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Background: Transfusion-transmitted malaria (TTM) is a public health problem in endemic and nonendemic areas. The Brazilian Ministry of Health (MH) requested the development of a nucleic acid amplification test (NAT) for the detection of Plasmodium spp. in public blood centers to increase blood safety., Study Design and Methods: The new Brazilian NAT kit named NAT PLUS HIV/HBV/HCV/Malaria Bio-Manguinhos was first implemented in HEMORIO, a public blood center in the city of Rio de Janeiro. Since October 1, 2022, this blood center has been testing all its blood donations for malaria in a pool of six plasma samples to detect Plasmodium spp. by real-time polymerase chain reaction (PCR)., Results: Since the implementation of the NAT PLUS platform until February 2023, HEMORIO has successfully received and tested 200,277 donations. The platform detected two asymptomatic donors in the city of Rio de Janeiro, which is a nonendemic region for malaria. Our analyses suggested a malaria from the Amazon region caused by Plasmodium vivax, in the first case, while an autochthonous transmission case by Plasmodium malariae was identified in the rural area of Rio de Janeiro state., Discussion: The NAT PLUS platform detects Plasmodium spp. in plasma samples with sensitivity capable of detecting subpatent infections. This is the first time worldwide that a group developed and implemented molecular diagnosis for Plasmodium spp. to be used by public blood centers to avoid TTM., (© 2024 AABB.)

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli . Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability., Competing Interests: The authors declare no conflict of interest. Funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2022 The Author(s). Published by Elsevier Ltd.)

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli . Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability., Competing Interests: The authors declare no conflict of interest. Funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2022 The Authors. Published by Elsevier Ltd.)

In response to the Zika epidemics in Brazil, the ZDC molecular assay (Bio-Manguinhos) was developed and registered at the Brazilian Regulatory Agency of Health Surveillance - ANVISA. The circulation of Zika (ZIKV) Dengue (DENV) and Chikungunya (CHIKV) viruses and their clinical similarities are challenges to correctly diagnose these viruses. The simultaneous detection of ZIKV, DENV and CHIKV is an important tool for diagnosis and surveillance. Here, we present the analytical and clinical performance evaluation of ZDC molecular assay (Bio-Manguinhos) at the public health laboratories three years after its registration at ANVISA. The clinical performance demonstrates the ZDC molecular assay (Bio-Manguinhos) has 100% sensitivity and 100% specificity to detect and discriminate ZIKV, CHIKV, and DENV from clinical plasma samples. The ZDC molecular assay (Bio-Manguinhos) results were highly reproducible and no cross-reactivity was seen during testing with a panel of other infectious agents. In conclusion, the ZDC molecular assay (Bio-Manguinhos) is an accurate and reliable tool to monitor Zika, dengue and chikungunya infections in countries like Brazil with simultaneous circulation of the three viruses., (Copyright © 2021 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Despite several available methodologies for Chagas disease (CD) serological screening, the main limitation of chronic CD diagnosis is the lack of effective tools for large-scale screening and point-of-care diagnosis to be used in different CD epidemiological scenarios. Taking into account that developing such a diagnostic tool will significantly improve the ability to identify CD carriers, we aimed at performing a proof-of-concept study (phase I study) to assess the use of these proteins in a point-of-care platform using serum samples from different geographical settings of Brazil and distinct clinical presentations. The diagnostic accuracy study was conducted on a panel of two WHO International Standards (IS) and 14 sera from T. cruzi -positive and 16 from T. cruzi -negative individuals. The results obtained with the test strips were converted to digital images, allowing quantitative comparison expressed as a relative band intensity ratio (RBI). The diagnostic potential and performance were also determined. Regardless of the geographical origin or clinical presentation, all sera with T. cruzi antibodies returned positive both for IBMP-8.1 and IBMP-8.4 chimeric antigens. The area under the ROC curve (AUC) values was 100% for both antigens, demonstrating an outstanding overall diagnostic accuracy (100%). Based on the data, we believe that the lateral flow assays based on these antigens are promising methodologies for screening CD., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Edimilson D. Silva et al.)

Background: Malaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy., Methods: In Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite., Results: Considering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM., Conclusion: The prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

[This corrects the article DOI: 10.1371/journal.pntd.0006871.].

Hepatitis C virus (HCV) infection is a worldwide health problem. Nowadays, direct-acting antiviral agents (DAAs) are the main treatment for HCV; however, the high level of virus variability leads to the development of resistance-associated variants (RAVs). Thus, assessing RAVs in infected patients is important for monitoring treatment efficacy. The aim of our study was to investigate the presence of naturally occurring resistance mutations in HCV NS3 and NS5 regions in treatment-naïve patients. Ninety-six anti-HCV positive serum samples from blood donors at the Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) were collected retrospectively in 2013 and evaluated in this study. HCV 1a (37.9%), 1b (25.3%), and 3a (36.8%) subtypes were found. The frequency of patients with RAVs in our study was 6.9%. The HCV NS5b sequencing reveled 1 sample with L320F mutation and 4 samples with the C316N/R polymorphism. The analysis of the NS3 region revealed the D168A/G/T (3.45%), S122G (1.15%), and V55A (2.3%) mutations. All samples from genotype 3a (36.8%) presented the V170 I/V non-synonymous mutation. In conclusion, we have shown that mutations in NS3 and NS5b genes are present in Brazilian isolates from therapy-naïve HCV patients.

Background: Canine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health., Methodology/principal Findings: To evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85-99%) vs. 83% (IC 95%; 69-92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49-97%) vs. 54% (IC 95%; 33-74%) for asymptomatic dogs and 94% (IC 95%; 83-99%) vs, 88% (IC 95%; 76-95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81-91%) of sensitivity, 94% (IC 95%; 90-97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59-74%), specificity of 87% (IC 95%; 81-92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52-66%), specificity of 98% (IC 95%; 95-99%) and accuracy of 80% (IC 95%; 76-84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63-77%), specificity of 99% (IC 95%; 97-100%) and accuracy of 86% (IC 95%; 83-89%)., Conclusion: Thus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil., Competing Interests: The authors have declared that no competing interests exist.

The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p = 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values., (Copyright © 2018 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Background: The history of the development and implementation of the Brazilian nucleic acid testing (NAT) platform to detect and discriminate among human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections in blood donors is described here. The results for the sensitivity, reproducibility, and NAT yield of the platform since program implementation are provided., Study Design and Methods: The Brazilian NAT HIV, HCV, and HBV kit was developed and evaluated with regard to analytical sensitivity, specificity, intralot and interlot reproducibility, interfering substances, and genotype and diagnostic sensitivity. Additionally, a sample of identified NAT-yield cases was characterized with regard to viral load., Results: The 95% limits of detection for HIV, HCV, and HBV were 68.02, 102.35, and 9.08 IU/mL, respectively. All replicates were detected with reproducibility assays between the acceptable values. A total of 13,610,536 blood donors was screened from 2010 to 2016, and 63 HIV-yield cases and 28 HCV-yield cases were detected. Among 5,795,424 blood donors screened for HBV from 2014 to 2016, 42 yield cases were found., Conclusion: The Brazilian NAT HIV, HCV, and HBV kit is an automated NAT system suitable for routine blood donor screening in a completely traceable process. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements set by the health ministry for blood donor screening. A significant number of transmission cases were prevented by the implementation of this important program., (© 2017 AABB.)