The main laboratorial test for the diagnosis of COVID-19 (Coronavirus disease 2019) is the detection of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) RNA by the RT-qPCR (real-time reverse transcription - polymerase chain reaction) technique. To optimize the diagnosis of COVID-19, we have developed a multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) technique, which targets the nucleocapsid protein of SARS-CoV-2. The multiplex RT-qPCR assay was compared with the US CDC singleplex protocol. Protocol 1 included 113 RNA samples previously tested for SARS-CoV-2 and Protocol 2, included 107 fresh RNA samples that were tested simultaneously by the singleplex and multiplex. Protocols 1 and 2 presented agreement between singleplex and multiplex RT-qPCR of 88.5% and 98,1%, respectively. After the validation of the multiplex RT-qPCR assay, this methodology was applied in the routine of the diagnostic laboratory and 2,015 samples were analyzed in the first month of the multiplex use. In this period, we found that the multiplex assay proved to be a practical approach which provided reliable results. In conclusion, the multiplex RT-qPCR using primers for targets N1 and N2 is comparable to singleplex.

The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

Variants of concern (VOCs) of SARS-CoV-2 are viral strains that have mutations associated with increased transmissibility and/or increased virulence, and their main mutations are in the receptor binding domain (RBD) region of the viral spike. This study aimed to characterize SARS-CoV-2 VOCs via Sanger sequencing of the RBD region and compare the results with data obtained via whole genome sequencing (WGS). Clinical samples (oro/nasopharyngeal) with positive RT-qPCR results for SARS-CoV-2 were used in this study. The viral RNA from SARS-CoV-2 was extracted and a PCR fragment of 1006 base pairs was submitted for Sanger sequencing. The results of the Sanger sequencing were compared to the lineage assigned by WGS using next-generation sequencing (NGS) techniques. A total of 37 specimens were sequenced via WGS, and classified as: VOC gamma (8); delta (7); omicron (10), with 3 omicron specimens classified as the BQ.1 subvariant and 12 specimens classified as non-VOC variants. The results of the partial Sanger sequencing presented as 100% in agreement with the WGS. The Sanger protocol made it possible to characterize the main SARS-CoV-2 VOCs currently circulating in Brazil through partial Sanger sequencing of the RBD region of the viral spike. Therefore, the sequencing of the RBD region is a fast and cost-effective laboratory tool for clinical and epidemiological use in the genomic surveillance of SARS-CoV-2.

Carbapenem-resistant Enterobacterales are a recognized health threat worldwide, due to high morbidity and mortality rates. After the COVID-19 pandemic, some geographic regions faced an increase in NDM-producing as well as NDM and KPC-co-producing Enterobacterales. Some of these species are intrinsically resistant to polymyxins, limiting significantly therapeutic options. This retrospective study analyzed 347 Enterobacterales resistant or intermediate to meropenem recovered between 2019 and 2023 from patients attending in a tertiary care hospital. The presence of carbapenemases and the susceptibility to ceftazidime-avibactam were assessed. Among members of Morganellaceae, 41.7% were resistant to meropenem while 58.2% were intermediate; most isolates (93.2%) produced NDM. On the other hand, among Serratia spp., resistance to meropenem was higher (78.7%) and isolates produced predominantly KPC (90.6%). Resistance to CAZ-AVI was not observed among isolates producing serine-carbapenemases. Our results reinforce the need for new therapeutic options to treat infections caused by gram-negative bacilli producing metallo-beta-lactamases, especially those intrinsically resistant to polymyxins.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy. AML classification is currently based on cytogenetic and molecular alterations as well as immunophenotyping, although risk stratification still relies primarily on cytogenetic findings. However, approximately 45% of AML patients present with a normal karyotype, which makes accurate risk classification and treatment stratification more challenging. Therefore, the identification of molecular prognostic markers described in the literature has become essential in routine diagnostic laboratories, enabling the more precise categorization of patients into risk groups. In this study, we present a simple, rapid, step-by-step multiplex PCR protocol combined with capillary electrophoresis for the detection of two of the most prevalent molecular alterations in AML: nucleophosmin 1 (NPM1) mutations and Fms-like tyrosine kinase 3 internal tandem duplications (FLT3/ITD). This protocol provides a practical workflow that can assist diagnostic laboratories in implementing and optimizing multiplex mutation detection in routine practice.