RESUMO
The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.
RESUMO
BACKGROUND: In the last decade, incidences of carbapenem-resistant Acinetobacter baumannii have been increasingly reported worldwide. Consequently, A. baumannii was included in the World Health Organization's new list of critical pathogens, for which new drugs are desperately needed. The objective of this research was to study the molecular epidemiology and antimicrobial susceptibility of clinical carbapenem-resistant A. baumannii isolated from Jordanian hospitals. METHODS: A total of 78 A. baumannii and 8 Acinetobacter spp. isolates were collected from three major hospitals in Jordan during 2018. Disc diffusion and microdilution methods were used to test their susceptibility against 19 antimicrobial agents. Multilocus sequence typing (MLST) was performed using the Pasteur scheme, followed by eBURST analysis for all isolates. PCR was used to detect ß-lactam resistance genes, blaOXA-23-like , blaOXA-51-like , and blaNDM-1 . RESULTS: Of the 86 tested isolates, 78 (90.6%) exhibited resistance to carbapenems, whereas no resistance was recorded to tigecycline or polymyxins. Based on the resistance profiles, 10.4% and 84.8% of isolates were classified into multidrug resistant (MDR) or extensively drug resistant (XDR), respectively. The most prevalent carbapenems resistance genes amongst isolates were blaOXA-51-Like (89.5%), followed by blaOXA-23-Like (88.3%) and blaNDM-1 (10.4%). MLST revealed the presence of 19 sequence types (STs), belonging to eight different international complexes. The most commonly detected clonal complex (CC) was CC2, representing 64% of all typed isolates. CONCLUSIONS: This is the first study to report the clonal diversity of A. baumannii isolates in Jordan. A high incidence of carbapenem resistance was detected in the isolates investigated. In addition, our findings provided evidence for the widespread of blaOXA-23-like harbouring carbapenem-resistant A. baumannii and belonging to CC2. The number of XDR isolates identified in this study is alarming. Thus, periodic surveillance and molecular epidemiological studies of resistance factors are important to improve treatment outcomes and prevent the spread of A. baumannii infections.
