Pulmonary vasculitis due to infection with Mycobacterium goodii: A case report.

A 57-year-old Caucasian woman suffered from dyspnea on exertion. One year following a supposed pulmonary embolism event, a chronic thromboembolic vasculopathy was diagnosed and a pulmonary thromboendarterectomy was performed. However, a granulomatous pulmonary arterial vasculitis was identified upon examination. DNA of Mycobacterium goodii was detected as the most likely causative agent. Anti-inflammatory and anti-mycobacterial therapy was initiated for more than 12 months. Regular PET-CT scans revealed improvement under therapy. The last PET-CT did not show any tracer uptake following 10 months of therapy.

Accuracy of Different Rapid Urease Tests in Comparison with Histopathology in Patients with Endoscopic Signs of Gastritis.

BACKGROUND AND AIM: According to several guidelines, both invasive and non-invasive tests can be used to detect Helicobacter pylori (H. pylori). Invasive methods include H. pylori culture, histological staining, rapid urease tests (RUTs) and PCR. Non-invasive methods include urease breath test, stool antigen and serum IgG testing. The aim of our study was to compare all commercially available RUTs and histology in Germany. MATERIAL AND METHODS: One hundred fifty patients were enrolled in our study, irrespective of proton pump inhibitors (PPIs) or antibiotic use. If the results of RUTs and histology were diverging, real-time PCR to detect H. pylori DNA was undertaken. RESULTS: We detected no differences in the sensitivity or specificity between the different RUTs. In PPI and/or antibiotic-treated patients, RUTs seemed to be more sensitive for the detection of H. pylori infection compared to histology. In addition to the cheaper price of RUTs, they are also quicker to process. We show that histological staining in patients with signs of gastritis is expensive and not necessary, if there are no additional histological questions besides H. pylori status. CONCLUSIONS: In conclusion, we consider RUTs to be cheap and fast alternatives to histology in patients with endoscopic signs of gastritis, independently of whether PPIs or antibiotic are used. Histological evaluation is expensive, time consuming and may be unnecessary in some cases.

Clinical implications of Mycobacterium chimaera detection in thermoregulatory devices used for extracorporeal membrane oxygenation (ECMO), Germany, 2015 to 2016.

Mycobacterium chimaera, a non-tuberculous mycobacterium, was recently identified as causative agent of deep-seated infections in patients who had previously undergone open-chest cardiac surgery. Outbreak investigations suggested an aerosol-borne pathogen transmission originating from water contained in heater-cooler units (HCUs) used during cardiac surgery. Similar thermoregulatory devices are used for extracorporeal membrane oxygenation (ECMO) and M. chimaera might also be detectable in ECMO treatment settings. We performed a prospective microbiological study investigating the occurrence of M. chimaera in water from ECMO systems and in environmental samples, and a retrospective clinical review of possible ECMO-related mycobacterial infections among patients in a pneumological intensive care unit. We detected M. chimaera in 9 of 18 water samples from 10 different thermoregulatory ECMO devices; no mycobacteria were found in the nine room air samples and other environmental samples. Among 118 ECMO patients, 76 had bronchial specimens analysed for mycobacteria and M. chimaera was found in three individuals without signs of mycobacterial infection at the time of sampling. We conclude that M. chimaera can be detected in water samples from ECMO-associated thermoregulatory devices and might potentially pose patients at risk of infection. Further research is warranted to elucidate the clinical significance of M. chimaera in ECMO treatment settings.

Injection anthrax--a new outbreak in heroin users.

BACKGROUND: Injection anthrax is a rare disease that affects heroin users and is caused by Bacillus anthracis. In 2012, there were four cases in Germany, one of which was fatal, as well as a small number of cases in other European countries, including Denmark, France, and the United Kingdom. Three cases among drug users occurred in Germany in 2009/2010, in the setting of a larger outbreak centered on Scotland, where there were 119 cases. CASE PRESENTATION AND CLINICAL COURSE: We present three cases of injection anthrax, two of which were treated in Regensburg and one in Berlin. One patient died of multi-organ-system failure on the day of admission to the hospital. The others were treated with antibiotics, one of them also with surgical wound debridement. The laboratory diagnosis of injection anthrax is based on the demonstration of the pathogen, generally by culture and/or by polymerase chain reaction, in material removed directly from the patient's wound. The diagnosis is additionally supported by the detection of specific antibodies. CONCLUSION: Injection anthrax may be viewed either as an independent disease entity or as a special type of cutaneous anthrax with massive edema, necrotizing fasciitis in many cases, and about 30% mortality. It has appeared in recent years among heroin users in various European countries. In patients with suggestive clinical presentation and a history of heroin use, anthrax infection must be suspected early, so that the appropriate diagnostic tests can be performed without delay. Timely treatment can be life-saving. It is therefore important that physicians--and the individuals at risk--should be well-informed about this disease.

"Mycobacterium avium subsp. hominissuis" in neck lymph nodes of children and their environment examined by culture and triplex quantitative real-time PCR.

"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.

Laboratory diagnosis of endophthalmitis: comparison of microbiology and molecular methods in the European Society of Cataract & Refractive Surgeons multicenter study and susceptibility testing.

PURPOSE: To investigate and compare the use of molecular biology with the use of traditional Gram stain and organism culture for the laboratory diagnosis of postoperative endophthalmitis. SETTING: Twenty-four ophthalmology units together with 9 microbiology laboratories and 2 European reference molecular biology laboratories. METHODS: A prospective randomized partially masked multicenter cataract surgery study recruited 16 603 patients. This resulted in 29 cases of presumed postoperative endophthalmitis. Gram stain and culture were performed in the local laboratory according to agreed protocols. Samples of aqueous and/or vitreous were transported to the first referenced molecular biology laboratory (Regensburg, Germany) for polymerase chain reaction (PCR) testing, and an extracted aliquot of DNA was then referred to the second laboratory (Alicante, Spain) for PCR. RESULTS: Of the 29 who presented with presumed postoperative endophthalmitis, 20 were classified as proven infective endophthalmitis with positive Gram stain, culture, or PCR. Fourteen patients were culture-positive; all but 1 of these was also positive by PCR. Six patients were positive by PCR but negative by Gram stain or culture. Nine patients were negative by both microbiology and PCR testing. CONCLUSIONS: Use of molecular biology technique increased the laboratory rate of identifying the pathogen by 20%, confirming the technique is very useful for the endophthalmitis specimen. Samples of both aqueous and vitreous should be collected and stored at -20 degrees C for PCR at the time of the diagnostic taps.

Helicobacter pylori in human oral cavity and stomach.

The oral cavity has been suspected as an extra-gastroduodenal reservoir for Helicobacter pylori infection and transmission, but conflicting evidence exists regarding the occurrence of H. pylori in the mouth, independently of stomach colonization. Ninety-four gastric biopsy patients were analysed for the concurrent presence of H. pylori in the mouth and stomach. Samples were collected from different areas within the mouth and H. pylori DNA was amplified by the polymerase chain reaction (PCR) and verified by sequencing. Helicobacter pylori-specific serology was performed, and stomach colonization was determined by culture. In addition, relevant dental and periodontal parameters, as well as general health parameters, were recorded. Helicobacter pylori was found in the stomach of 29 patients and in the oral cavity of 16 patients. In only six patients was the bacterium detected simultaneously in the stomach and mouth. Notably, the 10 patients in whom the bacterium was found solely in the mouth did not have serum antibodies to H. pylori. The occurrence of H. pylori in the mouth was found to be correlated neither to any general or oral health parameters, nor to any particular site of collection. This study shows that H. pylori can occur in the oral cavity independently of stomach colonization.

Mycobacterium monacense in a patient with a pulmonary tumor.

We report on a Mycobacterium monacense infection associated with a pulmonary tumor in a Chinese patient. To our knowledge, this is the first case of M. monacense described in a non-European patient with a tuberculosis-like disease. Further evaluation of the human pathogenic potential of M. monacense is needed.

Extranodal marginal zone B-cell lymphomas of the ocular adnexa: multiparameter analysis of 34 cases including interphase molecular cytogenetics and PCR for Chlamydia psittaci.

Extranodal marginal zone B-cell lymphomas of MALT type (MALT lymphomas) show site-dependent variations in their morphologic, phenotypic, and/or cytogenetic findings. This report describes a comprehensive analysis of 34 ocular adnexa MALT lymphomas, including interphase fluorescence in situ hybridization for MALT lymphoma-associated cytogenetic abnormalities and polymerase chain reaction for Chlamydia psittaci, which has recently been suggested to be associated with ocular adnexa lymphomas. A typical morphologic pattern was identified in 79% of cases, while overtly monocytoid cytology (12%), predominantly plasmacytic features (9%), and lymphoepithelial lesions (3%) were uncommon. Aberrant CD43 or CD5 expression was also uncommon (12% and 3%, respectively). Plasmacytic differentiation (41%) was associated with stage IV disease (P=0.036) and gains of chromosomes 3 and/or 18q (P=0.021) (79%). +3 was more frequent in the orbit than in lacrimal gland or conjunctiva (P=0.005). Each of 31 cases was negative for MALT1 translocations. IGH translocations were identified in 3 cases (10%), although the translocation partner gene could not be identified. Polymerase chain reaction assays targeting species-specific regions within the C. psittaci omp1 and omp2 genes were negative in each of 30 cases. This study identifies the characteristic morphologic, phenotypic, and cytogenetic findings in ocular adnexa MALT lymphoma, including a subset differing from those arising at other anatomic sites. The frequent presence of +3 and/or +18q suggests that these abnormalities may contribute to lymphomagenesis. The lack of C. psittaci in this series, in contrast to some prior reports, indicates that there may also be geographic heterogeneity in the pathogenesis of ocular adnexa MALT lymphoma.

Frontline: control of Anaplasma phagocytophilum, an obligate intracellular pathogen, in the absence of inducible nitric oxide synthase, phagocyte NADPH oxidase, tumor necrosis factor, Toll-like receptor (TLR)2 and TLR4, or the TLR adaptor molecule MyD88.

Anaplasma phagocytophilum is an obligate intracellular bacterium that is related to rickettsial organisms and replicates in the hostile environment of neutrophils. Previous studies with SCID mice suggested that T and/or B cells are required for its control in vivo. Here, we used mice deficient for Toll-like receptor (TLR)2 and TLR4, MyD88, tumor necrosis factor, inducible nitric oxide synthase, or phagocyte NADPH oxidase (gp91(phox-/-)) to define the pathways that are critical for the recognition and the killing of this pathogen. Whereas SCID mice developed a 60-fold higher bacterial load in the blood compared to wild-type mice and succumbed to infection, all other gene-deficient mouse strains were fully capable in overcoming a systemic infection with A. phagocytophilum. From these data we conclude that effector mechanisms that are crucial to the defense against numerous other intracellular pathogens are dispensable for the control of A. phagocytophilum.

Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients.

Toxoplasma reactivation is a serious complication in patients receiving allogenic stem cell transplantation. Real-time PCR assays allow a rapid diagnosis of toxoplasma infection; however, no comparative data are available on the performance of real-time PCR protocols under routine conditions. Therefore, the aim of this study was to amplify Toxoplasma gondii DNA from routine samples of allogenic stem cell recipients using two real-time PCR assays on a LightCycler, and using conventional nested PCR. Conventional nested PCR revealed T. gondii DNA in 16 samples. Only 12 of the 16 samples yielded a positive result in both real-time PCRs. The accuracy of the conventional PCR results was demonstrated by direct sequencing. Amplification and detection of the amplicon was completed in only 1 h using the real-time PCR assays. Thus, real-time PCR substantially accelerates the detection of T. gondii DNA in the majority of positive specimens; however, conventional nested PCR is required for detection of T. gondii DNA in some samples.

Purification and immunological characterization of recombinant antigens expressed in the form of insoluble aggregates (inclusion bodies).

This chapter describes a straightforward protein purification strategy for the specific separation of insoluble recombinant proteins (so-called "inclusion bodies") located in the E. coli cytoplasm and their subsequent recovery in form of soluble recombinant proteins. Optimization of this technique can overcome in some cases the application of tedious and yield-reducing standard protein purification procedures. Due to the different behavior of individual recombinant proteins during separation, solubilization, and purification, subsequent purification steps may be required to obtain recombinant proteins in such a purity, that they can be used as antigen components of immunological test systems.

Helicobacter pylori and the risk of benign and malignant biliary tract disease.

BACKGROUND: The etiology of tumors arising in the biliary tract remains unclear. Several previous studies have detected Helicobacter pylori organisms in bile from patients with gallstones or cholecystitis. The objective of this study was to determine whether there is an association between H. pylori in bile and biliary tract carcinoma. METHODS: The authors used polymerase chain reaction (PCR) assays to detect the presence of H. pylori in the stomach and bile from 89 patients: Sixty-three disease free patients had biliary calculi, 15 patients had carcinoma of the biliary tract, and 11 patients had neither gallstones nor carcinoma. Bile was considered to contain H. pylori only if the results of PCR determinations were positive in two or more samples assayed independently in two separate laboratories. RESULTS: There was a strong association between the presence of H. pylori in the stomach and in the bile (P < or = 0.01). Biliary H. pylori was associated with age but not with gender, and it was associated strongly with the clinical diagnosis. Patients with gallstones were 3.5 times as likely to have H. pylori in the bile compared with patients in a control group (95% confidence interval [95%CI], 0.8-15.8; P = 0.100), and H. pylori was 9.9 times more frequent in patients with biliary tract carcinoma compared with patients in the control group (95%CI, 1.4-70.5; P = 0.022). CONCLUSIONS: There is a strong association between biliary tract carcinoma and H. pylori in bile. If these results are confirmed by prospective studies, H. pylori may be responsible for a significant proportion of malignant biliary tract disease.

Corneal haze after photorefractive keratectomy for myopia: role of collagen IV mRNA typing as a predictor of haze.

PURPOSE: To develop a test based on the individual expression of collagen type IV synthesis in corneal epithelial cells to identify patients who have the potential for significant corneal haze after myopic photorefractive keratectomy (PRK). SETTING: Department of Ophthalmology and the Institute of Microbiology, University of Regensburg, Germany. METHODS: The individual synthesis of collagen type IV alpha3 mRNA was quantitatively measured in corneal epithelial cells of 34 eye (34 patients) with myopia ranging from -1.5 to -10.0 diopters (D) by a polymerase chain reaction (PCR) test. The corneal epithelial cells were collected before the PRK procedure. Collagen type IV alpha3 mRNA levels were correlated to postoperative haze and regression at 12 months. RESULTS: In all samples, collagen type IV alpha3 mRNA was detected; the mean was 1.47 (range 0.11 to 6.42). There was a correlation between haze and the amount of collagen type IV alpha3 mRNA; that is, eyes with haze had more collagen IV expression. In contrast, no correlation was observed between regression and the amount of collagen type IV alpha3 mRNA. CONCLUSIONS: The results show that collagen type IV alpha3 is an important factor in the development of corneal haze after PRK. Based on a quantitative PCR test, the individual collagen IV mRNA concentration in corneal epithelial cells could be measured. Further development could establish a screening test by which eyes with pronounced synthesis of collagen IV could be identified as being at high risk for haze after PRK.

Septicemia due to Acinetobacter junii.

Acinetobacter spp. are considered to be emerging nosocomial pathogens. Acinetobacter junii is a rare cause of disease in humans and was associated mainly with bacteremia in preterm infants and pediatric oncologic patients. In this report we describe a case of catheter-related infection by A. junii in an adult oncologic patient. Application of molecular methods for precise species identification of Acinetobacter spp. will help to further clarify their role as human pathogens.

Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays.

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 x 10(1) to 2.9 x 10(4) CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.