ESAT-6-ELISpot and interferon gamma in the diagnosis of pleural tuberculosis

Appropriate diagnostic methods for tuberculous pleural effusion are vital. The IFN-gamma tests using specific Mycobacterium Tuberculos is antigens in samples from the site of infection may be promising in diagnosis of tuberculosis. Objective we examined the ability of ELISpot test using circulating peripheral blood mononuclear cells [PBMC] and compartmentalized pleural fluid mononuclear cells [PFMC] for diagnosis of active TB infection in patients with tuberculous pleural effusion. Methods PBMC and PFMC-based ELISpot test for IFN-gamma test using specific M. tuberculosis antigen: Early Secretory Antigen Target-6 protein [ESAT-6] was used for diagnosis of active TB infection. Thirty-five patients with clinically suspected tuberculous pleural effusion were enrolled over a 12-month period. Results 11 patients out of 35 were positive by culture and PCR [31.4%]. Incubation of PBMC with ESAT-6 for 8 h showed sensitivity and specificity of 82% and 92%, respectively, for the PBMC-ELISpot as compared to PFMC-ELISpot that was 54% and 96% respectively. With 24 h incubation of ESAT-6 there was around 2.5 fold increase in the median number of spot forming cells [SFCs] in PFMC from 30 to 74, whereas there was minimal increase of median number of SFCs in PBMC from 55 to 60. Conclusion ESAT-6 - ELISpot using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-gamma in pleural effusion [PE] is another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap

Interferon gamma as a diagnostic marker for pleural tuberculosis

Background appropriate diagnostic methods for tuberculosis pleural effusion [TBPE] are vital. The IFN-gamma tests using specific M. Tuberculosis antigens in samples from the site of infection may be promising in diagnosis of tuberculosis

Objective we examined the ability of ELISpot test using circulating peripheral blood mononuclear cells [PBMC] and compartmentalized pleural fluid mononuclear cells [PFMC] for diagnosis of active TB infection in patients with TBPE

Methods PBMC and PFMC-based ELISpot test for IFN-gamma test using specific M. Tuberculosis antigen: Early Secretory Antigen Target-6 protein [ESA T-6] was used for diagnosis of active TB infection. Thirty-five patients with clinically suspected TBPE were enrolled over a 12-month period

Results eleven patients out of 35 were positive by culture and PCR [31.4%]. Incubation of PBMC with ESAT-6 for 8 hrs showed sensitivity and specficity of 82 % and 92 % respectively .for the PBMC-ELlSpot as compared to PFMC- ELlSpot that was 54% and 96 % respectively. With 24 hrs incubation of ESAT- 6 there was around 2.5 fold increase in the median number of spot forming cells [SFCs] in PFMC from 30 to 74, whereas there was minimal increase of median number of SFCs in PBMC from 55 to 60

Conclusion ESAT-6 - ELlSpot using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-gamma in pleural effusion [PE] is another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap

Evaluation of different techniques for detection of mecA-mediated resistance in staphylococcus aureus

Cefoxitin is a stronger inducer of mecA expression than oxacillin. It is being recommended by Clinical and Laboratory Standards Institute [CLSI] for detection of methicillin resistance in Staphylococcus aureus [MRSA] when using disk diffusion testing. This study was carried out to evaluate the performances of two methods for detection of mecA-mediated resistance in S. aureus strums: the automated Vitak 2 system [bi0Me'rieu.x] and the disk diffusion tests, using cefoxitin and oxacillin. Three hundred sixty strains of S. aureus isolated from different clinical samples were used in the study. Oxacillin susceptibility was tested by the Vitak 2 system with the AST-P580 card, [which includes oxacillin MIC and cefoxitin screen] and by disk diffusion methods using l-pg oxacillin disk and 30-pg cefoxitin disc. Oxacillin resistance was confirmed by PCR for the mecA gene "the gold standard method". The 360 clinical isolates of S. aureus studied were divided into 172 MRSA [mecA-positive] and 188 MSSA [mecA negative] isolates. The sensitivities and specificities for the methods evaluated were as follow: oxacillin disk diffusion, 97.7 and 95.2%, cefoxitin disk diffusion, 99.4 and 99.5%, Vitak 2 based oxacillin MIC, 98.8 and 95.2%, Vitak 2 cefoxitin screen, 100 and 99.5% and Vitak 2 Advanced Expert System[AES], 100 and 95.2%, respectively. The median times for methicillin testing were 8h for the Vitak 2 system versus 24h for disk diffusion methoa5.h conclusion all of the methods evaluated in the present study appeared to perform very well for the defection of 11dRSA. The results of cefoxitin were in concordance with the PCR for mecA gene. Cefoxitin test either cefoxitin disk diffusion or Vitak 2 cefoxitin screen, were the best performing tests for routine detection of mecA-mediated resistance in S.aureus and the tests can be used as an alternative to the technically demanding PCR, for detection of MRSA

Detection of aminoglycoside resistance genes by multiplex PCR among coagulase negative staphylococci isolated from Ventriculoperitoneal shunt infections

A Ventriculoperitoneal [VP] shunt infection is a cause of significant morbidity causing shunt malfunction and chronic ill health. This study was carried out to detect the causative pathogens associated with VP shunt infections, to assess the frequency of Coagulase negative Staphylococci [CoNS] as well as study their phenotypic resistance pattern to methicillin and different aminoglycosides [AGs], and genotypic pattern for gene encoding aminoglycosides modifying enzymes [AMEs] by multiplex PCR. This study was carried out in Medical Microbiology and Immunology and Neurosurgery Departments, Faculty of Medicine, Zagazig University during the period from June 2008 to June 2010. During this period 240 shunt procedures were carried on 175 infants and children with hydrocephalus. CoNS were identified using standard microbiological laboratory techniques. Antimicrobial susceptibility testing was performed using disc diffusion method with 4 aminoglycosides and methicillin. Multiplex PCR assay was used to identify AMEs encoding genes. 58 out of 240 shunt procedures cases showed shunt infections, [64%] caused by CoNS, [28%] by Gram negative rods [5%] by Staph aureus and [3%] by candida. 26 [70%] of CoNS isolates were methicillin resistant [MRCoNS]. 25 [67.5%] isolates of CoNS were resistant to at least one of the tested AGs and the highest resistance was to gentamicin and tobramycin [67.5%] for both followed by amikacin [27%] and streptomycin [19%]. As regard results of multiplex PCR aac [6'] le+aph [2' '] gene encoding for the bifunctional enzyme was the most common [54%] followed by ant[4]la gene encoding for the ANT[4'] - la enzyme [46%] and the aph [3']-llla gene encoding APH[3'] - Illa enzyme [40.5%]. In conclusion this study increased our knowledge about the causative pathogens of VP shunt infections, the phenotypic pattern of CoNS susceptibility to different AGs and the distribution of AMEs encoding genes in relation to methicillin susceptibility. The usage of appropriate antibiotic according to antimicrobial susceptibility testing at the probable time and the removal of the shunt apparatus are essential for successful treatment of VP shunt infection. Continued surveillance at both phenotypic and genotypic levels are recommended for monitoring the presence of other variant of the genes or new AGs resistance genes that may be produced within CoNS population

Prevalence of hepatitis G virus RNA and anti HGV-E2 among hemodialysis patients

Hepatitis G virus [HGV] is, like hepatitis C virus, a blood-borne virus and a member of the family of flaviviridae. HGV is distributed globally and is present in the volunteer blood donor population. Thus, for epidemiological reasons, HGV is of interest in hemodialysis [HD] patients, who are at risk of parenterally transmitted infections. To study HGV infection among hemodialysis patients in our locality, 50 HD patients and 25 apparently healthy volunteer blood donors were studied. All subjects' sera were tested for transaminases [ALT and AST], HBsAg, anti-HCV IgG, HCV-RNA, anti-HGV-E2 and HGV RNA. Anti-HGV-E2 was determined using an enzyme immunoassay; HGV RNA and HCV RNA were detected using reverse transcription polymerase chain reaction [RT-PCR]. HBsAg was detected in 10 [20%] of HD patients in comparison to none of the control. Anti-HGV-E2 and HGV- RNA were found in 18% and 42% of HD patients respectively, in comparison to 8% and 12% in the control group. The total prevalence of HGV infection in our HD patients was 60%. All the HGV-RNA positive sera were negative for anti-HGVE2 and vice versa. 15 of the HGV-RNA positive patients were followed up for 18 months, one of them showed anti-HGV-E2 seroconvertion with loss of viremia. HGV exposure did not correlate with age, gender, duration of HD therapy, or history of blood transfusions. HCV-RNA and HGV co-infection was found in 71.4% of HD patients. In addition, HGV infection was not found to cause significant elevation of ALT or AST enzymes levels in the group exposed to HGV. Although 18/21[85.7%] of HGV-RNA positive HD patients had history of blood transfusion, 3 [14.3%] HD patients who were HGV-RNA positive had neither history of transfusion nor other risk of parenteral exposure, supporting the hypothesis of nosocomial transmission. To conclude, patients on maintenance dialysis are at increased risk for HGV infection in our locality. In addition to transmission through blood transfusion, HGV may have been transmitted nosocomially, patient-to-patient, within the HD unit and the universal precautions should be strictly followed to prevent transmission of viruses among HD patients. The HGV RNA is considered a marker for current HGV viremia, while the anti-HGV-E2 is considered a marker for recovery from HGV infection. Detection of both antibody and viraemia is important to establish the real rate of HGV infection

Evaluation of different phenotypic methods for detection of methicillin [oxacillin] resistance in coagulase-negative staphylococci

Coagulase-negative staphylococci [CNS] are one of the major causes of nosocomial infections. Methicillin [oxacillin] resistant strains are particularly important because they narrow therapeutic options. Detecting methicillin resistance among CNS has been a challenge for years. The objective of this study was to evaluate the ability and accuracy of four phenotypic methods, disk diffusion [DD]; agar screening plate with 6 micro g of oxacillin per ml [OXA]; E-test and the MRSA-Screen latex agglutination test [Denka-Seiken, Tokyo, Japan], to determine the susceptibility of CNS to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard". One hundred and ninety seven strains of CNS of 7 species were analysed. 49.2% were mecA positive. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 93.8 and 93%, respectively; for the agar screen test 95.9 and 98%, respectively; for E-test, 100 and 95%, respectively; and for the slide latex agglutination test, 96.9 and 100%, respectively. The latex agglutination test sensitivity was increased to 100% when retested after induction. In conclusion, all of the phenotypic methods evaluated in the present study appeared to perform very well for the detection of oxacillin resistance in CNS. The MRSA-Screen latex agglutination test was not only the most sensitive, specific and accurate method but also rapid and technically simple method to be applied in routine laboratories for the detection of oxacillin resistance which is mediated by the mecA gene