Establishment of primary myoblast cell cultures from cryopreserved skeletal muscle biopsies to serve as a tool in related research & development studies.

BACKGROUND: Primary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes. METHODS AND RESULTS: This protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at -80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens. CONCLUSIONS: This protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.

Chromogenic culture media or rapid immunochromatographic test: Which is better for detecting Klebsiella pneumoniae that produce OXA-48 and can they be used in blood and urine specimens.

Our goal was to compare a rapid test (OXA-48K-SeT) and four different chromogenic media (CHROMagar KPC, CHROMagar mSuperCARBA, ChromID Carba and ChromID OXA-48) for the detection of OXA-48 producing Klebsiella pneumoniae isolates and spiked urine/blood samples with these bacteria. In total 100 K.pneumoniae isolates, including 60 OXA-48 positive, 15 other carbapenemase producing, 15 Extended spectrum betalactamases (ESBL) positive and 10 carbapenem sensitive K.pneumoniae were included in the study. After all samples were inoculated into all chromogenic media, temocillin discs were placed onto the media. OXA-48K-SeT was studied according to the manufacturer's instructions and the lower detection limit was determined. Sensitivities and specificities of all chromogenic media and rapid test were detected as 100%. All of the OXA-48 producers were found resistant to temocillin on all chromogenic media. The lower detection limit of the rapid assay was determined as 106 in both direct bacterial samples and in spiked urine/blood samples. As a result, four chromogenic culture media and OXA-48 K-SeT can be used safely for detection of OXA-48 positive K.pneumoniae isolates. Although direct clinical specimens were not used, our study suggests that this media and OXA-48 K-SeT may be used in patient samples like blood and urine. Further studies are needed to assess this suggestion.

The identification of carbapenemase types in Enterobacteriaceae by using molecular assay and phenotyping confirmation tests.

In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as control group were included in our study. The Xpert CARBA-R test was used as the molecular method. Antibiotic susceptibility tests were performed using combined discs, manually prepared with APBA (3-aminophenyl boronic acid), DPA (dipicolinic acid), EDTA (Ethylene diamine tetra acetic acid), cloxacillin supplements and Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors and temocillin discs. The modified Hodge test was performed on all isolates. OXA-48 gene was identified in 129 isolates , the NDM gene was identified in 10 isolates and VIM in one isolate. Thirty inaccurate results (30/185, 16%) were detected by using the manually prepared confirmation test. The sensitivity and specificity of this test were identified respectively 85% and 73%. Also, the sensitivity and specificity of the Mastdiscs Combi-D70C were identified as 100%. Negative results were detected in 3 NDM isolates with the use of a modified Hodge test. Sensitivity and specificity were calculated for the modified Hodge test respectively 97% and 100%. Finally, molecular methods provide results rapidly but they are not always easily accessible. The modified Hodge test can be used only for screening as a first step test and is not one of the tests that can identify the type of the carbapenemase. When carbapenem-resistant Enterobacteriaceae are detected, a commercial kit like Mastdiscs Combi-D70 may be preferred instead of the manually prepared phenotypic verification tests.

[Inappropriate use of serological tests for hepatitis B virus in Evliya Celebi Education and Research Hospital of Dumlupinar University, Kütahya]. / Dumlupinar Üniversitesi Evliya Çelebi Egitim ve Arastirma Hastanesinde hepatit B serolojik testlerinin uygunsuz kullanimi, Kütahya.

Since hepatitis B virus (HBV) infections may cause serious clinical symptoms and become chronic, the appropriate use and interpretation of laboratory tests in diagnosis is crucial. Implementation of serological tests for all HBV markers leads to high cost, labour force and time loss, so diagnostic algorithms are used for this purpose. The aims of this retrospective study were to detect the number of unnecessary test requests in HBV infection serology and financial burden as its consequence and to find out the reasons and ways of solutions. Three-years records of microbiology laboratory of Dumlupinar University Evliya Celebi Training and Research Hospital, Kutahya, Turkey, between 2011-2013 were analysed for the detection of unnecessary test requests and a total of 179.640 HBV serological test results were evaluated. Serological tests requested more than one on the same day and HBeAg, anti-HBe and anti-HBc IgM tests required from the patients with natural immunity or vaccinated ones were considered as unnecessary test requests. The main reasons of unnecessary test requests were detected as follows; unable to check the previous HBV test results in HIMS (Hospital Information Management System) owing to restricted of time, not being friendly user of HIMS, not trusting the test results, not knowing diagnostics algorithms, requiring of the tests by secretaries or other staff like nurses, not questioning HBV vaccination and requiring screening tests because of the defensive medicine. Survey questions prepared to search these reasons and awareness level of diagnostic algorithms were handed and asked to fill out 146 physicians working at clinics which made over 100 unnecessary requests in 3 years. Total numbers of unnecessary requested serological HBV tests were 5415 (3.01%) and total financial burden for our hospital in three years was 42.256 TL (18.373 USD) according to the notifications of Turkish Social Security Institution. It was determined that the most unnecessary test requests were HBeAg (44.86%), anti-HBe (37.75%) and anti-HBc IgM (37.41%). The most unnecessary test requests were made by internal medicine, obstetrics and gynaecology, general surgery and cardiology clinics, respectively. According to questionnaires filled by the 100 clinicians, the most common causes of unnecessary tests were hepatitis test requests from hospitalized patients for screening (96%), the tests required by secretary or nurse (62%), pre-surgical screening tests (53%) and lack of knowledge about diagnostic algorithms (42%). In conclusion, it is suggested that test requests should be restricted by using screening algorithms and in-service training should be provided, tests required by secretary or nurse should be stopped and previous test results should be conveyed to the clinicians actively through HIMS in order to prevent unnecessary test requests causing economic and workforce loss in HBV infection serology. These measures can provide early and true diagnosis for the patients and also will reduce the economic loss.