Detection of carbapenemase producers by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently applied in detection of carbapenemase-producing Gram-negative isolates. In the present study, we review the latest developments in this field.

Rapid identification of Gram-negative pathogens from positive blood cultures using MALDI-TOF MS following short-term incubation on solid media.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) enables the timely and reliable identification of microbes. The rapid identification of Gram-negative bacteria (GNB) in bloodstream infections is of critical importance. Several protocols have been proposed for the application of MALDI-TOF MS on samples from positive blood cultures (BCs) within the same day of BC positivity detection. The majority of these protocols include sample preparation steps with the use of chemicals or repeated centrifugations in order to avoid biases from human cells and proteins from the BC broth. These additional steps increase the hands-on processing time and the cost of identification. A different approach is to perform a MALDI-TOF MS analysis using biomass from briefly incubated subcultures on solid media. The present study discusses the findings of previous studies regarding the rapid identification of GNB from positive BC broth using MALDI-TOF MS following a short-term incubation period on solid media without any other additional steps or procedures.

Lactobacillus delbrueckii urinary tract infection in a male patient: a case report.

Introduction: Lactobacilli are Gram-positive rods, commensals of the normal human flora. Generally, these lactic acid-producing bacteria are considered contaminants, however over the last years their clinical relevance is reevaluated. Lactobacillus delbrueckii is very rarely isolated and only a few cases of L. delbrueckii urinary tract infections (UTIs) have been reported, mainly in females. Case report: We report the case of a L. delbrueckii UTI in an 82-year-old male suffering from benign prostate hyperplasia with repeated episodes of acute urinary retention over the last month before presenting to our hospital. The catheter urine culture grew >105 CFUs/mL of pure L. delbrueckii on Columbia CNA blood agar and on Trypticase soy agar. Identification was achieved by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), using VITEK MS (bioMérieux, France). The patient was successfully treated with cefixime for ten days. A follow-up urine culture performed 7 days after antibiotic discontinuation was sterile. Conclusions: To our knowledge the present is the second case of L. delbrueckii urinary tract infection in a male patient. Further cases are required to confirm the clinical significance of these unusual pathogens and their involvement in human urinary tract infections.

Female genital tuberculosis: a review.

Female genital tuberculosis is an uncommon type of tuberculosis that can lead to infertility. The present review describes the disease, reports available epidemiological data, and focuses on examinations and procedures necessary for the early diagnosis and the management of this curable disease.

[Comment] MALDI-TOF MS-based direct-on-target microdroplet growth assay: Latest developments.

The matrix-assisted laser desorption-ionization time-of-flight mass spectrometry direct-on-target microdroplet growth assay for the rapid susceptibility testing and the detection of the underlying antibiotic resistance mechanisms of microbia has been recently introduced. In the present study, we review the latest developments in the field.

Reduction in the processing of possible blood culture contaminants by the application of a selection criterion.

Possible blood culture (BC) contaminants are generally considered to be skin flora species including coagulase-negative Staphylococci (CNS), Corynebacterium species, Micrococcus species, Bacillus species and Propionibacterium acnes. Prior to October 1, 2016 all possible BC contaminants were fully processed (identification, susceptibility testing) in our laboratory. In order to reduce the laboratory workload from October 1, 2016 a possible contaminant was only processed if it was present in more than one BC pair drawn from the same patient within the same day. The two-year study period was divided in two periods namely period A from January 1, 2016 to September 30, 2016 (first 9 months) and period B from October 1, 2016 to December 31, 2017 (last 15 months). A series of indices (INs) were calculated including among others the Working Rate IN (WR) defined as the total isolates divided to the total number of BCs submitted per month and the CNS Rate (CNSR) defined as the total number of CNS processed divided to the total number of BCs submitted per month. A 23.08% reduction in the CNSR was noted (from 3.51% in period A to 2.70% in period B) whereas the overall WR was reduced from 7.19% in period A to 6.84% in period B. Furthermore, the total number of contaminants processed per month divided to the total number of isolates processed per month was reduced from 54.50% in period A to 42.41% in period B. The reduction in the INs recorded is of great value since it was achieved by the implementation of a simple criterion easily applicable and without any cost.

Evaluation of GenoType mycobacteria direct assay in comparison with Gen-Probe Mycobacterium tuberculosis amplified direct test and GenoType MTBDRplus for direct detection of Mycobacterium tuberculosis complex in clinical samples.

Three molecular assays were evaluated for the direct detection of Mycobacterium tuberculosis complex bacteria in 125 respiratory and 22 nonrespiratory samples. The overall sensitivities obtained were as follows: GenoType MTBDRplus, 97.9%; GenoType Mycobacteria Direct, 93.7%; Gen-Probe Mycobacterium tuberculosis Amplified Direct Test, 89.6%. The specificity of the assays used was 100%.

Molecular diagnostic tools in mycobacteriology.

Although the diagnosis of mycobacteriosis and susceptibility testing are still primarily based on conventional methods (staining, culture, biochemical analysis, proportional method), a series of molecular assays are increasingly introduced and incorporated in the workflow of clinical mycobacteriology laboratories worldwide. These assays are rapid and offer high sensitivities and specificities. In the present review, we describe the molecular assays concerning the early detection of Mycobacteria in clinical specimens, the identification of mycobacterial species, the detection of drug resistance and the typing for epidemiological investigations.

Miliary tuberculosis with no pulmonary involvement in myelodysplastic syndromes: a curable, yet rarely diagnosed, disease: case report and review of the literature.

BACKGROUND: Although tuberculosis is not uncommon among patients with myelodysplastic syndrome (MDS), only a few reports of such patients suffering from miliary tuberculosis (MT) exist. MT often presents as a fever of unknown origin and it is a curable disease, yet fatal if left untreated. CASE PRESENTATION: We report a case of MT with no clinical or laboratory indications of pulmonary involvement in a patient with MDS, and review the relevant literature. Mycobacterium tuberculosis was isolated from the liquid culture of a bone marrow aspirate. CONCLUSION: Even if the initial diagnostic investigation for a fever of obscure etiology is negative, MT should not be excluded from the differential diagnosis list. Since it is a curable disease, persistent and vigorous diagnostic efforts are warranted. In suspected cases, mycobacterial blood cultures should be collected as soon as possible after hospital admission and early bone marrow aspirate with mycobacterial cultures is advocated.

Analysis of the beta-lactam resistance phenotypes of Escherichia coli. An 8-year survey conducted in Greece.

There have been few studies, outside of France, on the resistance of Escherichi coli to beta-lactams by means of resistance phenotypes. In the present 8-year study from a tertiary Greek hospital, a statistically significant decrease in wild-type strains was noted, with a parallel increase in strains producing penicillinase. Of the total 6,089 isolates analyzed, 62.47% had no acquired resistance mechanisms while 35.70% produced penicillinase, 0.61% cefalosporinase and 0.94% extended-spectrum beta-lactamase. No overexpression of chromosomal cephalosporinase or synthesis of inhibitor-resistant enzymes was found. A shift in the pattern of penicillinase production was noted as, in the early years of the study, intermediate- and high-level penicillinase predominated whereas, in later years, low-level penicillinase prevailed.

Overexpression of VEGF and TGF-beta1 mRNA in Pap smears correlates with progression of cervical intraepithelial neoplasia to cancer: implication of YY1 in cervical tumorigenesis and HPV infection.

The screening of neo-angiogenesis related gene expression has uncovered many disrupted molecular pathways which may significantly confer to malignant transformation of various cell types including cervical cells. The objective of the present study was to delineate whether changes in certain gene expression profiles during the malignant conversion of the uterine cervix can be potentially used to predict the clinical course and outcome of the cervical pathology. Total RNA was isolated from Pap smears obtained from healthy females or patients diagnosed with low-grade squamous cervical intraepithelial lesions (LG-SIL), high-grade (HG)-SIL or cervical carcinoma. VEGF, TGF-beta1 and YY1 mRNA expression levels were assessed by QRT-PCR. Confirmation of YY1 protein discrepancy among cervical tissues of different histopathology was performed by immunohistochemistry. All tested genes showed statistically significant expression variations among the indicated groups. VEGF and TGF-beta1 mRNA overexpression was found to be associated with progression from low-grade to high-grade cervical intraepithelial neoplasia (CIN), while YY1 showed constitutively elevated transcript levels in CIN and cervical cancer compared to controls. At the protein level YY1 was also overexpressed in HG-SIL and cancer tissues compared to LG-SIL. Both YY1 transcript and protein overexpression were associated with HPV18- or HPV16-infected samples. Spearman analysis revealed a co-expression pattern for VEGF and TGF-beta1 mRNAs in normal cervix and LG-SIL; however, YY1 expression correlated negatively with VEGF and TGF-beta1 transcript levels upon the onset of the cervical neoplastic transformation. Our findings provide for the first time evidence for the implication of YY1 in uterine cervix carcinogenesis and suggest that VEGF, TGF-beta1 and YY1 could be useful biomarkers of cervical malignant transformation as well as potential targets for therapeutic approaches.

Major pathogen microorganisms except yeasts can be detected from blood cultures within the first three days of incubation: A two-year study from a University Hospital.

The knowledge of the expected time-to-positivity (TTP) of blood cultures by major pathogens is essential both clinically and economically. To this end, we conducted the present two-year study in our Institution, aiming to assess the TTP of all the major microorganisms including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetoacter baumannii, Enterococcii spp, Staphylococcus aureus and yeasts, to determine whether a 3-day interval is sufficient for their detection. The TTP for each case of strain isolation per patient was determined as the TTP of the first bottle among a set of bottles collected within the same period of time to be flagged as positive per patient. Based on our results, almost all major Gram-negative (99.30%), Gram-positive microbia (99.01%) and yeasts (98.85%) were detected within the first 5-days of incubation, leading to the solid conclusion that a 5-day period of incubation is adequate to detect almost all the major routine pathogens. By contrast, when a 3-day period was examined acceptable results were only found for Gram-negative (98.33%) and Gram-positive (98.51%) microbia. A significant proportion of yeasts (8.05%) could not be detected within this time frame. Therefore, regarding the yeasts, a 3-day incubation period cannot be considered as adequate and is not advocated.

Mycobacterium heraklionense sp. nov.: A case series.

Mycobacterium heraklionense sp. nov. (M. heraklionense) is a novel non-tuberculous mycobacterium belonging to the Mycobacterium terrae complex that has recently been described. It has a world-wide distribution. Recently, a case of tenosynovitis in an immunocompetent individual caused by M. heraklionense was reported, indicating that it has the ability to cause diseases. In the present study, in order to provide a more detailed profile of this mycobacterium and to obtain a more complete overall picture of its clinical significance, we report all available data regarding the initial 12 cases of its isolation. Of the 12 patients, 5 (42%) eventually died within a period of 3 months following the isolation of the mycobacterium. However, any connection between the presence of M. heraklionense and these deaths could not be documented. These 5 patients were all males with a mean age of 74.6 years suffering from serious underlying diseases, which most probably were the cause of death. Additional data from possible new cases of M. heraklionense isolation are anticipated.

Immunohistochemical expression of endoglin offers a reliable estimation of bone marrow neoangiogenesis in multiple myeloma.

PURPOSE: The aim of the present study was to evaluate CD105 tissue marker in the bone marrow (BM) of multiple myeloma (MM) patients. CD105 was evaluated using immunohistochemical method. An effort was made to correlate this marker with BM microvascular density (MVD) along with other known markers of angiogenesis in order to evaluate its clinical significance. METHODS: BM MVD was estimated by CD31. CD105 in BM was estimated by immunohistochemical method in 54 newly diagnosed patients with MM. Circulating levels of known angiogenic factors such as basic fibroblast growth factor (b-FGF) and soluble CD105 (sCD105) were measured by ELISA in the same group of patients. All these factors were also measured in 20 age- and sex-matched healthy controls. RESULTS: We found that CD105 MVD, along with the expected CD31 MVD, and serum levels of sCD105 and bFGF were increased, also in parallel with disease stage, and all were decreased after effective treatment. Moreover, CD105 MVD correlated with all the aforementioned markers of angiogenesis. CONCLUSIONS: Our results indicated that CD105 MVD is following the behavior of CD31 MVD in MM, suggesting being a valid marker of BM neoangiogenesis in MM. Its prognostic impact remains to be proven.

Molecular epidemiology of extended-spectrum beta-lactamases produced by clinical isolates in a university hospital in Greece: detection of SHV-5 in Pseudomonas aeruginosa and prevalence of SHV-12.

To assess the nature and diversity of various types of SHV and TEM derivatives in our hospital a survey was conducted. Sixty-seven extended-spectrum beta-lactamases (ESBL)-producing nosocomial pathogens, isolated over a 12-month period, were analyzed by means of PCR and direct sequencing. SHV-5 was the predominant ESBL found in our region (38 strains). Other less frequent variants included SHV-2 and SHV-12 with two and three isolates, respectively. For the first time, an outbreak of 11 Pseudomonas aeruginosa producing SHV-5 was encountered. All blaTEM-positive strains carried the non-ESBL TEM-1. The incidence of non-SHV non-TEM ESBLs was remarkably high as almost one out of three isolates harbored such an ESBL. The epidemiological and clinical impact of these findings must be carefully investigated and interpreted.

Spread of bla(VIM-1)-producing E. coli in a university hospital in Greece. Genetic analysis of the integron carrying the bla(VIM-1) metallo-beta-lactamase gene.

Bla(VIM-1) gene was detected in four Escherichia coli clinical isolates with both reduced susceptibility to carbapenems and an ESBL phenotype. The VIM-1 determinant was located within the variable region of a Class I integron along with a 6'-N-aminoglycoside acetyltransferase gene (aac(6')-Ib) and it could be transferred by conjugation. In all four clinical isolates the VIM-1 gene cassette presented a characteristic duplication of the 3' end coding 153 nucleotides followed by the first 14 nucleotides of the 59 base element (59be) that however did not seem to affect either the integrity of the coding sequence or the 59be of the gene cassette. These clinical isolates not only harbored the same Class I integron, but they also shared the same discrete ribotype-pattern, indicative for their clonal origin. Spread of carbapenem resistance genes among Enterobacteriaceae in hospital is a matter of great concern.

Is minocycline a solution for multidrug-resistant Acinetobacter baumannii?

Minocycline is an old, safe, second-line antimicrobial agent that has drawn attention over the last few years as a possible therapeutic option against multidrug-resistant Acinetobacter baumannii (MDR-AB) clinical isolates. Recent in vitro and in vivo results indicate that minocycline is a valid, alternative treatment option for minocycline-susceptible MDR-AB. Although effective alone, its administration as monotherapy should be avoided. Combinations with other antimicrobials can reduce the MIC of each component, present synergism and minimize the risk for drug resistance. Owing to its limited solubility in urine, it should be avoided for urinary pathogens. The present article reports all available information regarding its use as a therapeutic option against MDR-AB.

Confronting multidrug-resistant Acinetobacter baumannii: a review.

Multidrug-resistant Acinetobacter baumannii (MDR-AB) infections are difficult to treat owing to the extremely limited armamentarium. The present review reports all available treatment options against MDR-AB, including single molecules, combination schemes, and alternative modes of antimicrobial administration. Additionally, a group of recently reported peptides with anti-MDR-AB activity is described.

Report of 2 indigenous cases of leprosy from a European country: use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for identification of Mycobacterium leprae directly from a clinical sample.

In this article, we report on 2 indigenous cases of leprosy detected in a European country. We also report on the use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for rapid identification of Mycobacterium leprae directly from the clinical sample.