Jörg Wüllenweber

Rapid identification of microorganisms from positive blood cultures by MALDI‐TOF mass spectrometry subsequent to very short‐term incubation on solid medium

Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.

Autologous placental blood transfusion for the therapy of anaemic neonates

Almost 65% of all premature neonates with a birth weight <1,500 g receive at least one erythrocyte transfusion during their first weeks of life. In the present study, we examined the feasibility of autologous transfusions in neonates, using placental blood. Placental blood was obtained from 131 of 141 preterm and term infants using a special placental blood collecting system. Approximately 20 ml of placental blood per kilogram body weight could be harvested, irrespective of birth weight. One placental blood sample was contaminated with maternal erythrocytes; aerobe or anaerobe contamination was observed in any of the stored placental blood products (n = 119) after 35 days of storage. 19 of the 141 newborns needed allogeneic erythrocyte transfusions during the first 12 weeks of life. In 5 of these 19 patients, the amount of placental blood collected would have been enough to dispense with further allogeneic blood transfusions. After completion of the preclinical study, we transfused a total of 22 children, using autologous placental blood. 8 of the 10 infants with a birth weight between 1,000 and 2,000 g and 3 of 5 infants requiring surgical intervention directly after birth needed no further allogeneic blood transfusions. We, therefore, conclude that the collection and preparation of placental blood is feasible for clinical use. The target groups of neonates who are most likely to benefit are infants with a birth weight between 1,000 and 2,000 g and neonates requiring surgical intervention directly after birth.

Kytococcus schroeteri sp. nov., a novel Gram-positive actinobacterium isolated from a human clinical source.

A strain of a gram-positive, coccoid, yellow-pigmented bacterium was isolated from human blood. The bacterium was aerobic, non-encapsulated and non-motile. Phenotypically, the bacterium closely resembled Kytococcus sedentarius, but could be distinguished from this species by physiological tests and chemotaxonomic investigations. The peptidoglycan type is L-Lys-Glu2, variation A4alpha. The predominant menaquinones are MK-8 and MK-7. The major cellular fatty acids are iso-C17:1, iso-C17:0, iso-C15:0 and anteiso-C17:0. The strain contains catalase and does not produce acid from carbohydrates. The ability to hydrolyse Tween 80 and the lack of alpha-glucosidase activity are the most characteristic features. The results of comparative 16S rDNA analysis revealed that the strain represents a novel species within the genus Kytococcus, for which the name Kytococcus schroeteri sp. nov. is proposed. The type strain is strain Muenster 2000T (= DSM 13884T = CCM 4918T).

Rapid identification and susceptibility testing of Candida spp. from positive blood cultures by combination of direct MALDI-TOF mass spectrometry and direct inoculation of Vitek 2.

Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.

Processing of peripheral blood progenitor cell components in improved clean areas does not reduce the rate of microbial contamination

BACKGROUND: Microbial contamination of peripheral blood progenitor cell components (PBPCs) may cause severe complications in immunosuppressed recipients. Therefore, principles of Good Manufacturing Practice (GMP) are applicable for processing of PBPC components to reduce potential risks of contamination.

Prosthetic Valve Endocarditis due to Kytococcus schroeteri

To the Editor: Bacteria belonging to the former genus Micrococcus, the so-called micrococci, are usually regarded as contaminants from skin and mucous membranes. Nevertheless, micrococci have been reported as emerging pathogens in immunocompromised patients and have been described in severe infections (1–4). We describe what is, to our knowledge, the first case of prosthetic valve endocarditis caused by the newly described micrococcal species, Kytococcus schroeteri. Accurate identification of this species is of particular importance as kytococci—in contrast to other micrococcal species—are frequently resistant to penicillin and oxacillin (5).

Acceleration of Antimicrobial Susceptibility Testing of Positive Blood Cultures by Inoculation of Vitek 2 Cards with Briefly Incubated Solid Medium Cultures

ABSTRACT Briefly incubated agar cultures from positive blood cultures were used for antimicrobial susceptibility testing (AST) by Vitek 2. The cultivation time until inoculation was 3.8 h for Gram-positive cocci and 2.4 h for Gram-negative rods. The error rates were low, providing early and reliable AST without additional time or cost expenditure.

Pacemaker lead infection and related bacteraemia caused by normal and small colony variant phenotypes of Bacillus licheniformis.

Here, we report what we believe to be the first case of bacteraemia with small colony variants of Bacillus licheniformis related to a pacemaker lead infection by B. licheniformis displaying the normal phenotype. Arbitrarily primed PCR analysis showed a clonal strain. The infection was cured after the removal of the infected device.

Implementation of short incubation MALDI-TOF MS identification from positive blood cultures in routine diagnostics and effects on empiric antimicrobial therapy

BackgroundResults of blood culture (BC) diagnostics should be swiftly available to guide treatment of critically ill patients. Conventional BC diagnostics usually performs species identification of microorganisms from mature solid medium colonies. Species identification might be speed up by using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of biomass from shortly incubated solid media.MethodsThis single-center analysis compared the applicability of MALDI-TOF-based species identification from shortly incubated cultures in laboratory routine vs. conventional diagnostics and assessed its effects of on empiric antibiotic therapy.ResultsMedian time between detection of BCs as “positive” by incubators and further processing (e.g. microscopy) was 6xa0h 21xa0min. Median time between microscopy and result reporting to the ward was 15xa0min. Including 193 BCs, MALDI-TOF from shortly incubated biomass resulted in significantly faster (pu2009>u20090.001) species identification. Species results became available for clinicians after a median of 188xa0min (231xa0min for Gram-positive bacteria, 151xa0min for Gram-negative bacteria) compared to 909xa0min (nu2009=u2009192 BCs) when conventional diagnostics was used. For 152/179 bacteremia episodes (85%) empiric antibiotic therapy had already been started when the microscopy result was reported to the ward; microscopy led to changes of therapies in 14/179 (8%). In contrast, reporting the bacterial species (without antibiogram) resulted in therapeutic adjustments in 36/179 (20%). Evaluating these changes revealed improved therapies in 26/36 cases (72%).ConclusionsSpecies identification by MALDI-TOF MS from shortly incubated subcultures resulted in adjustments of empiric antibiotic therapies and might improve the clinical outcome of septic patients.

Progressive histoplasmosis with hemophagocytic lymphohistiocytosis and epithelioid cell granulomatosis: A case report and review of the literature

Histoplasmosis in central Europe is a rare fungal disease with diverse clinical presentations. Apart from acute pulmonary histoplasmosis and involvement of the central nervous system, the most serious clinical presentation is progressive disseminated histoplasmosis which is generally associated with severe immunodeficiency and, in particular, advanced human immunodeficiency virus infection. Here, we report on an immunocompetent female residing in a non‐endemic area, presenting with progressive disseminated histoplasmosis after a remote travel history to Thailand and Costa Rica. Diagnosis was delayed by several months due to misinterpretation of epithelioid cell granulomatosis of the intestine as Crohns disease and of similar lung lesions as acute sarcoidosis. Prompted by clinical deterioration with signs and symptoms consistent with hemophagocytic lymphohistiocytosis, a bone marrow aspiration was performed that documented hemophagocytosis and intracellular organisms interpreted as Leishmania sp., but later identified by molecular methods as Histoplasma capsulatum. Treatment with liposomal amphotericin B followed by posaconazole led to prompt clinical improvement and ultimately cure.