Improved Discrimination of Bacillus anthracis from Closely Related Species in the Bacillus cereus Sensu Lato Group Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Discrimination of highly pathogenic bacteria, such as Bacillus anthracis, from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to a collection of B. anthracis strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverse Bacillus strains of the Bacillus cereus sensu lato group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra of B. anthracis not present in closely related Bacillus species. We could identify 7, 10, 18, and 14 B. anthracis-specific biomarker candidates that were absent in B. cereus, B. mycoides, B. thuringiensis, and B. weihenstephanensis strains, respectively. Main spectra (MSP) of a defined collection of Bacillus strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains of B. anthracis and B. cereus yielded improved score values. The B. anthracis strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.

Yersinia pestis DNA from skeletal remains from the 6(th) century AD reveals insights into Justinianic Plague.

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.

Mobile diagnostics in outbreak response, not only for Ebola: a blueprint for a modular and robust field laboratory.

We established a modular, rapidly deployable laboratory system that provides diagnostic support in resource-limited, remote areas. Developed as a quick response asset to unusual outbreaks of infectious diseases worldwide, several of these laboratories have been used as part of the World Health Organization response to the Ebola virus outbreaks by teams of the 'European Mobile Lab' project in West Africa since March 2014. Within three days from deployment, the first European mobile laboratory became operational at the Ebola Treatment Unit (ETU) in Guéckédou, southern Guinea. Deployment in close proximity to the ETU decreased the turnaround time to an average of 4 h instead of several days in many cases. Between March 2014 and May 2015, more than 5,800 samples were tested in this field laboratory. Further EMLab units were deployed to Nigeria, Liberia and Sierra Leone in the following months of the Ebola outbreak. The technical concept of the EMLab units served as a blueprint for other mobile Ebola laboratories which have been set up in Mali, Côte d'Ivoire, Sierra Leone and other countries in West Africa. Here, we describe design, capabilities and utility of this deployable laboratory system for use in response to disease outbreaks, epidemiological surveillance and patient management.

Osteomyelitis caused by Burkholderia pseudomallei: relapse six years after pulmonary infection.

BACKGROUND: Burkholderia pseudomallei is highly endemic in Southeast Asia, whereas in Europe usually only few imported cases of melioidosis occur. CASE REPORT: In 2006, a 52-year-old male patient had been admitted to hospital with pneumonia after returning from a trip to Thailand. A blood culture isolate had been identified as Pseudomonas fluorescens and the patient had been treated with Piperacillin according to the antibiogram. Six years later the patient developed osteomyelitis of the leg and Burkholderia pseudomallei was identified as the causative agent. CONCLUSIONS: Misidentification of the cultural isolate in 2006 had led to inadequate therapy and to an unusually late relapse of melioidosis six years later.

Isolation, preliminary characterization, and full-genome analyses of tick-borne encephalitis virus from Mongolia.

Tick-borne encephalitis virus (TBEV) causes one of the most important inflammatory diseases of the central nervous system, namely severe encephalitis in Europe and Asia. Since the 1980s tick-borne encephalitis is known in Mongolia with increasing numbers of human cases reported during the last years. So far, however, data on TBEV strains are still sparse. We herein report the isolation of a TBEV strain from Ixodes persulcatus ticks collected in Mongolia in 2010. Phylogenetic analysis of the E-gene classified this isolate as Siberian subtype of TBEV. The Mongolian TBEV strain showed differences in virus titers, plaque sizes, and growth properties in two human neuronal cell-lines. In addition, the 10,242 nucleotide long open-reading frame and the corresponding polyprotein sequence were revealed. The isolate grouped in the genetic subclade of the Siberian subtype. The strain Zausaev (AF527415) and Vasilchenko (AF069066) had 97 and 94 % identity on the nucleotide level. In summary, we herein describe first detailed data regarding TBEV from Mongolia. Further investigations of TBEV in Mongolia and adjacent areas are needed to understand the intricate dispersal of this virus.

[Infection outbreak among German and Finish troups in Eastern Lapland during World War II - First description of hantavirus disease in the German language area]. / Infektionsausbruch bei deutschen und finnischen Soldaten während des 2. Weltkriegs im Östlichen Lappland ­ Erstbeschreibung der Hantavirus-Erkrankung im deutschen Sprachraum.

Eight decades ago, a report on "a swamp fever-like disease in German troups in Lapland" was published in this journal. The disease outbreak had occurred in 1942 and affected more than 1000 soldiers at the Finish front. The published, precise analysis of the clinical picture was obviously the first description of hantavirus disease in the German language area. Nowadays, hantavirus disease - in Central and Northern Europe also known as Nephropathia epidemica - is one of the most frequent notifiable virus diseases in Germany and Finland.

Identification and antimicrobial susceptibilities of Ochrobactrum spp.

Ochrobactrum (O.) anthropi is an opportunistic emerging pathogen closely related to the genus Brucella. Identification and differentiation from brucellae and other Ochrobactrum spp. using routine biochemical test systems is not reliable due to the high phenotypic similarity. In this study, antibiotic susceptibilities of 103 Ochrobactrum isolates were determined using Etest for 19 clinically relevant antimicrobial agents. Ochrobactrum strains were highly resistant to beta-lactam antibiotics, susceptible to ciprofloxacin, and 97.1% were susceptible to trimethoprim/sulfamethoxazole. It was also demonstrated that biochemical reaction profiles of the API and BD Phoenix 100 systems for identifying Ochrobactrum isolates can only be used on the genus level. Our in vitro data suggest that combinations of antimicrobial agents including ciprofloxacin and/or trimethoprim/sulfamethoxazole may be useful for empirical treatment of Ochrobactrum infections.

Outbreak of Query fever among Argentinean special police unit officers during a United Nations mission in Prizren, South Kosovo.

After an outbreak of Query fever (Q fever) in an Argentinean special police unit, 115 officers were investigated to evaluate the risk of infection with Coxiella burnetii after having been exposed to contaminated dust originating from a nearby barn harboring infected sheep. All officers were serologically tested and the medical history of potential risk factors was performed. The percentage of officers showing acute Q-fever seroconversion was found to be 51.3%. Forty-two individuals showed clinical symptoms, among them, 28 patients underwent medical care. No relevant risk factor was found. In areas of an unknown epidemiological situation, patients with unclear respiratory infections should be serologically tested for C. burnetii to offer the correct treatment and avoid possible chronic cases. Attention has to be drawn to choosing the site of a camp so as to protect troops from possible infectious disease. During a U.N. mission in Kosovo, we observed a Q-fever outbreak among the Argentinean special police unit. Our investigation was initiated to evaluate the incidence of C. burnetii infection and Q-fever manifestations in an entire population sharing the same exposure risk and to develop suitable measures to interrupt transmission.

Hyporesponsiveness of T cell subsets after cardiac surgery: a product of altered cell function or merely a result of absolute cell count changes in peripheral blood?

OBJECTIVE: The activity of the specific immune system and especially the function of T helper (TH) cells are reduced after cardiac surgery. This decrease is followed by an increase in TH2 cell activity and a delayed recovery of TH1 cell function (TH1/TH2 shift). Neither the underlying cause nor the relationship between the absolute numbers of T lymphocyte subpopulations, the state of activation of these cells and cytokine synthesis in cell culture has been clarified. We conducted a prospective study in order to test the hypothesis that the decrease in specific immunity is not caused by dilution effects but by functional alterations in T cell subsets. METHODS: Blood samples were obtained from 40 patients undergoing elective cardiac surgery with cardiopulmonary bypass (CPB) preoperatively (d0), immediately after surgery (dx), and on the 1st (d1), 3rd (d3) and 5th (d5) postoperative days. The samples were stimulated for 24h with staphylococcal enterotoxin B and lipopolysaccharide. Interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-5 concentrations were measured by flow cytometry using a cytokine bead array kit. We determined white blood cell counts, analysed lymphocyte populations, and assayed human leukocyte antigen (HLA)-DR expression on cluster of differentiation (CD)4+ and CD8+ lymphocytes. Cytokine concentrations were corrected to preoperative absolute numbers of T helper cells. RESULTS: Leukocyte counts were elevated during the entire postoperative course with a maximum on dx. Absolute lymphocyte counts and especially the T cell subpopulations significantly increased immediately after surgery, then decreased to a minimum on d1 and increased again until they returned to preoperative levels on d3. The release of IFN-gamma, IL-2 and IL-4 was significantly reduced from dx to d5 with a minimum on d1. IL-5 was significantly reduced on dx and d1. When the concentrations were corrected to preoperative TH lymphocyte levels, IL-2 and IL-5 synthesis was significantly reduced only on dx and IL-4 release only on dx and d1. By contrast, IFN-gamma synthesis decreased postoperatively and remained suppressed until d5 with a minimum on d1. Only on d1 did an increase in HLA-DR expression give evidence of a change in the state of TH cell activation. CONCLUSIONS: The number of immune cells of the specific and the non-specific immune system is not reduced in the immediate postoperative period. Haemodilution thus has no detectable effect on immune function at this time point. Beginning on d1, the function of specific immune cells, especially TH lymphocytes, is severely suppressed. This functional alteration appears not to be preceded by T cell activation during CPB. Although TH cell activity begins to increase on d1, cytokine synthesis is reduced. When cytokine synthesis is corrected to the absolute number of TH cells in culture, there is strong evidence for an increase in TH2 cell activity. On the whole, these results corroborate the hypothesis of a TH1/TH2 shift that is primarily caused by an alteration of TH1 function. Neither haemodilution nor a preceding activation plays a major role.

[Brucellosis, an overview and current aspects]. / Brucellose.

Although brucellosis, a zoonosis mostly associated with sheep, goats and cattle, is not endemic in Germany, it is a relevant imported infectious disease. If patients suffer from fever of unknown origin after a stay in highly endemic countries like the Mediterranean area or the Arab world, it is mandatory to formally exclude brucellosis. Cultural methods are the diagnostic gold standard, but due to special methodical and infrastructural requirements it is essential to inform the laboratory at suspicion of infection. The treatment of brucellosis is challenging and usually based on a long-term combination regime using doxycycline and rifampin for at least 6 weeks.

Pro-inflammatory cytokines after different kinds of cardio-thoracic surgical procedures: is what we see what we know?

OBJECTIVE: Due to the combination of local trauma, extracorporeal circulation (ECC), and pulmonary and myocardial reperfusion, cardiac surgery leads to substantial changes in the immune system and possibly to post-operative complications. Procedures without ECC, however, have failed to demonstrate clear advantages. We hypothesized that ECC is far less important in this context than the reperfusion/reventilation of the lung parenchyma and the surgical trauma. We therefore conducted a prospective observational study to compare immune reactions after cardiac operations with those after thoracic surgery. METHODS: Serum levels of pro-inflammatory interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-alpha as well as C-reactive protein (CRP), lipoprotein-binding protein (LBP) and procalcitonin (PCT) were measured pre-operatively (d0), at the end of the operation (dx), 6h after the operation (dx+), on the 1st (d1), 3rd (d3), and 5th (d5) post-operative days in 108 patients (pts) undergoing elective coronary artery bypass grafting (CAB) with ECC (n=42, CPB CAB), off-pump coronary artery bypass surgery (n=24, OP CAB) without ECC or thoracic surgery (n=42, TS). RESULTS: After cardiac surgery (CS), IL-6 and IL-8 increased and reached a maximum on dx+. IL-6 returned to baseline values at d3, whereas IL-8 remained elevated until d5. No difference was found between OP CAB and CPB CAB patients. In the TS patients, IL-6 increased later (dx+) and absolute levels were lower than in the CS patients. No increase in IL-8 was noted in the TS patients. Due to the high variation in the results obtained in all three groups, there was no significant change in TNF-alpha. A comparison of TS, OP CAB, and CPB CAB revealed that the CS patients had higher levels on d0, dx, d3, and d5. Serum levels of CRP, LBP, and IL-2R increased from dx+ to d5 in all groups and reached maximum values on d3. Whereas we found no difference in CRP and IL-2R between the groups, LBP levels were significantly higher from dx+ to d3 after OP CAB. PCT was elevated from dx+ to d3 in all pts. Similar levels were noted for the TS and OP CAB patients. The CPB CAB patients showed the highest levels. CONCLUSIONS: Surgical trauma and reperfusion injury appear to represent the predominant factors resulting in immunologic changes after cardiac surgery. Cardiopulmonary bypass (CPB) may be less important for immune response and acute-phase reactions than previously suspected. In addition, our data indicate a relationship between IL-6 synthesis and the degree of surgical trauma. IL-8 appears to be elevated only after cardiac surgery whereas PCT liberation depended on the use of ECC.

Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Phenotypical characterization of Mongolian Yersinia pestis strains.

Although Mongolia is regarded as one of the possible places of plague radiation, only few data are available from Mongolian Yersinia pestis strains. In this study a total of 100 Mongolian Y. pestis strains isolated from wild mammals and their parasites between the years 1960 and 2007 were analyzed for their phenotype. All strains grew well on selective Cefsulodin-Irgasan-Novobiocin agar and were positive for the F1-antigen, the F1-gene (caf1), and the plasminogen activator gene (pla). Biochemical analyses using the API20E® system identified 93% of the strains correctly as Y. pestis. The BWY in-house system consisting of 38 biochemical reactions was used to differentiate among Y. pestis subspecies pestis biovars Antiqua and Medievalis and also between the subspecies microtus biovars Ulegeica and Caucasica. Antibiotic susceptibility testing according to Clinical and Laboratory Standards Institute-guidelines identified one strain as being multiresistant. This strain was isolated from a wildlife rodent with no anthropogenic influence and thus suggests naturally acquired resistance.

Yersinia pestis lineages in Mongolia.

BACKGROUND: Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia. METHODOLOGY/PRINCIPAL FINDINGS: Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches. CONCLUSIONS/SIGNIFICANCE: We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.

Rickettsia raoultii, the predominant Rickettsia found in Mongolian Dermacentor nuttalli.

Since the year 2005, clinical patterns resembling tick-borne rickettsioses have been noticed in Mongolia. Epidemiological data regarding species of the aetiological agent, tick vector, prevalence, and distribution as well as incidence of human cases throughout Mongolia are still sparse to date. In order to identify Rickettsia species occurring in Mongolia, we investigated Dermacentor nuttalli (n=179) and Ixodes persulcatus (n=374) collected in 4 selected provinces. Rickettsia raoultii was the predominant Rickettsia (82% prevalence) found in D. nuttalli and was also detected in I. persulcatus (0.8%). The Rickettsia prevalence in D. nuttalli from different provinces varied between 70% and 97%. In addition, R. sibirica was identified in approximately 4% of D. nuttalli, but solely from Arkhanghai province. The results of this study extend the common knowledge about the geographic distribution of R. raoultii and its high prevalence in D. nuttalli. Although the pathogenicity of this Rickettsia is still unclear, it should be considered in Mongolian patients suspected of having tick-borne rickettsiosis.

Telemicrobiology: a novel telemedicine capability for mission support in the field of infectious medicine.

Infectious diseases are among the most common medical conditions suffered by soldiers while serving in missions away from their home countries. The diagnosis of these diseases requires special procedures and expertise, both of which are provided by field microbiological laboratories. In order to support the diagnostic process by means of telemedicine, a modification of the standard German Armed Forces telemedicine workstation was devised. A telemicrobiology module with special equipment, camera, and software has been designed and validated. This module, currently in use in two operational military theaters, has stood the test in routine practice. It allows the transmission of high-quality static images of microscopic specimens or overgrown nutrient media in a matter of seconds. The inclusion of experts in diagnostic analysis through the use of telemedicine improves diagnostic specificity by avoiding false positive results and, particularly in medical parasitology, allows a treatment-essential diagnosis without the dispatch of specimens to Germany. The recently designed telemicrobiology module has been proven, and is now deployed, providing a higher level of field diagnostic support than previously possible.

Is interferon gamma suppression after cardiac surgery caused by a decreased interleukin-12 synthesis?

BACKGROUND: The suppression of interferon gamma (IFN-gamma) synthesis after cardiac surgery is discussed as a cause of postoperative immunosuppression that predisposes to postoperative infectious complications. Because several studies have suggested that interleukin-12 (IL-12) production by monocytes and macrophages is reduced after cardiac surgery, this might cause a decrease in IFN-gamma release. To better understand these processes, we assessed the role of IL-12 in IFN-gamma synthesis in vitro before and after cardiac surgery. METHODS: Heparinized whole blood samples were obtained from 20 patients undergoing elective cardiac surgery preoperatively (day 0) and on the first (day 1), third (day 3), and fifth (day 5) postoperative days, and stimulated (24 hours) with staphylococcal enterotoxin B and lipopolysaccharide. Recombinant IL-12 was added at each time point investigated. Interferon-gamma, IL-12, IL-2, IL-4, and IL-5 concentrations and histocompatibility leukocyte antigen-DR (HLA-DR) expression on monocytes and macrophages were assayed by flow cytometry. RESULTS: The HLA-DR expression, IL-12 release, and IFN-gamma synthesis were significantly reduced on day 1, day 3, and day 5. Recovery began on day 3. Interleukin-12 caused a significant increase in IFN-gamma synthesis at each time point. When IL-12 was added, IFN-gamma synthesis returned to preoperative levels on days 3 and 5. CONCLUSIONS: The synthesis of IFN-gamma is significantly reduced after cardiac surgery. The application of IL-12 causes an increase in IFN-gamma synthesis before surgery and a return of IFN-gamma to preoperative levels within a few days after surgery. These findings suggest that postoperative suppression of IFN-gamma release is caused by a decrease in IL-12 synthesis. In addition, IL-12 has a mainly proinflammatory effect both before and after surgery.

Early diagnosis of Acanthamoeba infection during routine cytological examination of cerebrospinal fluid.

Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection.