الفهرس 
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Abstract
This study was done in microbiology unit, clinical pathology department, Assiut university hospital. The study included 120 isolates selected from 300 gram negative isolates, obtained from different clinical specimens (blood, urine, sputum, pus swab and other sterile fluids) from patients admitted to different departments.
All samples were taken under complete aseptic precautions in sterile container according to standard protocol for each sample.
All isolates were subjected to the following tests:
1. Identification of the organism
2. Antibiotic sensitivity test.
3. Phenotypic methods for detection of ESBL.
4. Detection of AmpC
a- Phenotypic method b- Genotypic method: PCR
Results:
Out of 300 Gram negative clinical isolates, 120 isolates (40%) were resistant to 2nd generation Cephalosporins (cefoxitin) as screening test for AMPC by using disc diffusion method and vitek 2 compact 15. Regarding the studied samples, 58.3% (70/120) of samples were isolated from sputum, 19.2% (23/120) from blood, 6.7 %( 8/120) from pus swab, 14.2% (17/120) from urine and 1.7% (2/120) from others.
Results of organism identification revealed: seventy five (62.5%) were Klebsiella pneumonae , thirty one (25.8%) were E.coli, ten (8.3%) were Enterobacter sp. (six (5%) were Enterobacter cloacae and four (3.3%) were Enterobacter aerogenes), three (2.5%) were Pseudomonas aeruginosa, and one (0.8%) was Acinetobacter bumannii.
Out of 120 isolates 14 (11.7%) were positive for Combination Disc Test of ESBL screening test while 17 isolates (14.2%) were positive for vitek ESBL screening test. If we consider that Vitek ESBL screening test is the imperfect reference test and the manual ESBL screening test (Combination Disc Test) is the new one under evaluation we can report that Kappa value (95% CI) = 0.667. This value of Kappa indicates the presence of good agreement between the two tests.
Regarding phenotypic tests which were performed on 120 cefoxitin resistance isolates, the disc approximation test was positive for 19 isolates (15.8 %). Boronic acid test using cefoxitin and 150µg/ml PBA was positive in 15 isolates (12.5%) while boronic acid test using ceftazidime and 600µg/ml PBA was positive only in 21 isolates (17.5 %).
The accuracy of disc approximation test was 77.5%. The test was fairly good negative test with a specificity 87.8% ,Negative Predictive Value (NPV)85.1%, and with sensitivity 31.8%, Positive Predictive Value (PPV) 36.8% . On other hand accuracy of Boronic acid test (150µg/ml) was 74.2% and was also a fairly good negative test with specificity 87.8%, NPV 81.9%, and sensitivity 13.6%, PPV 20%. The accuracy of boronic acid test (600µg/ml) was 86.6%. It was very good negative test with specificity 91.8 %, NPV 91.8%, and with sensitivity 63.6%, PPV 63.6%. Generally all were good negative tests but their sensitivity was low except boronic acid test (600µg/ml).
In our study, sensitivity and specificity of all three phenotypic methods were found to be inadequate in detecting pAmpC positivity, as they can lead to false positives and false negatives. But boronic acid test (600µg/ml) can be used as a good negative test to exclude the presence of AmpC producers, others are too time consuming or laborious for routine use.
Regarding PCR results, One hundred and twenty isolates among AmpC genes were identified in 22 of them, 6/22(27.3%) were kl. pneumoniae, 14/22 (63.6%) were E.coli, 1/22 (4.5%) was E.cloacae and 1/22 (4.5%) was p. aeruginosa.
Regarding AmpC genotypes, CIT only detected in 10/22 (45.5%), CIT and FOX together were detected in 10/22 (45.5%), CIT and DHA together were detected in 1/22 (4.5%), MOX only was detected in 1/22 (4.5%). No isolate was positive for EBC gene.
Six isolates of kl. Pneumoniae (27.3% ) were confirmed as being plasmid-mediated AmpC β-lactamase producers by the multiplex PCR, two isolates harbor bla CIT gene, two harbor bla FOX and CIT gene, one harbor bla MOX and one harbor bla CIT and DHA gene. Fourteen isolates of E.coli (63.6%) were confirmed as being plasmid-mediated AmpC β-lactamase producers, seven of them harbor bla CIT and 7 harbor bla FOX and bla CIT. One isolates of Enterobacter cloacae was confirmed as being plasmid-mediated AmpC β-lactamase producers, which harbor bla FOX and bla CIT. One isolate of P. aeroginosa was confirmed as being plasmid-mediated AmpC β-lactamase producers, which harbor bla CIT.
In our study CIT gene is the most dominant gene present by 45.5% alone and 45.5 with FOX gene and 4.5% with DHA gene.so Total prevalence of CIT gene in current study is 95.5%.