The effect of clinical characteristics on the performance of galactomannan and PCR for the diagnosis of invasive aspergillosis in febrile neutropenic patients.

Rapid diagnosis and early treatment of invasive aspergillosis is crucial for the management of the patients with haematological malignancy. We evaluated 358 sera from 78 febrile neutropenic episodes in patient with invasive aspergillosis (IA) (one proven, 17 probable, and 60 possible) and 83 episodes in patients with no IA according to the EORTC/MSG criteria. Patient's specimens were tested by Mycassay Aspergillus PCR (first commercial real-time PCR test) and in house real-time PCR to investigate the presence of Aspergillus DNA, and by ELISA for detect the galactomannan (GM) antigen. We systematically investigated the medical background that can be effective on the test results. The hospitalisation period was longer in proven/probable episodes when compared with no IA (P = 0.001) and possible episodes. With regard to duration of neutropenia, the differences between both proven/probable with no IA (P = 0.023) and possible with no IA (P = 0.002) were highly significant. Similarly, the rates of T cell suppressant therapy in group proven/probable and possible episodes were significantly higher than in no IA (P = 0.005). There are significant differences in the performance of GM and PCR-based tests among studies, and standardisation is required. Therefore, it can be useful to determine the effective factors on these tests. The use of larger volume of sera improved the performance of real-time PCR for detection of Aspergillus DNA in high-risk adult patients in the present study. Some host factors such as duration of neutropenia and administration of T cell suppressants related to the development of IA.

Potential of polymerase chain reaction and galactomannan for the diagnosis of invasive aspergillosis in patients with febrile neutropenia.

The incidence of invasive aspergillosis (IA) has increased over the last years, especially in immuncompromised patients with high mortality rates. Because of difficulties about the diagnosis; serological methods [galactomannan (GM) antigen test] and polymerase chain reaction (PCR) developed in recent years. MycAssay Aspergillus PCR performance in the diagnosis of IA was evaluated and compared with the GM and in-house PCR. This study was conducted with 358 serum samples obtained from 99 patient with febrile neutropenic episodes who were followed in haematology and bone marrow transplantation units. Patients were classified by the European Organization for the Research and Treatment of Cancer/Mycoses Study Group criteria, 18 of them is proven and probable IA. GM antigen test and two different real-time PCR; one of them is fist commercial PCR for IA; Mycassay Aspergillus and the other one is in-house real-time PCR performed. Sensitivity values were Mycassay Aspergillus PCR, in-house PCR, and GM 65.38%, 11.53% and 23.07%, respectively. The high sensitivity obtained from Mycassay Aspergillus PCR and sensitivity is increased by using a combination of diagnostic methods. GM antigen test and real-time PCR could be beneficial for early diagnosis and treatment of IA. For routine usage of PCR as diagnostic assay more studies needed in future.

Vancomycin and daptomycin minimum inhibitory concentration distribution and occurrence of heteroresistance among methicillin-resistant Staphylococcus aureus blood isolates in Turkey.

BACKGROUND: The aim of this study was to determine the distribution of vancomycin and daptomycin MICs among methicillin-resistant Staphylococcus aureus (MRSA) blood isolates, the prevalence of heterogeneous vancomycin-intermediate S. aureus (hVISA) and the relationship between hVISA and vancomycin MIC values. METHODS: A total of 175 MRSA blood isolates were collected from seven university hospitals in Turkey. All isolates were tested for susceptibility to vancomycin and daptomycin by reference broth microdilution (BMD) and by standard Etest method. BMD test was performed according to CLSI guidelines and Etest was performed according to the instructions of the manufacturer. All isolates were screened for the presence of the hVISA by using macro Etest (MET) and population analysis profile-area under the curve (PAP-AUC) methods. RESULTS: The vancomycin MIC50, MIC90 and MIC ranges were 1, 2, and 0.5-2 µg/ml, respectively, by both of BMD and Etest. The daptomycin MIC50, MIC90 and MIC ranges were 0.5, 1 and 0.125 -1 µg/ml by BMD and 0.25, 0.5 and 0.06-1 µg/ml by Etest, respectively. The vancomycin MIC for 40.6% (71/175) of the MRSA isolates tested was >1 µg/ml by BMD. No vancomycin and daptomycin resistance was found among MRSA isolates. Percent agreement of Etest MICs with BMD MICs within ±1 doubling dilution was 100% and 73.1% for vancomycin and daptomycin, respectively. The prevalence of hVISA among MRSA blood isolates was 13.7% (24/175) by PAP-AUC method. MET identified only 14 of the hVISA strains (sensitivity, 58.3%), and there were 12 strains identified as hVISA that were not subsequently confirmed by PAP-AUC (specificity, 92.1%). CONCLUSIONS: Agreement between BMD and Etest MICs is high both for vancomycin and daptomycin. Daptomycin was found to be highly active against MRSA isolates including hVISA. A considerable number of isolates are determined as hVISA among blood isolates. As it is impractical to use the reference method (PAP-AUC) for large numbers of isolates, laboratory methods for rapid and accurate identification of hVISA need to be developed.

[In vitro tigecycline and carbapenem susceptibilities of clinical Acinetobacter baumannii isolates]. / Acinetobacter baumannii klinik izolatlarinin tigesiklin ve karbapeneme karsi in vitro duyarliliklari

Acinetobacter baumannii is a frequent cause of nosocomial infections in most hospitals. Management of infections caused by these strains is difficult, as the strains often display multiple drug resistance, including carbapenem. Tigecycline which is a glycylcycline derivative has antimicrobial activity against many gram-positive and gram-negative organisms. In this study, in vitro activity of tigecycline and carbapenems against clinical isolates of A.baumannii strains were investigated. A total of 100 A.baumannii isolates were collected from hospitalized patients with documented nosocomial infections [pneumonia (n = 39), surgical wound infection (n = 32), bacteremia (n = 16), catheter infection (n = 6), urinary tract infection (n = 5), peritonitis (n = 1), eye infection (n = 1)] between October 2006 and June 2007. Only one isolate per patient was included to the study. Minimum inhibitory concentrations (MIC) of tigecycline were determined by E-test (AB Biodisk, Sweden). Carbapenem resistance of A.baumannii strains were determined by disk diffusion method. All of the 100 A.baumannii isolates (100%) were found susceptible to tigecycline (MIC values ≤ 2 µg/ml; MIC ranges: 0.032-1.5 µg/ml). Imipenem susceptibility test was performed for 95 strains, and 36 (37.9%) were found sensitive, 18 (18.9%) were intermediate sensitive, and 41 (43.2%) were resistant. Meropenem susceptibility test was performed for 87 strains, and 22 (25.3%) were found sensitive, 9 (10.3%) were intermediate sensitive, and 56 (64.4%) were resistant. Since tigecycline is found quite effective on nosocomial A.baumannii isolates, it may be considered as a treatment alternative in infections caused by carbapenem-resistant Acinetobacter spp.

[Is pneumatic sample transport system also a carrier for microorganisms?]. / Pnömatik sistem mikroorganizmalar için de tasiyici mi?

The purpose of this study was to evaluate the possible infection and contamination risk of the pneumatic system used in our hospital and to establish essential infection control measures. The study was conducted in a quaternary health care center with 1.000 bed capacity. A total of 614 specimens were taken 2 times weekly from the pneumatic transport system and its carriers at 22 wards, 5 intensive care units, 3 laboratories, 2 blood taking units, and pharmacy. Samples were also obtained from the fingertips of 33 subjects using the system, before and after contact with the carriers. A questionnaire that consisted of 8 questions was applied to 224 subjects who worked in those units, evaluating the degree of compliance to the obligations for the cleaning of the pneumatic system and carriers and their approach in case of visible pollution at the system. Bacterial growth was observed in 15.2% (45/296) of samples in the 1st week and 7.6% (18/238) of the samples in the 2nd week, making a total of 11.8% (63/534) bacterial growth. No growth was detected from the areas where the carriers were placed. Of these 69.8% were coagulase negative staphylococci, 11.1% diphteroids, 7.9% Acinetobacter Iwoffii, 4.8% Staphylococcus aureus, 4.8% Bacillus spp. and 1.6% Enterococcus durans. Acinetobacter baumannii and Aspergillus were detected at two fingertip samples taken before the contact with carriers, while again A. baumannii and Enterobacter cloacae were detected at the samples following contact. Moreover, 31.3% of the subjects noted that they cleaned the carriers only if any visible contamination was present. In addition, 14.3% reported that they have encountered broken or spilled up material in the system for more than 5 times, 10.3% reported that they followed the instructions in case of presence of infected material inside the carriers, 23.7% reported that they always washed their hands after any contact with the carriers, 9.8% noted that they always used gloves during contact with the system. Of the subjects 73.7% declared that they had no information about cleaning and decontamination procedures related to the system. These data revealed that the pneumatic system used in our hospital carried contamination risk and the rules for hygiene and disinfection regarding the pneumatic transport system has to be determined, implemented and checked in order to establish appropriate infection control measures.

[Short communication: In vitro activity of amphotericin B with fluconazole or voriconazole combinations against Candida albicans isolates]. / Kisa bildiri: Candida albicans izolatlarina karsi amfoterisin B ile flukonazol ve vorikonazol kombinasyonlarinin in vitro etkinligi.

The aim of this study was to investigate the in vitro interaction of amphotericin B in combination with fluconazole or voriconazole against Candida albicans clinical isolates by using a broth microdilution checkerboard assay and E-test. A total of 30 C. albicans strains isolated from blood, urine, sputum and pus samples were included to the study and the minimum inhibitory concentrations (MICs) of amphotericin B (AMB), fluconazole (FLU) and voriconazole (VOR) were determined by broth microdilution method and E-test. All strains tested for susceptibility were interpreted as susceptible by both methods (FLU MICs < 8 microg/ml, VOR and AMB MICs < 1 microg/ml). The rates of MIC agreement between two methods were as follows: AMB, 83%; FLU, 97%; VOR, 97%. AMB+ FLU and AMB+VOR combinations were tested by checkerboard broth microdilution and E-test methods. The combination test results were determined by using the fractional inhibitory concentration (FIC) index as synergistic, indifferent or antagonistic. AMB+FLU combination tested by checkerboard broth microdilution revealed synergy in one strain (3.3%) and antagonism in none, while the same combination tested by E-test revealed synergy in two (6.6%) and antagonism in four (13.3%) strains. The strains which exhibited synergy were different from eachother in two assays. This combination led to indifferent results in 23 (76.6%) of the strains. On the other hand AMB+VOR combination yielded synergistic results in two (6.6%) strains by both of the methods, however, these two strains were again different from eachother. No antagonism was detected by AMB+VOR combination while the combination was indifferent in 26 (86.6%) of the strains. Agreement between the checkerboard and E-test results was 87%. Although significant synergy was not detected in AMB+azole combinations, it was yet hopeful to obtain no antagonism. However, multi-center, large-scale, well standardized in-vitro and clinical studies about AMB and azole interaction which is a matter of debate, are necessary.

Methicillin-resistant S. aureus colonization in intensive care unit patients: Early identification and molecular typing.

INTRODUCTION: Early detection of methicillin-resistant Staphylococcus aureus (MRSA) in colonized patients is very important for infection control procedures to prevent MRSA spread. We aimed to monitor MRSA carriage in intensive care unit (ICU) patients and to evaluate the speed and efficiency of conventional culture, immunological, chromogenic, and molecular methods together with genotyping. METHODOLOGY: Nasal and axillar swab specimens were obtained from patients in the ICUs of the general surgery and neurosurgery wards in a tertiary hospital once a week over four weeks between December 2009 and July 2010. Oxacillin and cefoxitin disk diffusion tests, oxacillin agar screening test, latex agglutination test, chromogenic agar, and real-time polymerase chain reaction (PCR) tests were used for isolation and identification of MRSA. MRSA isolates were typed using ribotyping and pulsed-field gel electrophoresis (PFGE) typing. RESULTS: MRSA colonization was detected in 48 of 306 patients by real-time PCR. The MRSA colonization rate was 6.2%, 15.5%, and 38.5% at admission and in the first and second weeks, respectively. Sensitivity, specificity, positive and negative predictive values for all phenotypic tests were 98%, 99.6%, 98%, and 99.6%, respectively. The shortest handle time was observed in PCR. A total of three banding patterns were obtained from MRSA isolates by ribotyping, and PFGE analyses revealed 17 different pulsotypes varying from 11 to 18 distinct bands, showing high genetic diversity among the samples. CONCLUSION: Phenotypic MRSA screening tests in our study exhibited similar performances. The superiority of real-time PCR is its short turnaround time.

Evaluation of an automated system for the detection of carbapenem resistant Acinetobacter baumannii and assessment of metallo-ß-lactamase production using two different phenotyping methods.

We tested 200 clinical Acinetobacter baumannii isolates against imipenem and meropenem using the methods of broth microdilution, disk diffusion, agar dilution, MicroScan/WalkAway automated system. Very major errors were mostly obtained between MicroScan/WA system and disk diffusion test. MicroScan/WA system generated unacceptable errors. Combined disk and modified Hodge tests used for detection of metallo-ß-lactamase production were practical and useful.

The Evaluation of the Combination of Caspofungin with Voriconazole and Amphotericin B against Clinical Candida krusei Isolates by Etest and Disk Diffusion Methods.

Background: C. krusei is an opportunistic fungal pathogen that known of its intrinsic fluconazole resistance and its frequency is increasing especially among hematology patients. The increase in the frequency of high mortality fungal infections have accelerated the efforts of new drug development with broad spectrum, low toxicity and the studies including their combination. However, there is no standardized method to evaluate the activity of drug combinations. Aims: To evaluate the activity of caspofungin (CAS) with voriconazole (VOR) and amphotericin B (AMB) alone and in combination and the utility of Etest and disk diffusion methods for antifungal combinations. Methodology: The minimum inhibitory concentrations of VOR, CAS and AMB against 30 clinical C. krusei isolates were determined by using Etest, disk diffusion and reference broth microdilution methods. Combinations of CAS with VOR and CAS with AMB were evaluated using disk diffusion (three different ways) and Etest (two different ways) methods. Results: All isolates tested were susceptible to VOR and CAS in vitro by all three methods. Categorical agreements of Etest and disk diffusion methods with reference microdilutiontest were 100% for CAS and VOR (for each method), 86.7% and 50% for AMB, respectively. In the all ways of both combination methods, we did not observe distinctly antagonistic or synergic interaction. Conclusion: Etest and disk diffusion could be easy, convenient, and nontime-consuming alternative methods to evaluate the antifungal combinations. The combinations of CAS with VOR and AMB exhibited promising results because of an apparent antagonistic interaction was not detected in this study.