Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples.

The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.

Novel Genetic Constructs for Production of Recombinant HTLV-1/2 Antigens and Evaluation of Their Reactivity to Plasma Samples from HTLV-1-Infected Patients.

Human T-cell leukemia virus type 1 (HTLV-1) can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion, and organs for transplantation, as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity for recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV-1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints in expressing sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive samples from HTLV-negative samples using enzyme-linked immunosorbent assays. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens, and they demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV-1 infections.

Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil.

BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

A new Trypanosoma cruzi genotyping method enables high resolution evolutionary analyses.

BACKGROUND: Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS: To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS: We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION: As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve.

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.

Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures.

BACKGROUND: Trypanosomatids are a group of protozoan parasites that includes the etiologic agents of important human illnesses as Chagas disease, sleeping sickness and leishmaniasis. These parasites have a significant distinction from other eukaryotes concerning mRNA structure, since all mature mRNAs have an identical species-specific sequence of 39 nucleotides at the 5' extremity, named spliced leader (SL). Considering this peculiar aspect of trypanosomatid mRNA, the aim of the present work was to develop a Trypanosoma cruzi specific in vitro transcription (IVT) linear mRNA amplification method in order to improve parasite transcriptomics analyses. METHODS: We designed an oligonucleotide complementary to the last 21 bases of T. cruzi SL sequence, bearing an upstream T7 promoter (T7SL primer), which was used to direct the synthesis of second-strand cDNA. Original mRNA was then amplified by IVT using T7 RNA polymerase. T7SL-amplified RNA from two distinct T. cruzi stages (epimastigotes and trypomastigotes) were deep sequenced in SOLiD platform. Usual poly(A) + RNA and and T7-oligo(dT) amplified RNA (Eberwine method) were also sequenced. RNA-Seq reads were aligned to our new and improved T. cruzi Dm28c genome assembly (PacBio technology) and resulting transcriptome pattern from these three RNA preparation methods were compared, mainly concerning the conservation of mRNA transcritional levels and DEGs detection between epimastigotes and trypomastigotes. RESULTS: T7SL IVT method detected more potential differentially expressed genes in comparison to either poly(A) + RNA or T7dT IVT, and was also able to produce reliable quantifications of the parasite transcriptome down to 3 ng of total RNA. Furthermore, amplification of parasite mRNA in HeLa/epimastigote RNA mixtures showed that T7SL IVT generates transcriptome quantification with similar detection of differentially expressed genes when parasite RNA mass was only 0.1% of the total mixture (R = 0.78 when compared to poly(A) + RNA). CONCLUSIONS: The T7SL IVT amplification method presented here allows the detection of more potential parasite differentially expressed genes (in comparison to poly(A) + RNA) in host-parasite mixtures or samples with low amount of RNA. This method is especially useful for trypanosomatid transcriptomics because it produces less bias than PCR-based mRNA amplification. Additionally, by simply changing the complementary region of the T7SL primer, the present method can be applied to any trypanosomatid species.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

BACKGROUND: Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES: The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS: The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS: We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Genomic and phylogenetic evidence of VIPER retrotransposon domestication in trypanosomatids.

Transposable elements are important residents of eukaryotic genomes and eventually the host can domesticate them to serve cellular functions. We reported here a possible domestication event of the vestigial interposed retroelement (VIPER) in trypanosomatids. We found a large gene in a syntenic location in Leishmania braziliensis, L. panamensis, Leptomanas pyrrhocoris, and Crithidia fasciculata whose products share similarity in the C-terminal portion with the third protein of VIPER. No remnants of other VIPER regions surrounding the gene sequence were found. We hypothesise that the domestication event occurred more than 50 mya and the conservation of this gene suggests it might perform some function in the host species.

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

DENV-2 Outbreak Associated With Cosmopolitan Genotype Emergence in Western Brazilian Amazon.

Background: Dengue virus (DENV) causes an important disease and directly affects public health, being the arbovirus that presents the highest number of infections and deaths in the Western Brazilian Amazon. This virus is divided into 4 serotypes that have already circulated in the region. Methodology: Molecular characterization of a cohort containing 841 samples collected from febrile patients between 2021 and 2023 was analyzed using a commercial kit to detect the main arboviruses circulating in Brazil: Zika, DENV-1, DENV-2, DENV-3, DENV-4 and, Chikungunya. Subsequently, Sanger sequencing was performed for positive samples. Results: The cohort detected 162 positive samples, 12 for DENV-1 and 150 identified as DENV-2, indicating co-circulation of serotypes. The samples were subjected to sequencing and the analysis of the sequences that obtained good quality revealed that 5 samples belonged to the V genotype of DENV-1 and 46 were characterized as DENV-2 Cosmopolitan genotype-lineage 5. Conclusion: The results allowed us to identify for the first time the Cosmopolitan genotype in Rondônia, Brazilian Western Amazon, and its fast spread dispersion.

Validation of the NAT Chagas IVD Kit for the Detection and Quantification of Trypanosoma cruzi in Blood Samples of Patients with Chagas Disease.

In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 104 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs-TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.

Quantitative proteomics of Trypanosoma cruzi during metacyclogenesis.

Trypanosoma cruzi is the etiologic agent of Chagas disease, which is estimated to affect over eight million people around the world. Trypanosoma cruzi has a complex life cycle, involving insect and mammalian hosts and four distinct developmental stages: epimastigotes, metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. Metacyclogenesis is the process by which T. cruzi epimastigotes differentiate into metacyclic trypomastigotes and acquire infectivity, and involves differential gene expression associated with acquisition of virulence. In T. cruzi, gene expression regulation is achieved mainly posttranscriptionally. Therefore, proteomics-based approaches are extremely useful for gaining a better understanding of the changes that occur in the stage-regulated gene expression program of the parasite at the molecular level. Here, we performed an in-depth quantitative MS-based proteomic study of T. cruzi metacyclogenesis and quantified almost 3000 proteins expressed during the process of differentiation. To the best of our knowledge, this work is the most comprehensive quantitative proteomics study of different cell populations of T. cruzi available so far. We identified relevant proteins and pathways involved in the parasite's differentiation and infectivity acquisition, opening new perspectives for further studies that could, ultimately, lead to the identification of new targets for chemotherapy.

Hepatitis C virus quantification in serum and saliva of HCV-infected patients.

The hepatitis C virus (HCV) can be detected in blood and other bodily fluids, such as saliva, semen and gastric juices. The aim of this study was to compare the HCV viral loads in the serum and saliva of infected patients. Twenty-nine patients with detectable HCV RNA in their serum and saliva were included in this study. The HCV viral loads were determined through quantitative real-time polymerase chain reactions. The median viral RNA levels were 5.78 log10 copies in the serum and 3.32 log10 copies in the saliva. We observed that the salivary HCV viral load was significantly lower than the viral load in the serum. Further studies are required to understand the role of saliva in the diagnosis, management and potential transmission of HCV.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis.

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.

Performance of Chimeric Trypanosoma cruzi Antigens in Serological Screening for Chagas Disease in Blood Banks.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2-95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4-100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

Seroepidemiology of SARS-CoV-2 on a partially vaccinated island in Brazil: Determinants of infection and vaccine response.

Background: A vaccination campaign targeted adults in response to the pandemic in the City of Rio de Janeiro. Objective: We aimed to evaluate the seroprevalence of SARS-CoV-2 antibodies and identify factors associated with seropositivity on vaccinated and unvaccinated residents. Methods: We performed a seroepidemiologic survey in all residents of Paquetá Island, a neighborhood of Rio de Janeiro city, during the COVID-19 vaccine roll-out. Serological tests were performed from June 16 to June 19, 2021, and adjusted seropositivity rates were estimated by age and epidemiological variables. Logistic regression models were used to estimate adjusted ORs for risk factors to SARS-CoV-2 seropositivity in non-vaccinated individuals, and potential determinants of the magnitude of antibody responses in the seropositive population. Results: We included in the study 3,016 residents of Paquetá (83.5% of the island population). The crude seroprevalence of COVID-19 antibodies in our sample was 53.6% (95% CI = 51.0, 56.3). The risk factors for SARS-CoV-2 seropositivity in non-vaccinated individuals were history of confirmed previous COVID-19 infection (OR = 4.74; 95% CI = 3.3, 7.0), being a household contact of a case (OR = 1.93; 95% CI = 1.5, 2.6) and in-person learning (OR = 2.01; 95% CI = 1.4, 3.0). Potential determinants of the magnitude of antibody responses among the seropositive were hybrid immunity, the type of vaccine received, and time since the last vaccine dose. Being vaccinated with Pfizer or AstraZeneca (Beta = 2.2; 95% CI = 1.8, 2.6) determined higher antibody titers than those observed with CoronaVac (Beta = 1.2; 95% CI = 0.9, 1.5). Conclusions: Our study highlights the impact of vaccination on COVID-19 collective immunity even in a highly affected population, showing the difference in antibody titers achieved with different vaccines and how they wane with time, reinforcing how these factors should be considered when estimating effectiveness of a vaccination program at any given time. We also found that hybrid immunity was superior to both infection-induced and vaccine-induced immunity alone, and online learning protected students from COVID-19 exposure.