Establishing polymerase chain reaction-based diagnostic test for the detection of drug resistant Salmonella typhi in a hospital setting in the Philippines
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School of Tropical Medicine and Global Health, Nagasaki University, Japan
San Lazaro Hospital−Nagasaki University Collaborative Research Office, Manila, Philippines
Publication date: 2023-04-27
Popul. Med. 2023;5(Supplement):A139
With the increasing use of polymerase chain reaction-based technologies to diagnose diseases such as tuberculosis and severe acute respiratory syndrome coronavirus 2 in the Philippines, medical technologists are becoming more familiar with novel molecular techniques. However, development of polymerase chain reaction diagnostic assays based on the identified needs of healthcare facilities and surrounding communities remains uncommon. In this work, we outline the experience and challenges we faced when developing a set of conventional polymerase chain reaction-based diagnostic tests for Salmonella typhi and associated sulfonamide (Sul2), chloramphenicol (CatP), penicillin (Tem), and fluoroquinolone (gyrA) drug resistance genes in San Lazaro Hospital-Nagasaki University Collaborative Research Laboratory. This process involved three key components: procuring the necessary reagents, generating a positive control, and optimizing the polymerase chain reaction itself. To generate positive control, we re-cultured 21 archived isolates of Salmonella species on MacConkey agar and performed matrix assisted laser desorption ionization-time of flight mass spectrometry. Subsequently, genomic deoxyribonucleic acid was extracted from the identified colonies. Polymerase chain reaction optimization commenced by determining the optimal annealing temperatures. Good intensity of amplicon bands were observed at annealing temperatures 58°C for nested polymerase chain reaction and 45°C for conventional monoplex polymerase chain reaction. During optimization, we successfully detected flagellin gene (fliC) for Salmonella typhi and three resistance genes (gyrA, Sul2, and Tem) using positive control. Conversely, CatP gene was not detected. However, when we ran clinical samples, two of 18 Salmonella typhi polymerase chain reaction-positive specimens were positive for CatP gene. Our work may serve as a useful example for other laboratories and medical technology professionals in the country in responding to locally identified diagnostic needs. Moreover, early detection of resistance genes can inform both public health decision-making and individual clinical practice. Keywords: conventional polymerase chain reaction, Salmonella typhi, drug resistance
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