الفهرس 
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Abstract
SUMMARY
n recent years, an increase in the number of methicillin-resistant Staphylococcus aureus (MRSA) strains has become a serious clinical and epidemiological problem, as resistance to this antibiotic implies resistance to all β-lactam antibiotics. Thus, accuracy and promptness in the detection of methicillin resistance are of key importance in ensuring correct antibiotic treatment in infected patients and control of MRSA in the hospital environment. In addition, the early selection of an appropriate regime for the treatment of MRSA avoids the unnecessary use of vancomycin and subsequently the development of vancomycin resistance. Moreover, early detection reduces mortality, the length of hospitalization and costs associated with the infections caused by MRSA.
Several phenotypic methods for the detection of MRSA have been developed such as MIC determination (by agar dilution, broth dilution and E-test), the oxacillin screen agar method and the disc diffusion testing. However, phenotypic expression of resistance can vary depending on the growth conditions (e.g. temperature, osmolarity and culture medium supplements such as NaCl or sucrose) making susceptibility testing by standard microbiological methods potentially problematic.
The mecA gene is highly conserved in Staphylococcal strains. Its detection is considered the gold standard for detection of MRSA isolates. However, many laboratories throughout the world do not have the capacity to use molecular techniques to detect MRSA in routine clinical practice.
Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue dye, itself weakly fluorescent until it is irreversibly reduced to the pink colored and highly red fluorescent resorufin. Resazurin was first used to quantify bacterial content in milk by Pesch and Simmert in 1929. It was also used as an oxidation reduction indicator in bacterial and mammalian cell viability assays. It has been proposed for the determination of drug resistance and minimal inhibitory concentration of antimicrobial agents active against Mycobacterium tuberculosis.
The aim of the present study was to introduce the resazurin microplate assay (REMA) as a new colorimetric method for the identification of MRSA and to investigate its effectiveness as a rapid, sensitive and specific test for the detection of methicillin resistance among Staph.aureus clinical isolates.
The study included 100 Staph. aureus isolates that were collected from different clinical samples submitted to the Microbiology Laboratory for culture and susceptibility testing. Isolates from the same patient were excluded. The selected isolates were tested for their susceptibility to methicillin by the cefoxitin (30ug) disc diffusion method and the resazurin microplate assay. Detection of the mecA gene was done by the PCR.
In the present study, using the cefoxitin (30ug) disc diffusion method, 35 out of the 100 studied isolates (35%) were determined to be susceptible to methicillin or MSSA whereas 65/100 isolates (65%) were MRSA.
Also, using the resazurin microplate assay, 35/100 (35%) of the studied isolates were found to be MSSA whereas 65/100 (65%) were MRSA. Out of the 100 studied isolates 0% had cefoxitin MIC 0.5 ug/ml, 32/100 (32%) had cefoxitin MIC 2ug/ml, 3/100 (3%) had cefoxitin MIC 1ug/ml, 0/100 (0%) had cefoxitin MIC 4ug/ml, 31/100 (31%) had cefoxitin MIC 8ug/ml, 11/100 (11%) had cefoxitin MIC 16ug/ml, 4/100 (4%) had cefoxitin MIC 32ug/ml, 18/100 (18%) had cefoxitin MIC >32ug/ml.
Using the PCR, the mecA gene was detected in 64 out of the 100 studied isolates (64%) whereas 36/100 (36%) isolates were determined to be mecA negative.
There was a highly significant association between the results of the cefoxitin (30ug) disc diffusion method and the results of the PCR for the mecA gene (P<0.01). Only one isolate was found to be resistant to cefoxitin (30ug) disc and was found to be negative for the mecA gene by the PCR. On the other hand, all isolates that were sensitive to cefoxitin (30ug) disc were found to be negative for the mecA gene by the PCR.
Moreover, the study showed a highly significant association between the results of the resazurin microplate assay and the results of the PCR for the mecA gene (P<0.01). Only one isolate had a cefoxitin MIC value of >32ug/ml by the resazurin microplate assay
and was found to be negative for the mecA gene by PCR. On the other hand, all isolates that were sensitive to cefoxitin by the resazyrin microplate assay were found to be negative for the mecA gene by PCR.
A highly significant association was found between the results of the cefoxitin (30ug) disc diffusion method and the results of the resazurin microplate assay (P<0.01).
In the present study, the diagnostic performance of the cefoxitin (30ug) disc diffusion method and the resazurine microplate assay was calculated in reference to the PCR for the detection of the mecA gene which was considered as the gold standard. Accordingly, the cefoxitin (30ug) disc diffusion method was found to have 100% sensitivity, 97.2% specificity, 98.5% PPV and 100% NPV. Also, the resazurin microplate assay had a sensitivity of 100%, a specificity of 97.2%, PPV (98.5%) and NPV (100%).