Monitoring the Establishment of VOC Gamma in Minas Gerais, Brazil: A Retrospective Epidemiological and Genomic Surveillance Study.

Since its first identification in Brazil, the variant of concern (VOC) Gamma has been associated with increased infection and transmission rates, hospitalizations, and deaths. Minas Gerais (MG), the second-largest populated Brazilian state with more than 20 million inhabitants, observed a peak of cases and deaths in March-April 2021. We conducted a surveillance study in 1240 COVID-19-positive samples from 305 municipalities distributed across MG's 28 Regional Health Units (RHU) between 1 March to 27 April 2021. The most common variant was the VOC Gamma (71.2%), followed by the variant of interest (VOI) zeta (12.4%) and VOC alpha (9.6%). Although the predominance of Gamma was found in most of the RHUs, clusters of Zeta and Alpha variants were observed. One Alpha-clustered RHU has a history of high human mobility from countries with Alpha predominance. Other less frequent lineages, such as P.4, P.5, and P.7, were also identified. With our genomic characterization approach, we estimated the introduction of Gamma on 7 January 2021, at RHU Belo Horizonte. Differences in mortality between the Zeta, Gamma and Alpha variants were not observed. We reinforce the importance of vaccination programs to prevent severe cases and deaths during transmission peaks.

Performance differences among commercially available antigen rapid tests for COVID-19 in Brazil.

A rapid and accurate diagnosis is a crucial strategy for containing the coronavirus disease (COVID-19) pandemic. Considering the obstacles to upscaling the use of RT-qPCR, rapid tests based on antigen detection (Ag-RDT) have become an alternative to enhance mass testing, reducing the time for a prompt diagnosis and virus spreading. However, the performances of several commercially available Ag-RDTs have not yet been evaluated in several countries. Here, we evaluate the performance of eight Ag-RDTs available in Brazil to diagnose COVID-19. Patients admitted to tertiary hospitals with moderate or mild COVID-19 symptoms and presenting risk factors for severe disease were included. The tests were performed using a masked protocol, strictly following the manufacturer's recommendations and were compared with RT-qPCR. The overall sensitivity of the tests ranged from 9.8 to 81.1%, and specificity greater than 83% was observed for all the evaluated tests. Overall, slight or fair agreement was observed between Ag-RDTs and RT-PCR, except for the Ag-RDT COVID-19 (Acro Biotech), in which moderate agreement was observed. Lower sensitivity of Ag-RDTs was observed for patients with cycle threshold > 25, indicating that the sensitivity was directly affected by viral load, whereas the effect of the disease duration was unclear. Despite the lower sensitivity of Ag-RDTs compared with RT-qPCR, its easy fulfillment and promptness still justify its use, even at hospital admission. However, the main advantage of Ag-RDTs seems to be the possibility of increasing access to the diagnosis of COVID-19 in patients with a high viral load, allowing immediate clinical management and reduction of infectivity and community transmission.

Impact assessment of different DNA extraction methods for non-invasive molecular diagnosis of tegumentary leishmaniasis.

The aim of this study was to evaluate two methods of nucleic acid extraction (spin-column-based method - commercial kit and direct boil - DB) from swab sampling compared to biopsy sampling for the diagnosis of tegumentary leishmaniasis (TL), (cutaneous - CL and mucocutaneous - MCL forms). The impact of these nucleic acid extraction protocols on different types of PCR and LAMP techniques were compared regarding nucleic acid quality, molecular assays accuracy, indirect quantitation, and costs. The evaluated patients were 57 TL cases (36 CL and 21 MCL) and 34 non-cases. Swab samples extracted by the DB method showed a higher DNA degradation rate and worse DNA quality in comparison to the commercial kit. Molecular tests performed on biopsy samples showed identical or higher performance in all analysis, as compared to their own performance on swab samples for TL (CL and MCL). However, only the SSU rRNA TaqMan™ RT-PCR test showed a significant difference between the performance of biopsy and swab samples extracted by commercial kit. The kDNA-cPCR coupled with swab extracted by commercial kit showed the highest accuracy (95.6%) for TL diagnosis. The sensitivity of the LAMP-RT 18S method in swab samples extracted with a commercial kit (82.5%) was close to that found in biopsy samples (86%) for TL diagnosis. The DB extraction method presented the lowest cost. The use of swab as a minimally-invasive sampling method, associated with an efficient nucleic acid extraction protocol, may represent a low-cost alternative for the diagnosis of CL and MCL.

Diagnostic performance of commercially available COVID-19 serology tests in Brazil.

Timely and accurate laboratory testing is essential for managing the global COVID-19 pandemic. Reverse transcription polymerase chain reaction remains the gold-standard for SARS-CoV-2 diagnosis, but several practical issues limit the test's use. Immunoassays have been indicated as an alternative for individual and mass testing. OBJECTIVES: To access the performance of 12 serological tests for COVID-19 diagnosis. METHODS: We conducted a blind evaluation of six lateral-flow immunoassays (LFIAs) and six enzyme-linked immunosorbent assays (ELISAs) commercially available in Brazil for detecting anti-SARS-CoV-2 antibodies. RESULTS: Considering patients with seven or more days of symptoms, the sensitivity ranged from 59.5% to 83.1% for LFIAs and from 50.7% to 92.6% for ELISAs. For both methods, the sensitivity increased with clinical severity and days of symptoms. The agreement among LFIAs performed with digital blood and serum was moderate. Specificity was, in general, higher for LFIAs than for ELISAs. Infectious diseases prevalent in the tropics, such as HIV, leishmaniasis, arboviruses, and malaria, represent conditions with the potential to cause false-positive results with these tests, which significantly compromises their specificity. CONCLUSION: The performance of immunoassays was only moderate, affected by the duration and clinical severity of the disease. Absence of discriminatory power between IgM/IgA and IgG has also been demonstrated, which prevents the use of acute-phase antibodies for decisions on social isolation.

Laboratorial algorithm for serological diagnosis of visceral leishmaniasis using rK39-ICT, DAT-LPC and FC-Simplex IgG1.

The performance of distinct serological tests (rK39-ICT, IFAT, DAT-LPC, FC-Simplex IgG1) was assessed and a laboratorial algorithm was proposed for accurate diagnosis of VL. DAT-LPC and FC-Simplex IgG1 showed outstanding accuracy (AUC = 0.93) to identify VL patients. The use of a sequential serological algorithm (rK39-ICT screening followed by DAT-LPC or FC-Simplex IgG1) improved the global accuracy for VL (97.2%) diagnosis. An alternative approach for diagnosis of VL has been also assessed for interchangeable use of serum/whole blood lysate samples in DAT-LPC and FC-Simplex IgG1. Our data showed an outstanding agreement for the results obtained with whole blood lysate samples as compared to serum samples (DAT-LPC =100%; FC-Simplex IgG1 = 99%). Together, these findings provide insights to improve the current overall accuracy of VL diagnosis and present innovative laboratorial tests and alternative samples from use in public health services.

A prototype of the direct agglutination test kit (DAT-Canis) for the serological diagnosis of canine visceral leishmaniasis.

This report describes the stege I/II development of a new direct agglutination test (DAT) for the diagnosis of canine visceral leishmaniasis (CVL) using freeze-dried antigen produced Coomassie blue-stained Leishmania (Leishmania) infantum promastigotes. In stage I, 16 canine serum samples, collected from eight dogs carrying CVL and eight healthy dogs, were assessed with the DAT using 2-mercaptoethanol (2-ME), N-acetyl-cysteine (NAC), kaolin or NAC plus urea (NAC+U) to improve the assay conditions. Stage II assessed the diagnostic accuracy with 100 serum samples collected from dogs with symptomatic CVL and clinically healthy dogs, comparing the four different sample diluents. The CVL-DAT prototype kit showed equivalent performances when 2-ME, NAC or NAC+U were used: 97.1% sensitivity (CI: 83-99.8%), 97% specificity (CI: 88.5-99.5%) and a 97% diagnostic accuracy (CI: 90.8-99.2). With kaolin, a 94.1% sensitivity (CI: 79-99%), 97% specificity (CI: 88.5-99.5%) and 96% diagnostic accuracy were observed (CI: 89.5-98.7), with no statistically significant differences among the four reagents (p=1.0). The NAC plus urea in sample diluent decreased non-specific agglutination, promoted a better defined sharp-edged blue spot and was thus chosen as a component for the new DAT prototype to diagnose canine VL, designated DAT-Canis.

Comparison of parasitological, serological, and molecular tests for visceral leishmaniasis in HIV-infected patients: a cross-sectional delayed-type study.

The aim of this study was to evaluate the accuracy of invasive and non-invasive tests for diagnosis of visceral leishmaniasis (VL) in a large series of human immunodeficiency virus (HIV)-infected patients. In this delayed-type cross-sectional study, 113 HIV-infected symptomatic patients were evaluated by an adjudication committee after clinical follow-up to establish the presence or absence of VL as the target condition (reference test). The index tests were recombinant K39 antigen-based immunochromatographic test (rK39), indirect fluorescent antibody test (IFAT), prototype kit of direct agglutination test (DAT-LPC), and real-time polymerase chain reaction (qPCR) in peripheral blood. Compared with parasitological test and adjudication committee diagnosis or latent class model analyses, IFAT and rk39 dipstick test presented the lowest sensitivity. DAT-LPC exhibited good overall performance, and there was no statistical difference between DAT-LPC and qPCR diagnosis accuracy. Real-time PCR emerges as a less invasive alternative to parasitological examination for confirmation of cases not identified by DAT.

Validation of a direct agglutination test prototype kit for the diagnosis of visceral leishmaniasis.

BACKGROUND: A freeze-dried antigen was developed with Leishmania (L.) infantum and used for the production of a prototype direct agglutination test kit for the laboratory diagnosis of visceral leishmaniasis (VL), called DAT-LPC. On this study the diagnosis validity of this prototype was performed. METHODS: To evaluate the sensitivity and specificity 103 samples from Brazilian patients with VL and 110 samples from patients with other parasitic infections, and healthy subjects were assayed with DAT-LPC and DAT-KIT (Royal Tropical Institute, Amsterdam, NL). Additionally, the results of 103 samples of VL patients based on two agglutination tests were transformed in Log10 and correlated. RESULTS: The DAT-LPC showed a sensitivity of 99.0%, specificity of 98.2% and diagnosis validity of 98.6%, which were similar to those found by the DAT-KIT (p > 0.05). Moreover, there was positive correlation between the positive titers obtained by DAT-LPC and by DAT-KIT (Spearman correlation coefficient of 0.75 p = 0.0001). CONCLUSIONS: DAT-LPC showed thermal stability and diagnosis performance similar to those of the DAT-KIT. Our results suggest that DAT-LPC is a robust, simple, equipment-independent and efficient tool for the diagnosis of VL and should thus replace the IFAT as routine diagnostic test in the Brazilian public health system.