Hans-Jörg Linde

Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis

BackgroundTimely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC). We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis.MethodsBlood samples from patients with presumed sepsis were cultured with the Bactec 9240™ system (Becton Dickinson, Heidelberg, Germany) and aliquots subjected to analysis with the LightCycler® SeptiFast® (SF) Test (Roche Diagnostics, Mannheim, Germany) at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made.ResultsOf 101 blood samples from 77 patients, 63 (62%) yielded concordant negative results, 14 (13%) concordant positive and 9 (9%) were BC positive only. In 14 (13%) samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients). In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%).ConclusionThe addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.

Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis.

ABSTRACT Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day −2 to day +7. Chronic colitis was induced by transfer of CD4+ CD62L+ T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-γ], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-γ, 32,477 ± 6,377 versus 9,734 ± 1,717 [P = 0.004]; IL-6, 231 ± 35 versus 121 ± 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 ± 0.2 versus 1.9 ± 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.

Healthcare-associated outbreaks and community-acquired infections due to MRSA carrying the Panton-Valentine leucocidin gene in southeastern Germany

In response to several isolations of methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leucocidin gene (PVL-MRSA), the present study was conducted to document the spread of infection in a small region of southeastern Germany. During a 9-month period, two healthcare-associated outbreaks with PVL-MRSA occurred, affecting 83 patients, personnel and contacts of personnel, and 34 additional cases were detected in the community. The clinical spectrum ranged from colonization to skin infection and necrotizing pneumonia. The findings represent the largest number of PVL-MRSA cases detected in Germany so far, and demonstrate the potential of this emerging pathogen to spread within the community and in healthcare institutions.

Improved detection of microorganisms by polymerase chain reaction in delayed endophthalmitis after cataract surgery1

OBJECTIVE To evaluate whether the use of polymerase chain reaction (PCR) improves the identification of the causative pathogen in eyes developing delayed endophthalmitis after cataract surgery. DESIGN Prospective, noncomparative case series. PARTICIPANTS Consecutive series of 25 eyes with the clinical diagnosis of delayed endophthalmitis after cataract. MAIN OUTCOME MEASURE Presence of bacterial or fungal DNA in aqueous humor and vitreous samples. RESULTS In the aqueous humor the causative pathogen was identified in 84% (n = 21) of the eyes by PCR compared with 0% by diagnostic culture and 0% by microscopy. In the vitreous samples the pathogen was identified in 92% (n = 23) of the eyes by PCR compared with 24% by diagnostic culture (n = 6) and 0% by microscopy. CONCLUSIONS PCR is useful for the identification of the causative pathogen in delayed endophthalmitis and had a higher rate of positive identification of the causative organism than microscopy or diagnostic culture.

Direct Detection and Differentiation of Legionella spp. and Legionella pneumophila in Clinical Specimens by Dual-Color Real-Time PCR and Melting Curve Analysis

ABSTRACT A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.

Combined skin disinfection with chlorhexidine/propanol and aqueous povidone-iodine reduces bacterial colonisation of central venous catheters

ObjectiveCentral venous catheter (CVC)-related infections may be caused by micro-organisms introduced from the skin surface into deeper tissue at the time of CVC insertion. The optimal disinfection regimen to avoid catheter-related infections has not yet been defined. This study compares three different approaches.DesignProspective randomised trial.SettingA tertiary care hospital.Patients and participantsOne hundred nineteen patients scheduled electively to receive 140 CVCs.InterventionsSkin disinfection was performed with either povidone-iodine 10% (PVP-iodine), chlorhexidine 0.5%/propanol 70%, or chlorhexidine 0.5%/propanol 70% followed by PVP-iodine 10%. Prior to disinfection, a swab from the site of insertion was taken for culture. CVCs were removed if no longer needed or infection was suspected. All catheters were cultured quantitatively after removal.Measurement and resultsBacteria could be isolated from 20.7% of the catheter tips. Bacterial growth was found in 30.8% of the catheters placed after skin disinfection with povidone-iodine, in 24.4% after disinfection with propanol/chlorhexidine and in 4.7% after disinfection with propanol/chlorhexidine followed by povidone-iodine (p=0.006). In 15 cases, the same organism was isolated from the skin swab and the catheter tip. Ten of these paired isolates showed the same pattern in a pulsed-field gel electrophoresis analysis.ConclusionsSkin disinfection with propanol/chlorhexidine followed by PVP-iodine was superior in the prevention of microbial CVC colonisation compared to either of the regimens alone. These results support the concept that catheter infections can originate from bacterial translocation at the time of catheter insertion.

A bacteria-induced switch of sympathetic effector mechanisms augments local inhibition of TNF-α and IL-6 secretion in the spleen

It is believed that an inflammation‐induced activation of the CNS leads to an inhibition of overshooting immune responses to prevent extensive local cytokine secretion. However, immunosuppression by the sympathetic nervous system may be unfavorable when bacteria are present locally and when TNF‐α is necessary to overcome infection. We now report in a superfusion model, using mouse spleen slices, that although local Pseudomonas aeruginosa increased splenic TNF‐α and IL‐6 secretion severalfold over basal levels, electrically released neurotransmitters attenuated cytokine secretion to similar basal level as under bacteria‐free conditions. Bacteria reversed noradrenergic inhibitory effector mechanisms: Under bacteria‐free conditions, TNF‐α secretion was very low and IL‐6 secretion was mainly inhibited by α2‐adrenoreceptor ligation. In the presence of bacteria, TNF‐α and IL‐6 secretion were high and IL‐6 secretion was mainly inhibited by β‐adrenoreceptor ligation. The α‐ to β‐adrenoswitch of IL‐6 inhibition in the presence of bacteria was mediated by the prior adrenergic regulation of TNF‐α. In vivo, chemical abrogation of sympathetic inhibition reduced accumulation of bacteria in the spleen, which depended at least in part on TNF‐α. This suggests that activation of the sympathetic nervous system may be a forerunner for accumulation of bacteria in tissue and consecutively sepsis due to intensified inhibition of TNF‐α secretion.—Straub, R. H., Linde, H.‐J., Männel, D. N., Schölmerich, J., Falk, W. A bacteria‐induced switch of sympathetic effector mechanisms augments local inhibition of TNF‐α and IL‐6 secretion in the spleen. FASEB J. 14, 1380–1388 (2000)

Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in Germany associated with travel or foreign family origin

Until recently, Panton–Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) strains were considered to be rare in Germany [1, 2]. However, data collected retrospectively (122 cases in the period 1995–2003) and prospectively (127 cases in 2004) for the geographical region of southeast Bavaria indicate that PVL-positive MRSA might be more common than previously acknowledged [3]. In the study presented here, we evaluated 122 patients in whom PVL-positive MRSA was detected in 2004. Nineteen of these patients had either traveled to (or originated from) foreign countries (predominantly in the Middle East) prior to the onset of symptoms or they belonged to a family originating from a Mediterranean country. The emergence of PVL-positive MRSA has been noted worldwide in patients without common risk factors for nosocomial multidrug-resistant pathogens [4, 5]. Patients typically present with a history of multiple recurrent deepseated abscesses, often without an obvious point of entry [6]. Necrotizing pneumonia may also occur. In many cases, carrier status or infections are recorded in close contacts of the index case, such as family members, sexual contacts, fellow military recruits, contact-sport participants, or prison inmates. In the laboratory, PVL-positive MRSA can be distinguished from nosocomial MRSA negative for PVL by a number of phenotypic and genotypic characteristics. Oxacillin resistance is caused by the mecA gene as part of a specific mec-type IV or -type II staphylococcal cassette chromosome [7]. In contrast to MRSA, many PVL-positive MRSA strains isolated in Germany, France or Switzerland are also resistant to fusidic acid (often associated with the far1 gene coding for an efflux mechanism [2, 8]), whereas resistance to other classes of antibiotics is rare. In addition, most strains of PVL-positive MRSA harbor a specific set of pathogenicity factors (e.g., staphylococcal enterotoxin H, bacteriocin bca, and Panton–Valentine leukocidin) [9]. Although the clinical presentation of infection caused by S. aureus might depend on a variety of factors not yet fully understood [10, 11], PVL is likely to play a key role in skin-related infections and necrotizing pneumonia [6]. PVL is a pore-forming toxin causing lysis of granulocytes and macrophages [12, 13]. The presence of PVL genes is demonstrated by PCR testing for lukS/F [6]. In a prospective study starting in January 2004, we collected clinical and epidemiological data from 127 patients with PVL-positive MRSA using a standardized questionnaire in interviews with patients or their physicians. A case was considered community-acquired if, at the time of symptom onset, the patient or close contacts had no connection with a medical institution and none of the following risk factors: open wound, chronic disease, or antibiotic consumption in the preceding 6 months. Otherwise, the case was considered nosocomial. Altogether, 39 community-acquired and 88 nosocomial cases were observed. Of the 88 nosocomial cases, 75 were detected in one single outbreak that was unrelated to all of the other cases, as proven by epidemiological data, a uniform and unique pattern in pulsed-field gel-electrophoresis, and MLST type 22 (data not shown); these cases were excluded from further analysis in this report. In 19 patients acquisition of PVL-positive MRSA was linked to foreign countries through travel or family connections: nine patients had a history of travel and ten patients had family originating from the Mediterranean J. Maier . H. Melzl . U. Reischl . N. Lehn . H. Linde (*) Institut für Medizinische Mikrobiologie und Hygiene, Franz-Josef-Strauss-Allee 11, 93049 Regensburg, Germany e-mail: Hans-Joerg.Linde@klinik.uni-regensburg.de Tel.: +49-941-9446414 Fax: +49-941-9446402

In vivo increase in resistance to ciprofloxacin in Escherichia coli associated with deletion of the C-terminal part of MarR.

ABSTRACT We recovered two isolates (EP1 and EP2) of Escherichia coli from the same patient that had identical pulsed-field gel electrophoresis patterns but required different MICs of ciprofloxacin (CIP): 16 and 256 mg/liter for EP1 and EP2, respectively. Both isolates had mutations in the quinolone resistance-determining regions of GyrA (Ser83Leu and Asp87Tyr) and ParC (Ser80Ile), but not in those regions of GyrB or ParE. Isolate EP2 was also more resistant to chloramphenicol, tetracyclines, cefuroxime, and organic solvents. A deletion of adenine (A) 1821 was found in marR of isolate EP2, which resulted in an 18-amino-acid C-terminal deletion in the MarR protein. The causative relationship between ΔA1821 and the Mar phenotype was demonstrated both by the replacement of the wild-typemarR by marR ΔA1821 in isolate EP1 and by complementation with the wild-type marR intrans in isolate EP2. In isolate EP2 complemented with wild-type marR, susceptibility to chloramphenicol was restored completely, whereas susceptibility to CIP was restored only incompletely. Northern blotting demonstrated increased expression ofmarA and acrAB but not of soxS in isolate EP2 compared to EP1. In conclusion, the deletion of A1821 inmarR in the clinical isolate EP2 caused an increase in the MICs of CIP and unrelated antibiotics. Presumably, the C-terminal part of MarR is necessary for proper repressor function.

Diagnosis of infectious endophthalmitis after cataract surgery by polymerase chain reaction

Purpose: To ascertain whether the use of the polymerase chain reaction (PCR) technique leads to more rapid diagnosis of infectious endophthalmitis after cataract surgery. Setting: University Eye Clinic Regensburg, Germany. Methods: The aqueous humor and vitreous of 16 eyes with infectious endophthalmitis (10 acute, 6 delayed) were evaluated by microscopy, diagnostic culture, and PCR to detect the infectious agent. Results: Microscopy of the vitreous was positive in 3 eyes and the culture media results were positive in 7 eyes, all with acute endophthalmitis. Significantly fewer positive results were obtained in the aqueous humor. Using PCR, an infectious agent was detected in the aqueous humor of all 16 eyes and in the vitreous of 14. The vitreous sample was negative in 2 eyes with delayed endophthalmitis. Conclusions: Detection of the infectious agent was more successful using PCR than using conventional microbiological tests, especially in the diagnosis of delayed endophthalmitis where the pathogen was detected in the aqueous humor in all eyes.