BACKGROUND AND OBJECTIVE

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital and community-acquired infections, showing antimicrobial resistance (AMR), which is an increasing public health concern. One of the commonly-used methods to evaluate resistance include the Kirby-Bauer disk diffusion method. However, this test is found to be time-consuming, lacking in terms of mechanization and automation, alongside its non-applicability to certain antibiotics such as vancomycin. Thus, the Clinical Laboratory Standards Institute (CLSI) recommends using the broth microdilution method in the evaluation of antibacterial activities against S. aureus. A rapid laboratory identification of MRSA is important in the treatment of patients. Therefore, this study aims to optimize and evaluate the effectiveness of a rapid microplate assay using resazurin dye as a colorimetric indicator in determining antibacterial activity against clinical isolates of MRSA and methicillin-susceptible S. aureus (MSSA).

METHODS

Clinical isolates of MRSA and MSSA were obtained from the Philippine General Hospital (PGH) Microbiology Section, and American Type Culture Collection (ATCC) controls of both strains (ATCC 25923 and ATCC 43300) were acquired. These were then subjected to identification and confirmation procedures. A standardization of bacterial inoculum was performed by comparing its 24-hr growth in Mueller Hinton Broth to 0.5 McFarland Standard. The resazurin microplate assay (REMA) was set-up using two-fold serial dilution of control antibiotics such as oxacillin, vancomycin, and cefoxitin. Each plate was inoculated with standardized bacterial growth of controls and clinical isolates. To determine the time needed for the reduction of the resazurin dye, a qualitative assessment was conducted by comparing the reaction time between a 6.75 mg/mL dye with a 0.01 mg/mL dye. The plates were also subjected to different incubation times and dye concentrations, and the optical densities of the plates were compared using a microplate reader.

RESULTS

Results showed that there were no significant differences between the optical densities of the wells of those incubated for 5 hours and for 24 hours (p >0.05). Furthermore, there was a significant reduction in the reaction time of the dye (from 18 hours to 1 hour) when the dye concentration was reduced from 6.75 mg/mL to 0.01 mg/mL. The optimized REMA showed a significant difference between the minimum inhibitory concentrations (MICs) of the different antibiotics against the control and isolate strains of MRSA and MSSA, showing a W of -2.98 (pCONCLUSION

Based on the results presented, the researchers determined the optimal condition for the resazurin microtiter assay, which was 0.01 g/mL concentration of resazurin dye, at a 5-hour incubation period. This study has shown that an optimized REMA is an efficient and fast method to determine the antimicrobial activities of oxacillin, cefoxitin, and vancomycin against MRSA and MSSA.

Methicillin-resistant Staphylococcus Aureus

BACKGROUND AND OBJECTIVES

COVID-19 has quickly spread over the world and became an unprecedented burden on health care systems. COVID-19 diagnosis necessitates the use of precise testing methods such as RT-PCR. This method is generally reported as positive or negative, however, studies have shown its semi-quantitative capability through Ct values. This study determined an association that exists between the Ct values, clinical features, and laboratory findings among COVID-19 patients admitted in a tertiary infectious disease hospital in Manila, Philippines. This attempts to further explore the utility of RT-PCR in disease severity classification and diagnosis.

METHODS

This was an observational retrospective study that utilized a purposive sampling method, wherein patients were selected based on the DOH case definition of confirmed COVID-19, and were stratified according to disease severity. Baseline laboratory data of the patients were gathered from medical records covering the period of June 2021 to January 2022 using a Data Collection Form. Chi-square test was used to measure the degree of association between the groups and categorical variables. Regression Analysis was used to identify predictors for certain variables. SPSS Statistics for Windows, Version 25.0 was utilized for the statistical analysis.

RESULTS

The total WBC, neutrophil, lymphocyte and monocyte counts, serum urea, LDH, CRP and PTT were found to be predictors of COVID-19 severity. There was no significant difference observed between the disease severity and the patient’s clinical outcome. All routine laboratory tests that were taken at baseline (ORF Gene, N-Gene, Hematocrit, White Blood Cells, Neutrophil, Lymphocyte, Monocyte, Platelet Count, Urea, Creatinine, SGPT, SGOT, Na, K, LDH, Ferritin, C Reactive Protein, Procalcitonin, D-Dimer, PT, PTT) were not significant predictors of the clinical outcome. Although WBC, neutrophil, lymphocyte, and monocyte count, urea, LDH, CRP, and PTT were predictors  of disease severity. The study also reported that the odds of having severe to critical disease increases by 20.6% for every one unit increase in neutrophil count, and 17.4% for every one unit increase in lymphocyte count. Among the laboratory parameters, neutrophil count (p=0.010654063) and urea (p= 0.04149874 have direct relationship with the N gene Ct values while Orf gene Ct Values have direct relationship with lymphocyte count (p=0.01269027). Similarly, regression showed that as monocyte count, creatinine levels, and serum ferritin decrease, Ct values increase. Sex was found to not be a significant predictor of disease severity and clinical outcome. There was also no significant difference observed between the disease severity and the patient’s clinical outcome.

CONCLUSION

The study showed that the Ct values for both ORF and N genes were not significant predictors of both disease severity and clinical outcome. However, ORF gene Ct values have direct relationship with lymphocyte counts while N gene Ct values have direct correlation with neutrophil count and urea levels. Similarly, monocyte, creatinine, and ferritin are negatively correlated with Ct values. It is important to monitor the patient’s laboratory biomarkers in order to determine the proper course of treatment and management for each case.

Human ; Sars-cov-2 ; Covid-19

Background and Objective: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital and community-acquired infections, showing antimicrobial resistance (AMR), which is an increasing public health concern. One of the commonly-used methods to evaluate resistance include the Kirby-Bauer disk diffusion method. However, this test is found to be time-consuming, lacking in terms of mechanization and automation, alongside its non-applicability to certain antibiotics such as vancomycin. Thus, the Clinical Laboratory Standards Institute (CLSI) recommends using the broth microdilution method in the evaluation of antibacterial activities against S. aureus. A rapid laboratory identification of MRSA is important in the treatment of patients. Therefore, this study aims to optimize and evaluate the effectiveness of a rapid microplate assay using resazurin dye as a colorimetric indicator in determining antibacterial activity against clinical isolates of MRSA and methicillin-susceptible S. aureus (MSSA). Methods: Clinical isolates of MRSA and MSSA were obtained from the Philippine General Hospital (PGH) Microbiology Section, and American Type Culture Collection (ATCC) controls of both strains (ATCC 25923 and ATCC 43300) were acquired. These were then subjected to identification and confirmation procedures. A standardization of bacterial inoculum was performed by comparing its 24-hr growth in Mueller Hinton Broth to 0.5 McFarland Standard. The resazurin microplate assay (REMA) was set-up using two-fold serial dilution of control antibiotics such as oxacillin, vancomycin, and cefoxitin. Each plate was inoculated with standardized bacterial growth of controls and clinical isolates. To determine the time needed for the reduction of the resazurin dye, a qualitative assessment was conducted by comparing the reaction time between a 6.75 mg/mL dye with a 0.01 mg/mL dye. The plates were also subjected to different incubation times and dye concentrations, and the optical densities of the plates were compared using a microplate reader. Results: Results showed that there were no significant differences between the optical densities of the wells of those incubated for 5 hours and for 24 hours (p >0.05). Furthermore, there was a significant reduction in the reaction time of the dye (from 18 hours to 1 hour) when the dye concentration was reduced from 6.75 mg/mL to 0.01 mg/mL. The optimized REMA showed a significant difference between the minimum inhibitory concentrations (MICs) of the different antibiotics against the control and isolate strains of MRSA and MSSA, showing a W of -2.98 (p <0.05) using the Wilcoxon Rank-Sum non-parametric test. Furthermore, the REMA has shown better illustration of anti-MRSA and anti-MSSA activities as compared to the Kirby Bauer disk diffusion method. Conclusion Based on the results presented, the researchers determined the optimal condition for the resazurin microtiter assay, which was 0.01 g/mL concentration of resazurin dye, at a 5-hour incubation period. This study has shown that an optimized REMA is an efficient and fast method to determine the antimicrobial activities of oxacillin, cefoxitin, and vancomycin against MRSA and MSSA.
Methicillin Resistant Staphylococcus aureus