Comparative evaluation of the detection rate, workflow and associated costs of a multiplex PCR panel versus conventional methods in diagnosis of infectious gastroenteritis.

Introduction. Infectious gastroenteritis is a common reason for consulting a physician. Although most cases of gastrointestinal illness are self-limiting, the identification of the etiologic pathogen by stool specimen analysis is important in cases of more severe illness and for epidemiological reasons.Due to the broad range of causative pathogens, the conventional examination of a stool specimen is labour-intensive and usually requires different diagnostic methods. Multiplex PCR tests [e.g. BioFire Gastrointestinal (GI) Panel] allow the rapid detecting of up to 22 pathogens in one test.Hypothesis. Using a multiplex PCR panel to test stool specimens for infectious gastroenteritis pathogens can improve the detection rate, reduce the time-to-result and hands-on time and lower the costs of a microbiology laboratory.Aim. This study was aimed at evaluating the detection rate, the workflow and associated costs of stool specimen management using the BioFire GI Panel versus conventional methods.Methodology. Stool specimens were evaluated prospectively during the routine operation. Pathogen detection rate, hands-on time, time-to-result and material and personnel costs were determined for the BioFire GI Panel and conventional methods-the latter based on physician request and excluding viral testing.Results. Analysing 333 specimens collected between 2019 and 2020, the detection rate of enteropathogens was significantly higher with a positivity rate of 39.9 % using the multiplex PCR panel compared with 15.0 % using the conventional methods. The BioFire GI Panel presented results in a median time of 2.2 h compared with 77.5 h for culture and 22.1 h for antigen testing, noting that no tests were performed at weekends except for toxinogenic Clostridioides difficile. Based on list prices, the BioFire GI Panel was nine times more expensive compared with conventional methods, whereas hands-on-time was significantly lower using the BioFire GI Panel.Conclusion. Multiplex PCR panels are valuable tools for laboratory identification of infectious agents causing diarrhoea. The higher costs of such a multiplex PCR panel might be outweighed by the higher detection rate, ease of handling, rapid results and most likely improved patient management. However, these panels do not provide information on antimicrobial susceptibility testing. Therefore, if this is necessary for targeted therapy or if outbreak monitoring and control is required, specimens must still be cultured.

Antimicrobial Activity of Ceftolozane-Tazobactam, Ceftazidime-Avibactam, and Cefiderocol against Multidrug-Resistant Pseudomonas aeruginosa Recovered at a German University Hospital.

Multidrug-resistant (MDR) Pseudomonas aeruginosa increasingly causes health care-associated infections. In this study, we determined the activity of ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol against 223 MDR P. aeruginosa clinical isolates recovered from 2013 to 2017 at the University Hospital Frankfurt by using MIC test strips. Furthermore, we evaluated the presence of genes encoding major ß-lactamases, such as VIM, IMP, NDM, GIM, SPM, and KPC; the extended spectrum ß-lactamase (ESBL)-carbapenemase GES; and the virulence-associated traits ExoS and ExoU, as in particular ExoU is thought to be associated with poor clinical outcome. For MDR P. aeruginosa isolates, the MIC50/MIC90 values of ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol were 8/>256 mg/L, 16/>256 mg/L, and 0.25/1 mg/L, respectively. Cefiderocol showed the highest susceptibility rate (97.3%) followed by ceftazidime-avibactam (48.4%) and ceftolozane-tazobactam (46.6%). In 81 (36.3%) isolates, carbapenemase gene blaVIM was detected, and in 5 (2.2%) isolates, blaGES was detected (with a positive association of exoU and blaVIM). More than half of the isolates belong to the so-called international P. aeruginosa "high-risk" clones, with sequence type 235 (ST235) (24.7%) being the most prevalent. This study underlines that ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol are important options for the treatment of infections due to MDR P. aeruginosa, with cefiderocol currently being the most active available antipseudomonal ß-lactam agent. According to our clinical experience, the outcome of cefiderocol therapy (8 patients) was favorable especially in cases of MDR P. aeruginosa-associated complicated urinary tract infections. IMPORTANCE After testing ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol against a collection of 233 multidrug-resistant (MDR) Pseudomonas aeruginosa, we showed that cefiderocol is the most active antipseudomonal ß-lactam agent (susceptibility rates were 46.6%, 48.4%, and 97.4%, respectively). The most prevalent one was sequence type 235 (ST235) (24.7%), followed by ST244, ST175, and ST233, with all belonging to the top 10 P. aeruginosa high-risk clones with worldwide distribution. Our data indicate that during surveillance studies special attention should be paid to the MDR and highly virulent VIM- and ExoU-producing variant of ST235. Furthermore, in the case of infections caused by carbapenemase-producing MDR P. aeruginosa, cefiderocol is the preferred treatment option, while outcomes of complicated urinary tract infections and hospital-acquired pneumonia with cefiderocol were favorable.

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

Current prevalence of multidrug-resistant organisms in long-term care facilities in the Rhine-Main district, Germany, 2013.

Multidrug-resistant organisms (MDRO) and in particular multidrug-resistant Gram-negative organisms (MRGN) are an increasing problem in hospital care. However, data on the current prevalence of MDRO in long-term care facilities (LTCFs) are rare. To assess carriage rates of MDRO in LTCF residents in the German Rhine-Main region, we performed a point prevalence survey in 2013. Swabs from nose, throat and perineum were analysed for meticillin-resistant Staphylococcus aureus (MRSA), perianal swabs were analysed for extended-spectrum beta-lactamase (ESBL)-producing organisms, MRGN and vancomycin-resistant enterococci (VRE). In 26 LTCFs, 690 residents were enrolled for analysis of MRSA colonisation and 455 for analysis of rectal carriage of ESBL/MRGN and VRE. Prevalences for MRSA, ESBL/MRGN and VRE were 6.5%, 17.8%, and 0.4%, respectively. MRSA carriage was significantly associated with MRSA history, the presence of urinary catheters, percutaneous endoscopic gastrostomy tubes and previous antibiotic therapy, whereas ESBL/MRGN carriage was exclusively associated with urinary catheters. In conclusion, this study revealed no increase in MRSA prevalence in LTCFs since 2007. In contrast, the rate of ESBL/MRGN carriage in German LTCFs was remarkably high. In nearly all positive residents, MDRO carriage had not been known before, indicating a lack of screening efforts and/or a lack of information on hospital discharge.

Effects of storage temperature on hematopoietic stability and microbial safety of BM aspirates.

Bone marrow (BM) remains a common source for hematopoietic SCT. Due to the transcutaneous approach, contamination with skin bacteria is common. The delay between harvest and transfusion can be considerable, potentially allowing for bacterial proliferation. The optimal transportation temperature, specifically with respect to bacterial growth and consequences thereof for hematopoietic quality, remain undefined. For 72 h, 66 individual BM samples, non-spiked/spiked with different bacteria, stored at 20-24 °C room temperature (RT) or 3-5 °C (cold), were serially analyzed for hematopoietic quality and microbial burden. Under most conditions, hematopoietic quality of BM was equal or better at RT: Typical BM contaminants (P. acnes and S. epidermidis) and E. coli were killed or bacterial proliferation was arrested at RT; hematopoietic quality was not impacted by the contamination. However, several pathogenic bacteria not typically found in BM (S. aureus and K. pneumoniae) proliferated dramatically at RT and impaired hematopoietic quality. Bacterial proliferation was arrested in the cold. The overwhelming majority of BM samples, that is, those that are sterile or contaminated only with skin commensals, will benefit from transportation at RT. Those bacteria that proliferate and perturb hematopoietic quality are not typically found in BM. Our data support recommendations for RT transportation and storage of BM.

Koch's postulates and the pathogenesis of comparative infectious disease causation associated with Bartonella species.

In his homage to Lucretius ('Georgica'), Vergil is credited with stating: 'Felix qui potuit rerum cognoscere causas' ('Fortunate is he who knows the causes of things'). Based on numerous commentaries and publications it is obvious that clinicians, diagnosticians and biomedical research scientists continue to struggle with disease causation, particularly in the assessment of the pathogenic role of 'stealth pathogens' that produce persistent infections in the host. Bartonella species, because of their evolutionary ability to induce persistent intravascular infections, present substantial challenges for researchers attempting to clarify the ability of these stealth bacteria to cause disease. By studying the comparative biological and pathological behaviour of microbes across mammalian genera, researchers might be able more rapidly to advance medical science and, subsequently, patient care by undertaking focused research efforts involving a single mammalian species or by attempting to recapitulate a complex disease in an rodent model. Therefore, in an effort to further assist in the establishment of disease causation by stealth pathogens, we use recent research observations involving the genus Bartonella to propose an additional postulate of comparative infectious disease causation to Koch's postulates.

Prevalence, Spectrum and Antibiotic Susceptibility of Bacterial and Candida Colonization between the 21st and 33rd Week of Gestation in Women with PPROM - 5 Years' Experience in 1 Perinatal Center.

Objective: The aim of this study was to evaluate the prevalence, spectrum and antibiotic susceptibility of bacterial and Candida colonization of the vagina between the 21st and the 33rd week of gestation in women who had preterm premature rupture of membranes (PPROM). Study design: High vaginal swabs from 245 subjects with PPROM were analyzed in a retrospective cohort study using cultivation-dependent methods. Patients were additionally divided into two groups: women with PPROM between the 21st and 27th week of gestation (group A) and women with PPROM between the 28th and 33rd week of gestation (group B). A subgroup analysis comparing the two groups was done. Results: The prevalence of pathological bacterial colonization was similar in both study groups (40.8 vs. 41.4 %; p > 0.05), however, a difference in antibiotic susceptibility was noted, which did not reach statistical significance (resistance to ampicillin 71.4 vs. 52.5 %; cefuroxime 9.5 vs. 11.7 %; gentamicin 28.6 vs. 16.4 %; ciprofloxacin 5.0 vs. 5.4 %). In group A there was a statistically significant lower rate of Candida colonization (11.1 vs. 24.3 %; p = 0.04). Conclusion: In patients with early PPROM, the rate of Candida colonization (group A) is lower and there are indications of a difference in antibiotic susceptibility of the colonizing bacteria depending on gestational age. Larger study groups are required to confirm these preliminary results.

Extension of platelet shelf life from 4 to 5 days by implementation of a new screening strategy in Germany.

BACKGROUND: The Paul-Ehrlich-Institute analysed all fatalities due to bacterial infections between 1997 and 2007. Thereafter, the platelet shelf life was reduced to a maximum of 4 days after blood donation because the majority of all cases of severe transfusion-transmitted bacterial infections occurred with day 5 platelets. The current study compares the analytical sensitivity and the diagnostic specificity of four rapid bacterial detection procedures. METHODS: Nine transfusion-relevant bacterial strains were spiked in pooled platelets or apheresis platelets at a low concentration (10 CFU/bag). Samples were collected after day 3, day 4 and day 5 and investigated by four rapid bacterial detection methods (modified BacT/ALERT, Bactiflow, FACS method and 16s DNA PCR methods). RESULTS: Seven out of nine bacterial strains were adequately detected by BacT/ALERT, Bactiflow and PCR in apheresis platelets and pooled platelets after sample collection at day 3, day 4 and day 5. For three bacterial strains, analytical sensitivity was reduced for the FACS method. Two bacterial strains did not grow under the storage conditions in either pooled or apheresis platelets. CONCLUSIONS: A late sample collection on day 3, day 4 or day 5 after blood donation in combination with a rapid bacterial detection method offers a new opportunity to improve blood safety and reduce errors due to sampling., BacT/ALERT, Bactiflow or 16s ID-NAT are feasible for late bacterial screening in platelets may provide data which support the extension of platelet shelf life in Germany to 5 days.

Peliosis hepatis in cats is not associated with Bartonella henselae infections.

Peliosis hepatis is a vasculoproliferative disorder of the liver with infectious and noninfectious causes. In humans and dogs, Bartonella henselae has been linked to peliosis hepatis. Although domestic cats are the natural reservoir of B. henselae and although peliosis hepatis is common in this species, an association between this condition and infection with B. henselae has never been investigated in cats. In this study, 26 cases of peliosis hepatis in cats were tested for B. henselae infection by nested polymerase chain reaction and immunohistochemistry. The authors failed to detect B. henselae nucleic acid or antigen in any of the affected liver specimens. These findings suggest that, unlike in humans and dogs, peliosis hepatis in cats may not be significantly associated with a B. henselae infection.

Intracranial hemangiomas in a patient with POEMS syndrome.

POEMS syndrome is a rare multi-system disease with typical features of polyneuropathy, organomegaly, endocrinopathy, monoclonal plasmaproliferative disorder and skin changes. We describe a 44-year-old woman with polyneuropathy, hepatomegaly, IgA lambda-plasmacytoma, thrombocytosis, papilledema with elevated protein levels in cerebrospinal fluid and multiple cutaneous hemangiomas who was diagnosed with three intracranial lesions. Histology revealed capillary hemangiomas, one of them displaying partially glomeruloid features.

Non-tuberculous mycobacterial infection of the parotid gland in an immunosuppressed adult.

Infections of the parotid gland with non-tuberculous mycobacteria (NTM) are rarely described. Here, we report on an infection of the parotid gland caused by Mycobacterium avium and give a literature-based overview about this entity. In the light of a global increase of mycobacterial infections, unusual manifestations have to be considered and should be included in the differential diagnosis when dealing with solid lesions of uncertain aetiology in the head and neck region.

Rapid detection of methicillin-resistant staphylococci by real-time PCR directly from positive blood culture bottles.

For the rapid detection of methicillin-resistant staphylococci directly from blood cultures containing gram-positive cocci in clusters, we implemented a real-time (LightCycler) polymerase chain reaction (PCR) specific for the Staphylococcus aureus nuc gene encoding nuclease and the mecA gene encoding methicillin resistance. For the 475 positive blood cultures tested, the assay turned out to have 100% sensitivity and 100% specificity for the identification of methicillin-susceptible (n = 108) and methicillin-resistant (n = 34) S. aureus. When coagulase-negative staphylococci (CoNS) were included, the overall sensitivity for the detection of methicillin resistance was 93% and the specificity was 99%. Real-time PCR for nuc and mecA from blood culture bottles with staphylococci yields therefore a rapid (2-3 h) identification of S. aureus and CoNS including methicillin resistance.

Acute disseminated encephalomyelitis (ADEM) due to Mycoplasma pneumoniae infection in an adolescent.

A 17-year-old boy presented with a severe form of an acute disseminated encephalomyelitis (ADEM) with hemiparesis and coma after initial symptoms of a flu-like febrile infection 1 week previously. Titers against Mycoplasma pneumoniae were significantly increased in serum and cerebrospinal fluid (CSF). Detection of M. pneumoniae was achieved in the initial CSF sample using M. pneumoniae-specific PCR. The patient improved significantly on antimicrobial therapy with erythromycin and immunosupressive therapy with immunoglobulins and corticosteroids. This case report demonstrates a well-documented course of a central nervous system (CNS) infection resulting in the ADEM syndrome.

Evidence of a leading role for VEGF in Bartonella henselae-induced endothelial cell proliferations.

Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis (BA) and bacillary peliosis (BP). The pathomechanisms of these tumorous proliferations are unknown. Our results suggest a novel bacterial two-step pathogenicity strategy, in which the pathogen triggers growth factor production for subsequent proliferation of its own host cells. In fact, B. henselae induces host cell production of the angiogenic factor vascular endothelial growth factor (VEGF), leading to proliferation of endothelial cells. The presence of B. henselae pili was associated with host cell VEGF production, as a Pil- mutant of B. henselae was unable to induce VEGF production. In turn, VEGF-stimulated endothelial cells promoted the growth of B. henselae. Immunohistochemistry for VEGF in specimens from patients with BA or BP revealed increased VEGF expression in vivo. These findings suggest a novel bacteria-dependent mechanism of tumour growth.

Comparison of fluorescent in situ hybridization and conventional culturing for detection of Helicobacter pylori in gastric biopsy specimens.

In this study, we have investigated 201 gastric biopsy specimens obtained from dyspeptic patients for the presence of Helicobacter pylori. By means of fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescence-labeled oligonucleotide probes specific for H. pylori, this pathogen was detected in 63 biopsy specimens. By using conventional culturing, H. pylori was isolated from 49 of these 63 gastric biopsy specimens. In contrast, FISH failed to identify H. pylori in four samples from which the pathogen was cultured. The lowest sensitivity was obtained by using the urease test. H. pylori was detected indirectly by this method in 43 of 67 biopsy specimens, which were positive for the pathogen as determined by FISH and/or culturing. All 49 H. pylori isolates that were detected by FISH and culturing underwent antimicrobial susceptibility testing for clarithromycin, a macrolide drug that is a key component in the therapy of peptic ulcer disease caused by this pathogen. Clarithromycin susceptibility testing of cultured isolates was carried out by the E-test, whereas FISH was used on biopsy specimens to detect clarithromycin-resistant mutant strains. No discrepancies were found between these two methods. Thirty-seven strains were clarithromycin sensitive, and eight H. pylori isolates were resistant to the macrolide. From another four biopsy specimens, a mixture of clarithromycin-sensitive and -resistant strains was identified by both methods. Thus, FISH is a reliable technique for determining the clarithromycin susceptibility of this pathogen. Taken together, FISH is a more sensitive and rapid technique than culturing for detection of H. pylori in gastric biopsy specimens. However, in the microbiology routine diagnostic laboratory, the combination of both FISH and conventional culturing significantly increases the sensitivity in detection of H. pylori.

Cat scratch disease due to Bartonella henselae infection mimicking parotid malignancy.

An unusual Bartonella henselae infection presenting clinically as a putative parotid cancer was diagnosed based on serological tests, histomorphology and amplification of a 16S-rDNA sequence of Bartonella henselae. The patient improved greatly upon antibiotic treatment and did not require surgery. Although uncommon, infection with Bartonella spp., particularly Bartonella henselae, should be included in the differential diagnosis of parotid tumors.

Interferon consensus sequence binding protein confers resistance against Yersinia enterocolitica.

Interferon consensus sequence binding protein (ICSBP)-deficient mice display enhanced susceptibility to intracellular pathogens. At least two distinct immunoregulatory defects are responsible for this phenotype. First, diminished production of reactive oxygen intermediates in macrophages results in impaired intracellular killing of microorganisms. Second, defective early interleukin-12 (IL-12) production upon microbial challenge leads to a failure in gamma interferon (IFN-gamma) induction and subsequently in T helper 1 immune responses. Here, we investigated the role of ICSBP in resistance against the extracellular bacterium Yersinia enterocolitica. ICSBP(-/-) mice failed to produce IL-12 and IFN-gamma, but also IL-4, after Yersinia challenge. In addition, granuloma formation was highly disturbed in infected ICSBP(-/-) mice, leading to multiple necrotic abscesses in affected organs. Consequently, ICSBP(-/-) mice rapidly succumbed to acute Yersinia infection. In vitro treatment of spleen cells from ICSBP(-/-) mice with recombinant IL-12 (rIL-12) or rIL-18 in combination with a second stimulus resulted in IFN-gamma induction. In experimental therapy of infected ICSBP(-/-) mice, we observed that administration of rIL-12 induced IFN-gamma production which was associated with improved resistance to Yersinia. In contrast, treatment with rIL-18 failed to enhance endogenous IFN-gamma production but nevertheless reduced bacterial burden in ICSBP(-/-) mice. Although cytokine therapy with rIL-12 or rIL-18 ameliorated the course of Yersinia infection in ICSBP(-/-) mice, both cytokines failed to completely restore impaired immunity. Taken together, the results indicate that the transcription factor ICSBP is essential for efficient host immune defense against Yersinia. These results are important for understanding the complex host immune responses in bacterial infections.

Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures.

Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients.