Multiplex PCR for detection of bla CTX-M genes among the extended spectrum beta lactamase [ESBL] producing gram-negative isolates

Broad spectrum beta-Lactamase producing organisms are a growing world wide problem. Resistance has emerged ever to newer, more potent antimicrobial agents. Although there are several guidelines available for the phenotypic detection of ESBL producing bacteria. This remains a continuous issue. In this study, we used a multiplex PCR as a rapid method to identify bla CTX-M genes and discriminate between its groups that are responsible for ESBL production in members of Enterobacteriacae. Our study includes: 250 clinical isolates [23 sputum, 64 urine, 46 from blood, 28 from pus aspirates, 58 from entotracheal secretions, and 31 swabs from cellulitis, impetigo contagiosum [non bullous] and sycosis]. All isolates were biochemically identified, based on colony morphology, and was speciated by standard biochemical tests. ESBL enzyme production was confirmed by double disc synergy test according to CLSI guidelines. Multiplex PCR was performed for bla CTX-M of ESBL +ve isolates for detection and discrimination between groups. Our findings were as follows: out of 250 isolates; only 98 were proved to be resistant to different antibiotics by the disc diffusion method according to NCCLS: 3 of 53 [5.66%] Enterobacter. All from group [25/26]. 65 of 74 [87.8%] E.coli strains: -37 of which from groups [1] [CTX-M 15], 9 from group [1] [CTX-M-3], 8 from group [9] [CTX-M-14], 9 from group [9] [CTX M-9], 2 from group [25/26] [CTX-M 26]. 1 of 50 [2%] non fermenting gram -ve bacilli which is from group [25/26]. 29 of 73 Klebsiella strains [39.7%]: 19 from group [9] [CTX-M14] and 10 from group [9] [CTX-M 9]

Evaluation of simple screening tests in the diagnosis of non fermentative gram negative bacilli, a prospective study

Carbapenemases are B-lactamases which include serine B-lactamases andmetallo B-lactamases [MBLs]

Their production is the most important mechanisms of microbial resistance to [beta]-lactam antibiotics

Modified Hodge test is a phenotypic method for detection ofcarbapenemases. EDTA disk synergy [EDS] test is used for detection of MBLs. AmpC disk test is the commonly used tests for detection of AmpC [betaj-lactamases. Detection of genes coding for carbapenem resistance by PCR, usually give reliable and satisfactory results, but this method is of limited practical use for daily application in clinical laboratories because of the cost. This study was conducted over a period of ten months [July 2012 to April 2013] at the Medical Microbiology and Immunology Department, Faculty of Medicine, Tanta University. A total of 110 Acinetobacter species and 120 Pseudomonas species were included in this study

These organisms were isolated from specimens like aspirated synovial fluid from knee infective arthritis, sputum, tracheal aspirate, pus, urine, blood, pleural fluid and ascitic fluid of patients admitted to Internal Medicine, Chest, ENT, Orthopaedic and Urology Departments, Faculty of Medicine, Tanta University

The Acinetobacter species and Pseudomonas species were identified and screened for meropenem resistance by Kirby-Bauer method. The meropenem resistant strains were subjected to modified Hodge test for detection of carbapenemases. EDTA disk synergy [EDS] test was done with simultaneous testing of two different [beta]-lactams [meropenem and ceftazidime], for detection of metallo-[beta]-lactamases in the meropenem resistant isolates. AmpC disk test was also done for the meropenem resistant strains for detection of AmpC [betaj-lactamases. Of the 110 clinical isolates of Acinetobacter species, 82 were A. baumannii, while 28 were A. Iwoffli. Among the 120 Pseudomonas isolates screened, 91 were Ps. aeruginosa, while the remaining 29 were other Pseudomonas spp. Forty two [51.0%] A. baumannii, 8 [31.8%] A. Iwoffii, 29 [31.8%] Ps. aeruginosa and 8 [27.6%] Pseudomonas spp. were found resistant to meropenem. Among the 29 meropenem resistant Ps. aeruginosa, 13 [44.8%] were AmpC [beta]-lactamase producers, 15 [51.7%] were MBL producers by EDTA disk synergy test, but only 10 [34.4%] were positive for carbapenemases by modified Hodge test. Of the 8 meropenem resistant Pseudomonas spp., 5 [62.5%] were AmpC [beta]-lactamase producers, 2 [25.0%] were MBL producers by EDTA disk synergy test, but only 1 [12.5%] was positive for carbapenemases by modified Hodge test

Among the 42 meropenem resistant A. baumannii, 32 [76.2%] were AmpC [beta]-lactamase producers, 3 [7.1%] were MBL producers, but only 2 [4.8%] was positive for carbapenemases by modified Hodge test

Of the 8 meropenem resistant A. Iwoffii, 3 [37.5%] were AmpC [beta]-lactamase producers, and 2 [25.0 %] were positive for MBL that were detected only using EDTA-ceftazidime combination and no carbapenemases were detected by modified Hodge test. EDS is a more sensitive diagnostic method for detection of MBLs. The modified hodge test is not considered a useful screening test for carbapenemases as many MBL producing isolates were not detected by this test, while EDS is a more sensitive diagnostic method for detection of MBLs. AmpC B- lactamase is a major factor for carbapenemases resistance among the isolates in the hospital

Microscopic observation drug susceptibility [MODS] as a rapid, simple low-cost diagnostic tool in cases with MDR tuberculosis

Multidrug resistant tuberculosis [MDR-TB], which is tuberculosis [TB] resistant to at least isoniazid and rifampicin, is a major threat to TB control worldwide. Early detection of MDR-TB allows initiation of an appropriate treatment. Diagnosis of MDR-TB requires microbiologic evaluation of the Mycobacterium tuberculosis isolates drug susceptibility. The microscopic observation drug susceptibility [MODS] is a relatively low-cost, simple liquid culture method that relies on the microscopic detection of cording growth that is characteristic of Mycobacterium tuberculosis. We undertook a prospective study to evaluate the performance of MODS. Testing for the direct detection of M. tuberculosis drug resistance in specimen from patients with suspected pulmonary TB at risk for drug resistance. The study was carried out on 150 individuals. Samples at risk for drug resistance were cultured positive for M. tuberculosis by both MODS and LJ. methods. By the comparator LJ. testing method, 57 isolates [38.0%] were resistant to both INH and RIF, 19 [12.7%] were resistant to INH alone, 4 [2.7%] were RIF monoresistant, and 70 [46.6%] were susceptible to both INH and RIF. The performance parameters of the MODS assay are shown. MODS test results are shown for undiluted MODS inocula and for inocula diluted 1:10. The MODS assay indicated some specimens to be resistant than the comparator method indicated to be susceptible. This trend was slightly more marked when undiluted inocula were used for MODS. The time interval of 14 days was chosen as the susceptibility or resistance status indicated by the MODS assay is stable for at least 2 weeks after the detection of growth and because this strategy optimized efficiency by eliminating the need for daily microscopic examination of all drug-containing wells. Median times to the availability of susceptibility results were 21 days [interquartile range, 17 to 24 days] for the MODS method and 49 days [interquartile range, 46 to 55 days] for the LJ. method [P, <0.001 by paired t tes]

The MODS assay provides low-cost, safe and highly sensitive detection of TB greatly faster than existing gold standards and automated methods with concurrent highly accurate identification ofmultidrug resistant [MDR] strains

Helicobacter pylori infection and hyperemesis gravidarum: possible causal relationship with evaluation of a new immunoassay in stool compared with standard diagnostic techniques

The aim of the present study was to investigate any possible association between infection with Helicobacter pylori [H. pylori] and hyperemesis gravidarum [HG]. Moreover; evaluation of different methods used in the diagnosis of H. pylori during pregnancy aiming to present a simple non-invasive and reliable method. 68 pregnant women with hyperemesis gravidarum and 72 control pregnant women were enrolled in the study. All participants were examined both for H. pylori serum immunoglobulin G antibodies [HpIgG Ab], showing chronic infection, and H. pylori stool antigens [HpSA], and showing active gastrointestinal colonization. Serologically positive H. pylori infection was detected in 59 [86.8%] subjects of the hyperemesis gravidarum group and in 32 [44.4%] of the controls [P<0.01]. HpSA was detected in 45.6% of patients with hyperemesis gravidarum, whereas only 5.6% of patients in the control group were positive for this specific antigen [P<0.001]. The new stool immunoassay test had a sensitivity of 96% [95% confidence interval 90.6% to 100%], specificity of 93% [85.1% to 99.5%], positive predictive value of 92%, and negative predictive value of 96%. In conclusion, this study supports the studies suggesting an association between H. pylori and HG. Infection with H. pylori should be kept in mind in cases of HG in pregnant women. The findings of the current study have, also, demonstrated that HpSA as a relatively simple, inexpensive and time saving noninvasive test is a reliable method for detection of active H. pylori infections in pregnant women with hyperemesis gravidarum. This stool immunoassay represents a new, accurate, and non-invasive method for H pylori infection that overcomes the limitations of existing tests