Erythrocyte morphology is one of the most sensitive indicators of toxic impact of various environmental factors on fish. Present investigation deals with the assessment of deleterious effects of heavy metals, cadmium and copper on the structural alterations of RBCs of a freshwater air-breathing catfish, Heteropneustes fossilis (Bloch). On exposure of the fish to the toxicant at concentrations of cadmium (2.4 ppm) and copper (0.3 ppm) for a period of 30 days, the erythrocytes exhibited marked alterations in structure such as lobopodial projections, shrinkage of cell membrane and clumping of cells. Different types of alterations observed were echinocytes, spherocytes, acanthocytes, crenated, rhomboidal and racket shaped cells. The cellular damage of fish erythrocytes on treatment with cadmium and copper lead to physiological malfunctioning and hence RBCs SEM study might be utilized as a sensitive indicator of metal pollution.

Rarely, zoonotic Taenia species other than Taenia solium cause human cysticercosis. The larval stages are morphologically often indistinguishable. We therefore investigated 12 samples of suspected human cysticercosis cases at the molecular level and surprisingly identified one Taenia crassiceps and one Taenia serialis (coenurosis) infection, which were caused by tapeworm larvae normally infecting rodents and sheep via eggs released from foxes and dogs.

Introduction. The aim of the study was the evaluation of panfungal PCR protocols with subsequent sequence analysis for the diagnostic identification of invasive mycoses in formalin-fixed, paraffin-embedded tissue samples with rare tropical mycoses. Materials and Methods. Five different previously described panfungal PCR/sequencing protocols targeting 18S and 28S ribosomal RNA gene fragments as well as internal transcribed spacer 1 and 2 fragments were evaluated with a collection of 17 formalin-fixed, paraffin-embedded tissue samples of patients with rare and/or tropical invasive mycoses, comprising chromoblastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, mycetoma/maduromycosis, and rhinosporidiosis, in a proof-of-principle analysis. Results. The primers of the panfungal PCRs readily and predominantly reacted with contaminating environmental fungi that had deposited on the paraffin blocks. Altogether three sequence results of histoplasmosis and mycetoma samples that matched the histological assessment were associated with sample age <10 years and virtually without PCR inhibition. Conclusions. The high risk of amplifying environmental contaminants severely reduces the usefulness of the assessed panfungal PCR/sequencing protocols for the identification of rare and/or tropical mycoses in stored formalin-fixed, paraffin-embedded tissues. Histological assessment remains valuable for such indications if cultural differentiation is impossible from inactivated sample material.

Background Puumala virus (PUUV) is the most important hantavirus species in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute renal injury (AKI) with thrombocytopenia and frequently gastrointestinal symptoms. Methods 456 patients with serologically and clinically confirmed NE were investigated at time of follow-up in a single clinic. The course of the NE was investigated using medical reports. We identified patients who had endoscopy with intestinal biopsy during acute phase of NE. Histopathological, immunohistochemical and molecular analyses of the biopsies were performed. Results Thirteen patients underwent colonoscopy or gastroscopy for abdominal pain, diarrhea, nausea and vomiting during acute phase of NE. Immunohistochemistry (IHC) revealed PUUV nucleocapsid antigen in 11 biopsies from 8 patients; 14 biopsies from 5 patients were negative for PUUV nucleocapsid antigen. IHC localized PUUV nucleocapsid antigen in endothelial cells of capillaries or larger vessels in the lamina propria. Rate of AKI was not higher and severity of AKI was not different in the PUUV-positive compared to the PUUV-negative group. All IHC positive biopsies were positive for PUUV RNA using RT-PCR. Phylogenetic reconstruction revealed clustering of all PUUV strains from this study with viruses previously detected from the South-West of Germany. Long-term outcome was favorable in both groups. Conclusions In patients with NE, PUUV nucleocapsid antigen and PUUV RNA was detected frequently in the intestine. This finding could explain frequent GI-symptoms in NE patients, thus demonstration of a more generalized PUUV infection. The RT-PCR was an effective and sensitive method to detect PUUV RNA in FFPE tissues. Therefore, it can be used as a diagnostic and phylogenetic approach also for archival materials. AKI was not more often present in patients with PUUV-positive IHC. This last finding should be investigated in larger numbers of patients with PUUV infection.

Background: Two months after a vacation in Thailand, a 54-year-old alcohol-addicted diabetes patient was admitted to hospital presenting with a febrile, therapy-refractory urinary tract infection. Burkholderia pseudomallei was cultured from urine but misidentified as Burkholderia cepacia. Application of inappropriate chemotherapy provoked systemic spread of bacteria from urinary tract abscesses. Septic shock and respiratory failure resulted in a fatal outcome.Results: Correct identification at the species level was achieved by 16S rRNA gene sequencing. Postmortem, B. pseudomallei was additionally detected in prostatic tissue by PCR and fluorescence in situ hybridization.Conclusions: Biochemical differentiation of bacteria of the genus Burkholderia is not reliable at the species level. Molecular approaches should be added if an infection with B. pseudomallei is probable according to clinical anamnesis. The recommended dosage of antibiotic drugs should be at the upper limit if abscess formation is likely.

Zusammenfassung Kasuistik: Ein 54-jähriger Diabetespatient mit bekanntem Alkoholabusus wurde mit therapierefraktärem, fieberhaftem Harnwegsinfekt nach Thailandaufenthalt stationär aufgenommen. Nach Fehldiagnose von Burkholderia pseudomallei als Burkholderia cepacia im Urin kam es unter insuffizienter antibiotischer Therapie zur systemischen Ausbreitung der abszedierenden Harnwegsinfektion, woran der Patient im septischen Schock mit respiratorischem Versagen verstarb. Ergebnisse: Die korrekte Spezies-Identifizierung erfolgte durch 16S rRNA-Gen-Sequenzierung. Zudem gelang post mortem der Nachweis von Burkholderia pseudomallei aus Prostatagewebe mittels PCR und Fluoreszenz-in-situ-Hybridisierung. Schlussfolgerung: Die biochemische Differenzierung von Bakterien des Genus Burkholderia ist auf Speziesebene unzuverlässig. Wenn die klinische Anamnese eine B. pseudomallei Infektion wahrscheinlich erscheinen lässt, sollten molekulare Identifikationsverfahren ergänzt werden. Bei Verdacht auf abszedierende Prozesse sollte die Antibiotika-Dosierempfehlung am oberen Rand des Dosisspektrums gewählt werden.

A 40-year old patient developed a painless swelling in the left zygomatico-temporal region. Magnetic resonance images and computed tomograms showed a non-specific soft tissue mass in the tumour region, but no invasion of bone. After application of antibiotics, the tumour reduced in size but a firm mass of about 3 cm in maximum diameter persisted under therapy. Surgical exploration revealed a distinct firm mass adhering to the superficial layer of the temporal muscle. Histological and molecular biological investigations demonstrated fragments of Dirofilaria repens in the centre of the lesion. Clinical follow-up was uneventful and additional investigations excluded further manifestations of the parasite. D. repens infections are extremely rare in northern Europe, but recent reports about the increase of human dirofilariasis in northern parts of Europe should alert the clinician to include helminthoses in the differential diagnosis of atypical space-occupying lesions of the maxillofacial regions.

To the Editor: Pentastomiasis is a parasitic zoonotic disease with an incremental number of reported human infections caused by larval stages (nymphs) of pentastomes (1–3). The vermiform parasites are in their own phylum and are related to branchiuran crustaceans (2). Most human infections with these parasites are caused by Armillifer armillatus (2), a parasite endemic to western and central Africa. Most cases are reported from the Congo region and Nigeria, and occasionally infections in African immigrants to Europe and North America have been reported (4,5). Imported cases to Germany have not been reported. A. grandis, a related parasite from central Africa, has been rarely found (6), but A. moniliformis, a pentastome species from Asia, has recently reemerged and caused a human infection after ≈40 years in Malaysia (1). Adult Armillifer spp. inhabit the respiratory tract of large snakes (Python spp.). These dioecious parasites produce large amounts of ova that are shed into the environment by snake feces and secretions. When intermediate hosts, such as rodents or other small mammals, ingest ova, larvae hatch, migrate to the viscera, encyst, and molt several times (3). Humans become accidental intermediate hosts after uptake of environmental parasite ova or by consumption of contaminated snake meat. We report an infection with A. armillatus in an African immigrant to Germany that was diagnosed by histopathologic analysis and confirmed by PCR. In 2005, a 23-year-old man from Togo who had immigrated to Germany 3 years earlier showed development of acute myeloid leukemia. He subsequently underwent stem cell transplantation, which was followed by graft versus host disease. The patient died of sudden intracerebral hemorrhage and leukencephalopathy. His medical history also included α-thalassemia and a heterocygotous sickle cell trait, chronic hemolytic anemia, splenomegaly, and cardiomyopathy. He had been treated for schistosomiasis and filariasis. An autopsy specimen showed several living pentastome nymphs of ≈2 cm in size, which were found in the subscapular region of liver parenchyma. A presumptive diagnosis of visceral pentastomiasis caused by A. armillatus nymphs was made in accordance with the origin of the patient and the geographic distribution of the parasite. Microscopic slides from patient specimens were retrieved from an archive and reanalyzed (Figure, Technical Appendix Figure). A pentastome-specific PCR targeting the 18S rRNA gene (2,7) was conducted after DNA extraction from formalin-fixed tissue on a remaining unstained microscope slide. The resulting 383-bp amplicon was sequenced, and BLAST analysis (www.ncbi.nlm.hih.gov/blast) confirmed 100% identity with A. armillatus (GenBank accession no. {type:entrez-nucleotide,attrs:{text:HM756289.1,term_id:306569736}}HM756289.1) and 99% homology with A. agkistrodontis ({type:entrez-nucleotide,attrs:{text:FJ607339.1,term_id:223049411}}FJ607339.1) and A. moniliformis ({type:entrez-nucleotide,attrs:{text:HM048870.1,term_id:297242398}}HM048870.1). Figure Oblique cross-section of liver of a patient (immigrant) from Togo, showing a well-preserved Armillifer armillatus nymph in a subcapsular location. The annulated parasite is encapsulated by its shed cuticle (exuvia) and dense fibrosis. Consistent with ... Visceral pentastomiasis in humans is often asymptomatic and an incidental finding during surgery (1,3) or autopsy (8,9). In a large autopsy series from Malaysia, a pentastomiasis prevalence of 45.5% was found in adult Aborigines (8). In Nigeria, a rate of 33% was seen during autopsies of patients who had died of malignancies (9). However, a few severe and even lethal cases have been described for heavy A. armillatus and A. grandis infections in persons from Africa (4,6). Diagnosis is achieved by gross pathologic and histopathologic analyses. Nymphs are found in the serosa around the liver and spleen, in liver parenchyma, mesenterium, intestine wall, and abdominal lymph nodes. The lungs or pleura are occasionally infected (3). Radiographic analysis may show typical C-shaped chest or abdominal calcifications (10). Species identification is performed by counting annulations (A. armillatus 18–22, A. grandis >25) and measuring the size of larval parasites (3). Recently, PCR has been used for diagnosis in veterinary infections (2,7). For the patient in our study, molecular analysis identified human pentastomiasis by using a formalin-fixed microscope slide that had been stored for 7 years. A difference of 2 nt each was seen when the amplified nucleotide sequence was compared with database sequences of A. agkistrodontis and A. moniliformis. However, there is no database entry in GenBank for A. grandis, the geographically closest Armillifer species. Serologic assays have been developed for identification of A. armillatus (2), but no serum was available for retrospective analysis. In special settings, such as tropical snake farming and pet keeping, pentastomiasis may be a public health concern (2). However, most infections have been linked to consumption of undercooked snake meat or other snake products (1). Most immigrants who were given a diagnosis of visceral pentastomiasis were from Nigeria or the Congo region, and diagnoses were made after death. Molecular analysis is particularly valuable when only autoptic paraffin-embedded patient material is available. For industrialized countries, where experience in morphologic identification of unusual parasite species is limited, molecular analysis is a valuable diagnostic tool. Our case-patient constitutes a record of imported Armillifer species pentastomiasis to Germany. Because of increasing international migration, more cases of pentastomiasis are likely to be seen. Technical Appendix Figure: Transverse section of an Armillifer armillatus larva from the liver of a patient (immigrant) from Togo. Typical for pentastomid lesions, the parenchyma shows focal hemorrhage around the parasite and no inflammatory cellular reaction (1). There is also focal destruction of the trabecular liver parenchyma. Subcuticular gland cells of the parasite are visible, and the intestine is clearly discernable in the center (hematoxylin and eosin stain, original magnification ×20). Click here to view.(132K, pdf)

Background: Two months after a vacation in Thailand, a 54-year-old alcohol-addicted diabetes patient was admitted to hospital presenting with a febrile, therapyrefractory urinary tract infection. Burkholderia pseudomallei was cultured from urine but misidentified as Burkholderia cepacia . Application of inappropriate chemotherapy provoked systemic spread of bacteria from urinary tract abscesses. Septic shock and respiratory failure resulted in a fatal outcome. Results: Correct identification at the species level was achieved by 16S rRNA gene sequencing. Postmortem, B. pseudomallei was additionally detected in prostatic tissue by PCR and fluorescence in situ hybridization. Conclusions: Biochemical differentiation of bacteria of the genus Burkholderia is not reliable at the species level. Molecular approaches should be added if an infection with B. pseudomallei is probable according to clinical anamnesis. The recommended dosage of antibiotic drugs should be at the upper limit if abscess formation is likely.

countries where incidence of MDR TB is low.

Mucosal mononuclear (MMC) CCR5+CD4+ T cells of the gastrointestinal (GI) tract are selectively infected and depleted during acute HIV-1 infection. Despite early initiation of combination antiretroviral therapy (cART), gut-associated lymphoid tissue (GALT) CD4+ T cell depletion and activation persist in the majority of HIV-1 positive individuals studied. This may result from ongoing HIV-1 replication and T-cell activation despite effective cART. We hypothesized that ongoing viral replication in the GI tract during cART would result in measurable viral evolution, with divergent populations emerging over time. Subjects treated during early HIV-1 infection underwent phlebotomy and flexible sigmoidoscopy with biopsies prior to and 15-24 months post initiation of cART. At the 2(nd) biopsy, three GALT phenotypes were noted, characterized by high, intermediate and low levels of immune activation. A representative case from each phenotype was analyzed. Each subject had plasma HIV-1 RNA levels <50 copies/ml at 2(nd) GI biopsy and CD4+ T cell reconstitution in the peripheral blood. Single genome amplification of full-length HIV-1 envelope was performed for each subject pre- and post-initiation of cART in GALT and PBMC. A total of 280 confirmed single genome sequences (SGS) were analyzed for experimental cases. For each subject, maximum likelihood phylogenetic trees derived from molecular sequence data showed no evidence of evolved forms in the GALT over the study period. During treatment, HIV-1 envelope diversity in GALT-derived SGS did not increase and post-treatment GALT-derived SGS showed no substantial genetic divergence from pre-treatment sequences within transmitted groups. Similar results were obtained from PBMC-derived SGS. Our results reveal that initiation of cART during acute/early HIV-1 infection can result in the interruption of measurable viral evolution in the GALT, suggesting the absence of de-novo rounds of HIV-1 replication in this compartment during suppressive cART.

Objective To describe and validate fluorescence in situ hybridization (FISH), a new method of Leishmania spp. identification. FISH allows for a rapid detection of target organisms by specific binding of fluorescently labelled oligonucleotide probes to ribosomal RNA. Methods Two genus-specific, fluorescently labelled Leishmania spp. FISH probes were designed and evaluated with a panel of 18 Leishmania spp. and six Trypanosoma spp. including well-defined strains and clinical isolates. In addition, the FISH probes were tested in comparison with Giemsa staining in formalin-fixed, paraffin-embedded tissues of five mice that had been artificially infected with Leishmania major strains, leading to concordant results. Finally, 11 tissue samples of patients with cutaneous leishmaniasis, four tissue samples of patients with visceral leishmaniasis, and one native bone marrow sample of a patient with visceral leishmaniasis were analysed with FISH and Giemsa staining. Results Concordant results were achieved by FISH and Giemsa staining in 15/16 specimens. Conclusion This analysis provides proof of principle that FISH is a suitable method for the rapid and easy detection of Leishmania spp. in formalin-fixed, paraffin-embedded tissue samples. Because of the good contrast of Leishmania spp. in tissue, FISH facilitates the identification of these organisms in tissue samples even by less experienced investigators.

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.

Human dirofilariasis is a zoonotic infectious disease caused by the filarial nematodes of dogsDirofilaria repensandDirofilaria immitis. Depending on the species involved, human infections usually manifest as one cutaneous or visceral larva migrans that forms a painless nodule in the later course of disease. Dirofilariae are endemic in the Mediterranean, particularly in Italy. They are considered as emerging pathogens currently increasing their geographical range. We present one of the few known cases of human dirofilariasis caused byD. repensin Romania. The patient developed unusual and severe clinical manifestations that mimicked pathological conditions like cellulitis or deep venous thrombosis.

BACKGROUND:Targeting antigens encoded by DNA vaccines to dendritic cells (DCs) in the presence of adjuvants enhances their immunogenicity and efficacy in mice.METHODOLOGY/PRINCIPAL FINDINGS:To explore the immunogenicity of this approach in non-human primates, we generated a single chain antibody to the antigen uptake receptor DEC-205 expressed on rhesus macaque DCs. DNA vaccines encoding this single chain antibody fused to the SIV capsid protein were delivered to six monkeys each by either intramuscular electroporation or conventional intramuscular injection co-injected or not with poly ICLC, a stabilized poly I: C analogue, as adjuvant. Antibodies to capsid were induced by the DC-targeting and non-targeting control DNA delivered by electroporation while conventional DNA immunization at a 10-fold higher dose of DNA failed to induce detectable humoral immune responses. Substantial cellular immune responses were also observed after DNA electroporation of both DNAs, but stronger responses were induced by the non-targeting vaccine. Conventional immunization with the DC-targeting DNA at a 10-fold higher dose did not give rise to substantial cellular immune responses, neither when co-injected with poly ICLC.CONCLUSIONS/SIGNIFICANCE:The study confirms the potent immunogenicity of DNA vaccines delivered by electroporation. Targeting the DNA via a single chain antibody to DEC-205 expressed by DCs, however, does not improve the immunogenicity of the antigens in non-human primates.

We report a case of subcutaneous sparganosis in a 68-year-old female Japanese immigrant in Germany. The patient complained of a painless erythema caudal of the umbilicus with a palpable subcutaneous cherry-sized lump. Polymerase chain reaction on formalin-fixed parasite tissue identified Spirometra erinaceieuropaei as the causative agent; the proliferative form of sparganosis, which is caused by the branching and disseminating Sparganum proliferum, could, thus, be excluded. From the excised sparganum, an immunofluorescence test was established and revealed an antibody response directed against the parasite's tegument. Histological key features of the plerocercoid that facilitate diagnosis with different stains are presented.