Microbiological monitoring of continuous positive airway pressure and resuscitation equipment in very-low birth weight infants.

BackgroundTo investigate the colonization of respiratory equipment and the rate of respiratory infections of very-low birth weight (VLBW) infants.MethodsThe prospective study includes 26 VLBW infants on continuous airway pressure (CPAP) from September until December 2012. Swabs from respiratory equipment and colonization/infections were evaluated.ResultsA total of 603 swabs was cultured with 298 isolates; 59% of cultures from CPAP equipment (n=337; 95% confidence interval (CI; 54;64)) and 19% from ambu bags (n=51; 95% CI (14;24)) were positive. Overall, 181/201 CPAP prongs and masks hosted 221 microorganisms. Colonization on days 3 and 7 were 93% and 87%, respectively, with an increase in pathogens and a decrease in skin flora (79% vs. 68%). Comparing the 58 paired swab results from days 3 and 7 showed an increase in Gram-negative bacteria (P=0.014). Eighteen infants had positive weekly screening results, with similar colonization of CPAP equipment, dominated by Enterobacteriacae. Pneumonia was diagnosed in two infants.ConclusionOf the CPAP equipment close to the patient, 90% was colonized with microorganisms increasing during 1 week of CPAP. The pathogens were dominated by gastrointestinal bacteria, and persisted over weeks. Frequent cleaning did not prevent pneumonia, although pneumonia rates were rare.

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 Crohn's 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.

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

BACKGROUND: Results 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. METHODS: This 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. RESULTS: Median time between detection of BCs as "positive" by incubators and further processing (e.g. microscopy) was 6 h 21 min. Median time between microscopy and result reporting to the ward was 15 min. Including 193 BCs, MALDI-TOF from shortly incubated biomass resulted in significantly faster (p > 0.001) species identification. Species results became available for clinicians after a median of 188 min (231 min for Gram-positive bacteria, 151 min for Gram-negative bacteria) compared to 909 min (n = 192 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%). CONCLUSIONS: Species 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.

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.

Acceleration of antimicrobial susceptibility testing of positive blood cultures by inoculation of Vitek 2 cards with briefly incubated solid medium cultures.

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.

A new triplex real time PCR which distinguishes between MRSA, MSSA, and mecA coagulase negative strains by means of melting point analysis using SYTO 9.

BACKGROUND: A new screening method was developed for the detection and identification of methicillin resistant staphylococcus aureus (MRSA) from sterile sites or mixed flora samples (inguinal or nose swabs). METHODS: After rapid treatment of samples, the method consists of two steps, template DNA preparation by a simple and rapid boiling procedure and a multiplex real time PCR. The triplex PCR system simultaneously detects the following targets, (i) the integration site for the open reading frame X (orfX) of the staphylococcal cassette chromosome mec type I - V (SCCmec), (ii) the mecA gene which codes for the penicillin-binding protein PBP-2a, and (iii) an internal control (IC) which can be amplified with the SCCmec primer system. A new buffer system, which contains the fluorescent dye SYTO 9, allows a reproducible real time PCR for the discrimination of the above mentioned PCR products by means of a high resolution melting point analysis (HRM). RESULTS: This new PCR system distinguishes between MRSA, MSSA, and mecA positive but coagulase-negative staphylococci (CoNS) strains. An internal control confirms the integrity of the PCR run. The HRM shows three melting points specific for each amplification product. 78.75 degrees C for the mecA gene, 83.15 degrees C for the SCCmec/orfX fragment and 88.25 degrees C for the internal control. CONCLUSIONS: This new multilocus MRSA PCR system is a fast and inexpensive alternative to commercially available test systems and costs only five to six euros.

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.

Simultaneous tuberculous meningoencephalitis in two siblings.

UNLABELLED: The high morbidity and mortality of tuberculous meningoencephalitis (TBM) warrants an early diagnosis and treatment. BCG vaccine has been proven to reduce the incidence of disseminated disease in children. We report on two siblings (2-year-old boy and 4-year-old girl) with simultaneous TBM, whose parents originated from Kosovo, Albania, but presently reside in Germany. Early diagnosis of TBM was delayed, and at first the misdiagnosis of viral meningoencephalitis was made. Antituberculosis treatment was not initiated despite profound hyponatremia, hydrocephalus, and signs of inflammatory cerebral disease. After establishing the diagnosis of TBM, the boy died from antituberculosis, drug-induced hepatic failure; the sister survived with severe neurological deficits. Contact tracing revealed that TB had been transmitted by a household contact person with proven pulmonary TB who had refused antituberculosis treatment. A thorough contact investigation including tuberculin skin testing to identify children at risk for TB in the vicinity of this patient was not carried out. These case reports demonstrate an unusual simultaneous occurrence of TBM in a brother and sister. It draws attention to the importance of TBM as a differential diagnosis in children with suspected viral meningoencephalitis. CONCLUSIONS: To prevent severe neurological sequelae, early antituberculosis therapy should be considered in infants and children with a clinical impression of meningitis in the context of cerebrospinal fluid white blood cell count of less than 500 cells/microl and lymphocytic predominance, hyponatremia, and possible hydrocephalus. This notion is especially true for children originating from high-endemicity countries for TB. A rigid implementation of antituberculosis treatment of infected individuals and contact tracing is mandatory in order to prevent dissemination of TB in the community. The use of BCG vaccine should be considered in children at high risk for TB infection because of its potential to reduce disseminated TB.

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).

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.

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. STUDY DESIGN AND METHODS: It was investigated in a retrospective study whether the microbial contamination of PBPC components could be reduced after processing in improved clean areas according to the "Manufacture of Sterile Medicinal Products." Starting in 1994, a total of 1478 autologous and allogeneic PBPC components have been collected and processed into 3149 cryopreservation bags at the Department of Transfusion Medicine. Sterility testing was performed for all bags. Until December 1998, 783 PBPC components were processed at a clean bench only (group I). Thereafter, 695 PBPC components have been processed at a clean bench located in a clean area with an airlock system for personnel and equipment (group II). RESULTS: In group I, 16 of 1555 bags (1.03%) showed positive results in the first sterility testing. In group II, 21 of 1594 bags (1.32%) were positive (p = NS). The clinical follow-up was inconspicuous. CONCLUSION: Microbial contamination of PBPC components could not be reduced by installation of improved clean area conditions.