A prolonged outbreak of Salmonella Infantis associated with pork products in central Germany, April-October 2013.

One of the largest and longest Salmonella outbreaks in Germany within the last 10 years occurred in central Germany in 2013. To identify vehicles of infection, we analysed surveillance data, conducted a case-control study and food traceback. We identified 267 cases infected with Salmonella Infantis with symptom onset between 16 April and 26 October 2013 in four neighbouring federal states. Results of our study indicated that cases were more likely to have eaten raw minced pork from local butcher's shops [odds ratio (OR) 2·5, 95% confidence interval (CI) 1·1-5·8] and have taken gastric acid-reducing or -neutralizing medication (OR 3·8, 95% CI 1·3-13) than controls. The outbreak was traced back to contaminated raw pork products found in different butcher's shops supplied by one slaughterhouse, to pigs at one farm and to an animal feed producer. Characterization of isolates of human, food, animal, feed, and environmental origin by phage-typing and pulsed-field gel electrophoresis confirmed the chain of infection. Insufficient hygiene standards in the slaughterhouse were the most probable cause of the ongoing transmission. We recommend that persons taking gastric acid suppressants should refrain from consuming raw pork products. Improving and maintaining adequate hygiene standards and process controls during slaughter is important to prevent future outbreaks.

Puumala virus outbreak in Western Thuringia, Germany, 2010: epidemiology and strain identification.

In 2010, the highest annual number of human Puumala virus (PUUV) infections was reported in Germany since hantavirus surveillance started in 2001. The increase in annual case numbers was especially marked in western Thuringia. We combined results of case-based hantavirus surveillance in humans and serological and molecular investigations in the rodent reservoir to describe the epidemiological situation and to identify the putative outbreak strain. A 5-fold increase in notified hantavirus cases compared to the previous annual maximum was observed in western Thuringia in 2010. Disease incidence varied tremendously within a small geographical area with case patients' places of residence clustering around beech-dominated broad leaf forest patches. Investigations in the rodent reservoir revealed a novel Puumala virus (PUUV) subtype, which is clearly distinct from strains collected in other PUUV endemic regions of Germany. It can be assumed that in regions in western Thuringia where hantavirus cases occurred in 2010 or previous outbreak years, PUUV has been present in the environment for a long time. Further studies are needed to elucidate the population dynamics and hantavirus prevalence of the rodent reservoir and driving ecological factors.

Multilocus microsatellite markers for molecular typing of Candida glabrata: application to analysis of genetic relationships between bloodstream and digestive system isolates.

Candida glabrata has emerged as the second most common etiologic agent, after Candida albicans, of superficial and invasive candidiasis in adults. Strain typing is essential for epidemiological investigation, but easy-to-use and reliable typing methods are still lacking. We report the use of a multilocus microsatellite typing method with a set of eight markers on a panel of 180 strains, including 136 blood isolates from hospitalized patients and 34 digestive tract isolates from nonhospitalized patients. A total of 44 different alleles were observed, generating 87 distinct genotypes. In addition to perfect reproducibility, typing ability, and stability, the method had a discriminatory power calculated at 0.97 when all 8 markers were associated, making it suitable for tracing strains. In addition, it is shown that digestive tract isolates differed from blood culture isolates by exhibiting a higher genotypic diversity associated with different allelic frequencies and preferentially did not group in clonal complexes (CCs). The demonstration of the occurrence of microevolution in digestive strains supports the idea that C. glabrata can be a persistent commensal of the human gut.

[Mycoserology--did we move on? Aspergillus]. / Mykoserologie--sind wir weitergekommen? Aspergillus.

Diseases caused by Aspergillus spp. are difficult to diagnose and thus require supplementary serological assays. This is the result of a selective review of the relevant literature with special regard to recent guidelines. In addition to conventional diagnostic tools (radiology, microscopy, culture) the measurement of the following serological markers is recommended, depending on the clinical type of aspergillosis: Invasive and chronic necrotising aspergillosis: Aspergillus-galactomannan antigen. Test format: EIA using the rat MAb EB-A2. Cut-off 0.5 (index). Monitoring of high risk patients: Twice weekly. Aspergillus-IgG (test format EIA) as confirmatory assay after recovery of the leukocyte function under therapy. Aspergilloma: Aspergillus IgG. Test format: EIA. Allergical aspergillosis: Aspergillus IgE. Test format: RAST. Galactomannan antigen detection rates high in the diagnosis of invasive aspergillosis. The evaluation of Aspergillus nucleic acid amplification assays is pending.

[Performance of the Candida mannan antigen detection in patients with fungemia]. / Performance des Candida-Mannan-Antigennachweises bei Patienten mit Fungamien.

For several years, the Platelia Candida mannan antigen enzyme immunoassay (Candida EIA) has been commercially available as a diagnostic test for invasive candidosis. We evaluated the Candida EIA with patients with proven fungemia caused by yeasts from which at least one serum sample was available. Fifty-nine patients with 121 serum samples were included in the study. Sixty-one different yeast strains were isolated from positive blood-cultures. The Candida EIA was positive (n = 35) or borderline positive (n = 8) in 43 of 59 patients with fungemia, resulting in an overall sensitivity of 73%. For the different yeast species, the following sensitivities were calculated: Candida albicans 30 of 39 (77%), Candida glabrata 7 of 11 (64%), Candida parapsilosis 1 of 3, Candida tropicalis 2 of 2, Candida kefyr 2 of 2, Candida lipolytica 0 of 1, Candida lusitaniae 1 of 1, Candida krusei 1 borderline positive of 1, Saccharomyces cerevisiae 1 of 1. In six patients the antigen levels over time were evaluable. In three cases the antigen was positive 3-4 days before the day the blood culture was drawn, in one case on the same day, and in two cases 2 and 5 days afterwards. In conclusion, the Candida EIA was suitable for the detection of fungemia due to the major facultatively pathogenic yeast species. The test was positive in about half of the patients before blood cultures became positive. In these cases, it contributed to an early diagnosis of invasive candidiasis.

[Experience with the Platelia Candida ELISA for the diagnostics of invasive candidosis in neutropenic patients]. / Erfahrungen mit dem Platelia Candida ELISA zur Diagnostik invasiver Candida-Infektionen bei neutropenischen Patienten.

Invasive Candida infections (IC) belong to the most important opportunistic fungal infections in immunocompromised patients. IC is difficult to diagnose, because clinical symptoms are nonspecific and cultural methods lack sensitivity or specificity. We evaluated the Platelia Candida enzyme immunoassay (Candida EIA) for the diagnosis of IC in patients with haematological malignancies. A total of 62 neutropenic patients with 469 serum samples were included in the study. Candida colonization was monitored by weekly cultures of mouth washings, urine, and stool samples. Yeasts were grown from samples of 42 patients (68%), mainly Candida albicans (50%), followed by Candida glabrata (23%) and Candida krusei (20%). According to the criteria of the EORTC/NIH, the patients were categorized: (1) 3 patients with proven IC; (2) 6 patients with probable IC; (3) 34 patients colonized with Candida; (4) 19 patients without Candida colonization and without IC. In the patient categories (1) to (4), 3/3 (100%), 3/6 (50%), 20/34 (59%), and 7/19 (37%) patients were Candida EIA positive (>0.5 ng/ml) in at least one serum sample. The sensitivity of the assay for the detection of proven IC was 100%, for proven and probable IC 67%, the specificity was 49% for both groups. An increase of the cut-off value to 2.0 ng/ml raised the specificity to 61%, but lowered the sensitivity to 56%. In conclusion, the Platelia Candida EIA does not discriminate between Candida colonization and probable invasive infection in haematological patients.

[Antibody detection in patients with invasive aspergillosis]. / Antikorper-Nachweis bei invasiver Aspergillose.

The clinical significance of Aspergillus antibody assays for the diagnosis of invasive aspergillosis (IA) is unclear. In two studies, three different antibody assays were evaluated with patients suffering from proven IA: (i) a commercial haemagglutination test (HAT), (ii) a commercial enzyme immunoassay (EIA) for IgG, IgM, and IgA, and (iii) an experimental mitogillin enzyme immunoassay for IgG, IgM, and IgA. In the first study, 99 serum samples from 26 patients with IA and 22 serum samples from 22 control patients were tested with all the three tests. Ten of the 26 patients (38%) reacted positively in at least one antibody assay. The highest sensitivity was generated by the detection of IgG using the EIA formats (22 and 21%, respectively), the HAT had a sensitivity of 8%. IgM type antibodies were detected in only two patients; no IgA type antibodies were detected. The specificities of the IgG EIA and the HAT were 72 and 85%, respectively. Antibody detection was the single positive laboratory test in two patients with proven and probable IA. In the second study, antibody test results of 60 patients with proven IA were retrospectively evaluated. Fourteen patients (23%) tested positive in the EIA and/or in the HAT. Investigations of the antibody levels in individual immunocompromised patients over time revealed that IgG production started after a mean of 10.8 days after diagnosis of IA. To conclude, antibodies against Aspergillus were detected in 23% of patients with IA. The antibody production started in successfully treated immunosuppressed patients after a mean of 10.8 days after the onset of infection. In particular, the detection of IgG-antibodies with an EIA can be useful for the confirmation of the diagnosis of IA and for the monitoring of the treatment of IA.

[Fungemia after oral treatment with Saccharomyces boulardii in a patient with multiple comorbidities]. / Fungämie nach oraler Gabe von.

HISTORY AND CLINICAL FINDINGS: A 48-year-old diabetic with multiple co-morbidities presented with generalized micro- and macroangiopathy including peripheral artery disease stage IV with necroses in several digits of both feet. He was admitted to the department of surgery for the insertion of femoropopliteal bypasses. INVESTIGATIONS: Infectious parameters were elevated (CRP 66.1 mg/l, sedimentation rate 90/96), accompanied by anemia (Hb 7.1 mmol/l), leukocytosis (14.8 Gpt/l) and thrombocytosis (514 Gpt/l). Body temperature was normal (36.8 degrees C). With insulin treatment the patient became nearly normoglycemic (HbA1c 6.8 %). TREATMENT AND FOLLOW UP: After receiving different broad-spectrum antibiotics over seven weeks the patient developed Clostridium difficile toxin-positive diarrhea that resolved after administration of oral metronidazole and Saccharomyces boulardii (Perenterol ((R))). Three days after bypass insertion, both legs had to be amputated due to infection and beginning sepsis. The condition of the patient improved. However, eight days after bypass-insertion the patient developed a toxic megacolon and sepsis. Blood cultures yielded the growth of Saccharomyces cerevisae. Despite of intensive care treatment the patient died five days later from to multi-organ failure. CONCLUSION: S. boulardii (synonym: S. cerevisiae) is considered an non-pathogenic probiotic yeast, and live yeast cells are used for supportive therapy of diarrhea. The present case and a review of the literature demonstrate that fungemia and sepsis are rare complications of the administration of S. boulardii in immunocompromised patients. For this reason the therapeutic usage of probiotics should be carefully considered regarding its risk-benefit potential.

Local production of Aspergillus fumigatus specific immunoglobulin E in nasal polyps.

BACKGROUND: Aspergillus spp. play a significant role in the etiology of immunoglobulin (Ig)E mediated allergic fungal sinusitis (AFS). It is unclear whether Aspergillus spp. are also involved in nasal polyps without the characteristic clinical features of AFS. OBJECTIVES: The frequency of Aspergillus spp. and Aspergillus-specific IgE in nasal lavages and serum of patients with severe nasal polyps (n = 33) without clinical features of AFS should be investigated. STUDY DESIGN: Prospective study. METHODS: An aliquot of nasal lavage fluid was treated with dithiothreitol and examined for Aspergillus fumigatus by culture and an Aspergillus-specific polymerase chain reaction (PCR) assay. An additional aliquot of nasal fluid and serum of the same patient were tested for specific IgE (Unicap, Pharmacia, Freiburg, Germany) to recombinant Aspergillus fumigatus allergen (rAspf) 1 to 6. RESULTS: All patients had negative skin prick tests for Aspergillus fumigatus. Four of 33 (12%) lavage samples were positive for Aspergillus spp. by PCR. In one of these samples, rAspf-specific IgE was detected but none in the serum. Nasal lavage and serum samples of the remaining 29 patients were negative for rAspf-specific IgE. CONCLUSIONS: Aspergillus spp. detection is rare in patients with severe nasal polyps without characteristic clinical features of AFS. Specific IgE in nasal secretions may be elevated in patients with negative skin prick tests and serum IgE. In these cases, immunologic mechanisms similar to AFS may be involved. Fungal etiology has been proposed to underlie severe nasal polyps in general. However, Aspergillus spp. seem not to play a significant role.

Rate of transmission and endogenous origin of Candida albicans and Candida glabrata on adult intensive care units studied by pulsed field gel electrophoresis.

We determined the relative roles of endogenous origin and patient-to-patient transmission in Candida colonization of patients on adult intensive care units (ICU). A total of 48 Candida albicans and 18 Candida glabrata strains from various clinical samples of 28 long-term patients, hospitalized in two neurological ICUs between April and June 1999, were typed using pulsed field gel electrophoresis (PFGE). Three patients were co-colonized by both C. albicans and C. glabrata strains. Twenty-four C. albicans and 17 C. glabrata karyotypes were defined. The colonization was found to be polyclonal in six C. albicans and five C. glabrata patients. Twenty-six patients (93%) carried strains, which were not detected in other patients hospitalized at the same time, i.e. they were colonized by unique C. albicans and C. glabrata strains. Only two patients, who were hospitalized during the same period of time, although in different rooms of the same ICU, shared strains with an identical PFGE type, indicating possible patient-to-patient transmission. Patient-to-patient transmission of yeasts played a minor role on these ICUs.

Karyotyping of Candida albicans and Candida glabrata from patients with Candida sepsis.

The aim of this study was to determine the relatedness of Candida strains from patients suffering from Candida septicaemia by typing of Candida isolates from blood cultures and different body sites by pulsed field gel electrophoresis (PFGE using a contour-clamped homogenous electric field, CHEF). We studied 17 isolates of Candida albicans and 10 isolates of Candida glabrata from six patients. Four patients suffered from a C. albicans septicaemia, one patient from a C. glabrata septicaemia, and one patient had a mixed septicaemia with C. albicans and C. glabrata. Eight isolates from blood cultures were compared with 19 isolates of other sites (stool six, urine four, genital swab four, tip of central venous catheter three, tracheal secretion one, sputum one). PFGE typing resulted in 10 different patterns, four with C. albicans and six with C. glabrata. Five of the six patients had strains of identical PFGE patterns in the blood and at other sites. Seven isolates of a 58-year-old female with a C. glabrata septicaemia fell into five different PFGE patterns. However, they showed minor differences only, which may be due to chromosomal rearrangements within a single strain. Thus it appears, that the colonizing Candida strains were identical to the circulating strains in the bloodstream in at least five of six patients.

Broad spectrum herbal therapy against superficial fungal infections.

Skin disease associated with keratinized tissues in animal and human beings has been investigated. The essential oil of Eucalyptus pauciflora in vitro showed strong antifungal activity at 1.0 microl/ml against human pathogenic fungi, viz. Epidermophyton floccosum, Microsporum audouinii, M. canis, M. gypseum, M. nanum, Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum. The oil has heavy doses of inoculum potential at 1.0 microl/ml. Moreover, it did not exhibit any adverse effects on mammalian skin up to 5% concentrations. Further, we formulated the oil in the form of ointment 'BSHT' (broad spectrum herbal therapy) (1% v/v) and subjected it to topical testing on patients attending the outpatient department of M.L.N. Medical College, Allahabad. Fifty patients were selected on the basis of KOH-positive results and diagnosed as either tinea pedis, tinea corporis or tinea cruris. After the second week of treatment, all patients were KOH-negative. At the end of medication, 60% of patients recovered completely and 40% showed significant improvement from the disease. No KOH-negative cases of relapse were observed when patients were re-examined after 2 months following the end of treatment. Thus, the ointment can be exploited commercially after undergoing successful multicenter clinical trials, which are in progress.

[Evaluation of a panfungal pcr assay for the diagnosis of pneumocystis carinii pneumonia (pcp)]. / Evaluation eines universellen Pilz-PCR-Assays zur Diagnostik der Pneumocystis carinii-Pneumonie (PCP).

We compared a universal fungal PCR assay with fluorescence microscopy for the diagnosis of Pneumocystis carinii pneumonia. 82 bronchoalveolar lavages (BALs) of 64 immunocompromised patients with atypical pneumonia and 50 BALs of 50 immunocompetent adults without lung disease were examined. 10 immunocompromised patients were clinically and/or histologically proven to suffer from PCP. For fluorescence microscopy, sensitivity and specificity in detecting P. carinii were 80.0% and 98.1%, for the PCR assay 100.0% and 96.2%, respectively. The PCR assay is a useful method for the diagnosis of PCP and is recommended as an additional test to microscopical methods.

Identification of contaminating fungal DNA sequences in Zymolyase.

When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the Saccharomyces sensu stricto complex and Kluyveromyces lodderae. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.

Disseminated Penicillium marneffei infection in an HIV-positive female from Thailand in Germany.

We report the case of a 33 year old Thai female, who was married in Germany for eight years and used to travel to Thailand every year for several weeks. She presented with abdominal and back pain, prolonged fever, generalized lymphadenopathy, and a recent history of oral thrush. She was diagnosed HIV positive with initial CD4 counts of 18/microliter and an HI virus load of 59,000 copies/ml. Antiviral therapy was installed with zidovudin, lamivudin, and efavirenz. Abdominal CT scans revealed greatly enlarged abdominal lymph nodes. Fine needle aspirates of cervical and retroperitoneal lymph nodes, sputum samples, blood samples, and a bone marrow biopsy were microscopically positive for Penicillium marneffei and grew P. marneffei. The isolates were sensitive to amphotericin B, flucytosine, itraconazole, and fluconazole. Both universal and specific fungal polymerase chain reaction assays were positive in various samples. Serum Aspergillus galactomannan antigen, which is known to crossreact with P. marneffei, was elevated and subsequently used for monitoring of therapy. With antifungal treatment (intravenous amphotericin B 0.6 mg/kg/d for two weeks, oral itraconazole 400 mg/d for 10 weeks and 200 mg/d as maintenance therapy), the fever declined in 6 days, the size of the enlarged lymph nodes gradually decreased in the CT scans, and the initial abdominal and back pain vanished.

Preoperative diagnosis of Mycobacterium avium lymphadenitis in two immunocompetent children by polymerase chain reaction of gastric aspirates.

BACKGROUND: Analysis of gastric aspirates is a routine procedure for detection of Mycobacterium tuberculosis in pediatric pulmonary tuberculosis. However, identification of nontuberculous mycobacteria in gastric aspirates of immunocompetent children is not thought to be clinically significant. METHODS: A PCR method was devised for the detection of M. avium in clinical specimens. The method is based on the amplification of a M. avium-specific DNA fragment present in the 3'-end of the repetitive element IS1245. Surgically removed lymphatic tissue was analyzed prospectively by microscopy, culture and PCR in 13 children admitted to our hospital with suspected mycobacterial lymphadenitis. In 4 of these children 1 to 4 gastric aspirates were obtained before surgical treatment and submitted to the same analysis. RESULTS: We report the detection of M. avium in the gastric aspirates of two children with cervical lymphadenitis before surgical intervention by a novel PCR method. The subsequently surgically removed lymph nodes were also positive by PCR and culture. In one child cultures of both sources grew M. avium. The isolates could be identified as the same strain by DNA fingerprinting. The PCR assay was almost twice as sensitive as culture in detecting M. avium. CONCLUSIONS: Our findings suggest the possibility for noninvasive diagnosis of cervical lymphadenitis caused by nontuberculous mycobacteria before surgery. In addition detection of M. avium in gastric aspirates without evidence of fistula formation provides new insights into the pathogenesis of mycobacterial infection and disease in immunocompetent children.

[Fungal nucleic acid detection for invasive aspergillosis]. / Pilz-Nukleinsäure-Nachweis bei invasiven Aspergillosen.

A universal PCR-assay for the detection of fungal DNA was compared with microscopy and culture for the diagnosis of invasive aspergillosis using 78 samples from 42 patients. Eighteen patients were suffering from invasive aspergillosis, 5 patients were colonized with Aspergillus in the respiratory tract, 19 patients did not show any sign of aspergillosis. Samples from 6 of the 18 patients with invasive aspergillosis were microscopically positive with true mycelia, 15 of 18 grew Aspergillus in culture, 16 of 18 were PCR-positive. The combination of microscopy and culture led to the diagnosis in 17 of 18 patients, the combination of microscopy and PCR in 16 of 18 and the combination of culture and PCR in all the 18 patients. For 3 of 18 patients, PCR was the diagnostic key: in 2 biopsies the histologically detected fungal elements were identified as Aspergillus, in 3 bronchial lavages from 1 patients nothing but PCR was positive for Aspergillus. Four out of 5 culture positive patients with Aspergillus colonization were also PCR positive; one out of 19 patients without aspergillosis was culture positive, 3 out of 19 were falsely PCR positive. Candida colonization in the upper respiratory tract or Pneumocystis carinii pneumonia did not lead to false positive Aspergillus-PCR results. In conclusion, the evaluated fungal PCR-assay can supplement conventional methods for the diagnosis of invasive aspergillosis.

Hydroxylation of collagen type I: evidence that both lysyl and prolyl residues are overhydroxylated in osteogenesis imperfecta.

The composition of the collagens secreted into the media of fibroblast cultures of 39 patients with osteogenesis imperfecta (OI) was the same in controls and OI cultures. An abnormal migration pattern of collagens upon SDS-PAGE was evident in one third of the cultures investigated. Lysyl and prolyl hydroxylation of HPLC-purified alpha 1(I) chains was elevated in about 60% of cultures. The degree of hydroxylation was highest in the lethal forms. The extent of lysyl and prolyl hydroxylation showed a strong correlation (r = 0.74, P < 0.001). While high levels of hydroxylation are frequently observed in OI patients, a direct correlation between lysyl or prolyl hydroxylation and fracture rate or growth retardation could not be established.

Lysyl hydroxylation in collagens from hyperplastic callus and embryonic bones.

Tissue from two patients with osteogenesis imperfecta suffering from a hyperplastic callus was studied. Although collagen type I from the compact bone and the skin and fibroblast cultures of these patients showed normal lysyl hydroxylation, collagen types I, II, III and V from the callus tissue were markedly overhydroxylated. Furthermore, the overhydroxylation of lysine residues covered almost equally the entire alpha 1 (I) collagen chain, as demonstrated by the analysis of individual CNBr-derived peptides. In addition, collagen type I was isolated from femoral compact bone of 33 individuals who died between the 16th week of gestational age and 22 years. Lysyl hydroxylation rapidly decreased in both collagen alpha 1 (I) and alpha 2 (I) chains during fetal development, and only little in the postnatal period. The transient increase in lysyl hydroxylation and the involvement of various collagen types in callus tissue argue for a regulatory mechanism that may operate in bone repair and during fetal development.