applied on RNA extracted from antennae and mouthparts from single or pooled individuals. The 454 library yielded 278 Mb. Fifteen Illumina libraries were constructed and yielded nearly 360 million RNA-seq single reads and 46 million RNA-seq paired-end reads for nearly 45 Gb. For the 454 reads, we used three assemblers, Newbler, CAP3 and/or MIRA and for the Illumina reads, the Trinity assembler. Ten assembly workflows were compared using these programs separately or in combination. To compare the assemblies obtained, quantitative and qualitative criteria were used, including contig length, N50, contig number and the percentage of chimeric contigs. Completeness of the assemblies was estimated using the CEGMA pipeline. The best assembly (57,657 contigs, completeness of 80 %, < 1 % chimeric contigs) was a hybrid assembly leading to recommend the use of (1) a single individual with large representation of biological tissues, (2) merging both long reads and short paired-end Illumina reads, (3) several assemblers in order to combine the specific advantages of each.

Risk factors for hepatitis C virus infection in Inland Brazil: An analysis of pooled epidemiological sectional studies

In order to assess the contribution of different parenteral routes as risk exposure to the hepatitis C virus (HCV), samples from nine surveys or cross-sectional studies conducted in two Brazilian inland regions were pooled, including a total of 3,910 subjects. Heterogeneity among the study results for different risk factors was tested and the results were shown to be homogeneous. Anti-HCV antibodies were observed in 241 individuals, of which 146 (3.7%, 95% CI?=?3.24.4) had HCV exposure confirmed by immunoblot analysis or PCR test. After adjustment for relevant variables, a correlation between confirmed HCV exposure and injection drug use, tattooing, and advance age was observed. In a second logistic model that included exposures not searched in all nine studies, a smaller sample was analyzed, revealing an independent HCV association with past history of surgery and males who have sex with other males, in addition to repeated injection drug use. Overall, these analyses corroborate the finding that injection drug use is the main risk factor for HCV exposure and spread, in addition to other parenteral routes. J. Med. Virol. 84:756762, 2012. (C) 2012 Wiley Periodicals, Inc.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Brossi P.M., Baccarin R.Y.A. & Massoco C.O. 2012 Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro? Pesquisa Veterinaria Brasileira 32(12):1355-1360. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Butanta, Sao Paulo, SP 5508-210, Brazil. E-mail: baccarin@ usp.br Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)(4) - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients

The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking Fc gamma RIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

β-Glucan Antigenemia Anticipates Diagnosis of Blood Culture–Negative Intraabdominal Candidiasis

Rationale: Life-threatening intraabdominal candidiasis (IAC) occurs in 30 to 40% of high-risk surgical intensive care unit (ICU) patients. Although early IAC diagnosis is crucial, blood cultures are negative, and the role of Candida score/colonization indexes is not established. Objectives: The aim of this prospective Fungal Infection Network of Switzerland (FUNGINOS) cohort study was to assess accuracy of 1,3-β-d-glucan (BG) antigenemia for diagnosis of IAC. Methods: Four hundred thirty-four consecutive adults with abdominal surgery or acute pancreatitis and ICU stay 72 hours or longer were screened: 89 (20.5%) at high risk for IAC were studied (68 recurrent gastrointestinal tract perforation, 21 acute necrotizing pancreatitis). Diagnostic accuracy of serum BG (Fungitell), Candida score, and colonization indexes was compared. Measurements and Main Results: Fifty-eight of 89 (65%) patients were colonized by Candida; 29 of 89 (33%) presented IAC (27 of 29 with negative blood cultures). Nine hundred twenty-one sera were analyzed (9/patient): median BG was 253 pg/ml (46–9,557) in IAC versus 99 pg/ml (8–440) in colonization (P < 0.01). Sensitivity and specificity of two consecutive BG measurements greater than or equal to 80 pg/ml were 65 and 78%, respectively. In recurrent gastrointestinal tract perforation it was 75 and 77% versus 90 and 38% (Candida score ≥ 3), 79 and 34% (colonization index ≥ 0.5), and 54 and 63% (corrected colonization index ≥ 0.4), respectively. BG positivity anticipated IAC diagnosis (5 d) and antifungal therapy (6 d). Severe sepsis/septic shock and death occurred in 10 of 11 (91%) and 4 of 11 (36%) patients with BG 400 pg/ml or more versus 5 of 18 (28%, P = 0.002) and 1 of 18 (6%, P = 0.05) with BG measurement less than 400 pg/ml. β-Glucan decreased in IAC responding to therapy and increased in nonresponse. Conclusions: BG antigenemia is superior to Candida score and colonization indexes and anticipates diagnosis of blood culture–negative IAC. This proof-of-concept observation in strictly selected high-risk surgical ICU patients deserves investigation of BG-driven preemptive therapy.

Repeated measurements of cerebral blood flow in the left superior temporal gyrus reveal tonic hyperactivity in patients with auditory verbal hallucinations: a possible trait marker

Background: The left superior temporal gyrus (STG) has been suggested to play a key role in auditory verbal hallucinations (AVH) in patients with schizophrenia. Methods: Eleven medicated subjects with schizophrenia and medication-resistant AVH and 19 healthy controls underwent perfusion magnetic resonance (MR) imaging with arterial spin labeling (ASL). Three additional repeated measurements were conducted in the patients. Patients underwent a treatment with transcranial magnetic stimulation (TMS) between the first 2 measurements. The main outcome measure was the pooled cerebral blood flow (CBF), which consisted of the regional CBF measurement in the left STG and the global CBF measurement in the whole brain. Results: Regional CBF in the left STG in patients was significantly higher compared to controls (p < 0.0001) and to the global CBF in patients (p < 0.004) at baseline. Regional CBF in the left STG remained significantly increased compared to the global CBF in patients across time (p < 0.0007), and it remained increased in patients after TMS compared to the baseline CBF in controls (p < 0.0001). After TMS, PANSS (p = 0.003) and PSYRATS (p = 0.01) scores decreased significantly in patients. Conclusions: This study demonstrated tonically increased regional CBF in the left STG in patients with schizophrenia and auditory hallucinations despite a decrease in symptoms after TMS. These findings were consistent with what has previously been termed a trait marker of AVH in schizophrenia.

Differential influence of maternal and fetal pregnancy factors on the in-vitro induction of human regulatory T cells: a preliminary study.

PROBLEM Given the important role of regulatory T cells (Treg) for successful pregnancy, the ability of soluble maternal and fetal pregnancy factors to induce human Treg was investigated. METHOD OF STUDY Peripheral blood mononuclear cells (PBMCs) or isolated CD4+CD25‒ cells were cultured in the presence of pooled second or third trimester pregnancy sera, steroid hormones or supernatants from placental explants, and the numbers and function of induced CD4+CD25+FOXP3+ Treg were analysed. RESULTS Third trimester pregnancy sera and supernatants of early placental explants, but not sex steroid hormones, induced an increase of Tregs from PBMCs. Early placental supernatant containing high levels of tumour necrosis factor-α, interferon-γ, interleukins -1, -6 and -17, soluble human leucocyte antigen-G, and transforming growth factor-β1, increased the proportion of Treg most effectively and was able to induce interleukin-10-secreting-Treg from CD4+CD25‒cells. CONCLUSIONS Compared with circulating maternal factors, placental- and fetal-derived factors appear to exert a more powerful effect on numerical changes of Treg, thereby supporting fetomaternal tolerance during human pregnancy.

Expression of TNF-$\alpha$ protein and C -FOS and TNF mRNA during human peripheral blood monocyte maturation and activation

Human peripheral blood monocytes (HPBM) were isolated by centrifugal elutriation from mononuclear cell enriched fractions after routine plateletapheresis and the relationship between maturation of HPBM to macrophage-like cells and activation for tumoricidal activity determined. HPBM were cultured for various times in RPMI 1640 supplemented with 5% pooled human AB serum and cytotoxicity to $\sp{125}$IUDR labeled A375M, a human melanoma cell line, and TNF-$\alpha$ release determined by cytolysis of actinomycin D treated L929 cells. Freshly isolated HPBM or those exposed to recombinant IFN-$\gamma$(1.0 U/ml) were not cytolytic and did not release TNF-$\alpha$ into culture supernatants. Exposure to bacterial lipopolysaccharide (LPS, 1.0 $\upsilon$g/ml) stimulated cytolytic activity and release of TNF-$\alpha$. Maximal release of TNF-$\alpha$ protein occurred at 8 hrs and returned to baseline by 72 hrs. Expression of TNF-$\alpha$ protein was determined by Western blotting. Neither freshly isolated nor IFN-$\gamma$ treated HPBM expressed TNF protein at any time during in vitro culture. LPS treated HPBM maximally expressed the 17KD TNF-$\alpha$ protein at 8 hrs, and protein was not detected after 36 hrs of in vitro culture. Expression of TNF-$\alpha$ mRNA was determined by Northern blotting. Freshly isolated HPBM express TNF-$\alpha$ mRNA which decays to basal levels by 6 hrs of in vitro culture. IFN-$\gamma$ treatment maintains TNF-$\alpha$ mRNA expression for up to 48 hrs of culture, after which it is undetectable. LPS induces TNF-$\alpha$ mRNA after 30 minutes of exposure with maximal accumulation occurring between 4 to 8 hrs. TNF mRNA was not detected in control HPBM at any time after 6 hrs or IFN-$\gamma$ treated HPBM after 48 hrs of in vitro culture. A pulse of LPS the last 24 hrs of in vitro culture induces the accumulation of TNF-$\alpha$ mRNA in HPBM cultured for 3, 5, and 7 days, with the magnitude of induction decreasing approximately 10 fold between 3 and 7 days. Induction of TNF-$\alpha$ mRNA occurred in the absence of detectable TNF-$\alpha$ protein or supernatant activity. Maturation of HPBM to macrophage-like cells controls competence for activation, magnitude and duration of the activation response. ^

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Levels of 12 perfluorinated chemicals in pooled Australian serum, collected 2002-2003, in relation to age, gender, and region

Pooled serum samples from 3802 Australian residents were analyzed for four perfluoroalkylsulfonates, seven perfluoroalkylcarboxylates, and perfluorooctanesulfonamide (PFOSA). Serum was collected from men and women of five different age groups and from rural and urban regions in Australia. The highest mean concentration was obtained for perfluorooctane sulfonate (PFOS, 20.8 ng/mL) followed by perfluorooctanoic acid (PFOA, 7.6 ng/mL), perfluorohexane sulfonate (PFHxS, 6.2 ng/mL), perfluorononanoic acid (PFNA, 1.1 ng/mL), and PFOSA (0.71 ng/mL). Additional four PFCs were detected in 5-18 % of the samples at concentrations near the detection limits (0.1-0.5 ng/mL). An increase in PFOS concentration with increasing age in both regions and genders was observed. The male pool levels of some of the age groups compared to females were higher for PFOS, PFOA, and PFHxS. In contrast, PFNA concentrations were higher in the female pools. No substantial difference was found in levels of PFCs between the urban and rural regions. The levels are equal or higher than previously reported serum levels in Europe and Asia but lower compared to the U. S. A. These results suggest that emissions from production in the Northern Hemisphere are of less importance for human exposure.

Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia.

Background: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent. Objective: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection. Patient and Methods: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene). Results: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result. Conclusion: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.