Hintergrund: Infektionen mit dem Respiratory Syncytial Virus (RSV) sind die häufigste virale Ursache für respiratorische Erkrankungen bei Säuglingen und Kleinkindern. Reinfektionen treten lebenslang auf. Es wurden zwei Typen (A und B) und mehrere Genotypen beschrieben. Die vorliegenden Daten über die molekulare Epidemiologie von RSV in Deutschland sind nur begrenzt. Material und Methoden: Zwischen Januar 2002 und Juli 2006 wurden 221 Nasenrachensekrete (NRS) von Kindern, welche in der Universitätskinderklinik Würzburg behandelt wurden, durch Routine-Untersuchung mit einem Immunfluoreszenztest auf RSV-Antigen positiv befunden. Die phylogenetische Analyse wurde aus Restmaterial von 211 NRS durchgeführt, indem die zweite variable Region des G-Gens amplifiziert und sequenziert wurde. Ergebnisse: Insgesamt war die Prävalenz von Typ A-Viren mit 69,5 % größer als die der Typ B-Viren mit 30,5 %. RSV Typ A war das dominierende Virus in allen Saisons außer in der Saison 2002-2003. Über den gesamten Beobachtungszeitraum traten drei A-Genotypen (GA2, GA5 und GA7) und vier B-Genotypen (GB3, SAB3, BA und ein neuer Genotyp) auf. Die Genotypen GA2, GA5, SAB3 und BA waren am häufigsten im Umlauf und in beinahe allen Saisons prävalent. Unter den B-Genotypen nahm der Anteil des Genotyps BA von 25 % (2002) auf 92 % (2005-2006) zu. Drei Typ B-Sequenzen wurden einem neuen Genotyp zugeordnet, welcher BWUE benannt wurde. Es wurde eine Reinfektion mit demselben Genotyp (GA5) bei einem Kind beobachtet, welches im Alter von 12 und 28 Monaten mit einer RSV-Infektion hospitalisiert war. Schlußfolgerung: Die Ergebnisse unserer Studie stehen in Einklang mit der molekularen Epidemiologie von RSV in anderen geographischen Regionen. Wir beobachteten sowohl Genotypen, welche über mehrere Saisons prävalent waren, als auch Genotypen, welche über den beobachteten Zeitraum zunehmend dominanter werdend andere Genotypen verdrängten. Zudem wurde ein neuer B-Genotyp entdeckt. / Title: Molecular epidemiology of respiratory syncytial virus infections in Underfranconia between 2002 and 2006 Background: The respiratory syncytial virus (RSV) es the most common viral cause of respiratory infections in infants and children. Reinfections occur lifelong. Two RSV subtypes (A and B) and several genotypes have been described. The available data on the molecular epidemiology of RSV in Germany are only limited. Material and methods: Between January 2002 and July 2006, 211 respiratory samples of infants and children treated in the Children’s Hospital of the University Würzburg were found to be positive for RSV antigen by routine testing with immunofluorescensce assays. Phylogenetic analysis was performed on 211 of these samples by amplification and sequencing of the second variable region of the RSV G gene. Results: The type distribution of the 211 RSV positive samples was 69,5 % type A and 30,5 % type B. RSV type A was the predominating virus in all seasons except for the winter season 2002/03. Over the whole observation period, three different A-genotypes (GA2, GA5, GA7) and four different B-genotypes (GB3, SAB3, BA and novel genotype) were detected. The RSV genotypes GA2, GA5, SAB3 and BA were most frequently found and were prevalent in almost all seasons. Among the B-genotypes, the proportion of the genotype BA increased from 25 % in 2002 to 91 % in 2005/06. Three type B sequences were assigned to a novel genotype, which was tentatively named BWUE. One reinfection with the same genotype (GA5) was observed in a child who was hospitalised with RSV infection at the age of 12 and 28 months. Conclusion: The results of our study are in agreement with the molecular epidemiology of RSV in other geographical regions. We observed both genotype persistence and genotype shifting during the observation period. In addition, we detected a novel B genotype.

Tuberculosis is a major public health problem in Ethiopia. The aims of this study were (i) to investigate the recovery rate of M. tuberculosis from smear positive single morning sputum specimens subjected to long-term storage at -20°C, (ii) to assess the level and risk factors for first- and second-line anti-TB drug resistance, (iii) to evaluate the performance of the GenoType®MTBDRplus and GenoType®MTBDRsl assays for drug susceptibility testing compared to the BacT/ALERT 3D system as reference method, (iv) to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among M. tuberculosis isolates, and (v) to study the population structure and transmission dynamics of M. tuberculosis isolates from patients in Amhara region, Northwest Ethiopia. The median specimen storage time was 132 days. Of 319 specimens, 90.0% were culture positive. The length of time of sputum storage had no significant effect on the recovery rate of M. tuberculosis. Of 260 M. tuberculosis isolates, 15.8% were resistant to at least one first-line drug, 5.0% were multidrug resistant (MDR) and 3.5% were resistant to all first-line drugs. Any resistance to INH, RMP, streptomycin (STM), EMB and pyrazinamide (PZA) was 13.8%, 5.8%, 10.0%, 7.3% and 4.6%, respectively. All isolates were susceptible to second-line drugs. The GenoType®MTBDRplus assay had a sensitivity of 92% and specificity of 99% to detect INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType®MTBDRsl assay had a sensitivity of 42% and specificity of 100% to detect EMB resistance. According to the molecular methods, mutations conferring resistance to INH, RMP, or EMB were detected in 13.5%, 5.8%, and 3.1% of the isolates, respectively, while mutation conferring MDR was present in 5.0% of the isolates. Of 244 M. tuberculosis isolates, 59.0% were classified as known lineages; Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%) lineage. Interestingly, 31.6% of the isolates were grouped in to four previously undefined phylogenetic lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating high rate of recent transmission. Similarly, 66.7% of MDR strains were grouped in clusters.

Tuberculosis is a major public health problem in Ethiopia. The aims of this study were (i) to investigate the recovery rate of M. tuberculosis from smear positive single morning sputum specimens subjected to long-term storage at -20°C, (ii) to assess the level and risk factors for first- and second-line anti-TB drug resistance, (iii) to evaluate the performance of the GenoType®MTBDRplus and GenoType®MTBDRsl assays for drug susceptibility testing compared to the BacT/ALERT 3D system as reference method, (iv) to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among M. tuberculosis isolates, and (v) to study the population structure and transmission dynamics of M. tuberculosis isolates from patients in Amhara region, Northwest Ethiopia. The median specimen storage time was 132 days. Of 319 specimens, 90.0% were culture positive. The length of time of sputum storage had no significant effect on the recovery rate of M. tuberculosis. Of 260 M. tuberculosis isolates, 15.8% were resistant to at least one first-line drug, 5.0% were multidrug resistant (MDR) and 3.5% were resistant to all first-line drugs. Any resistance to INH, RMP, streptomycin (STM), EMB and pyrazinamide (PZA) was 13.8%, 5.8%, 10.0%, 7.3% and 4.6%, respectively. All isolates were susceptible to second-line drugs. The GenoType®MTBDRplus assay had a sensitivity of 92% and specificity of 99% to detect INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType®MTBDRsl assay had a sensitivity of 42% and specificity of 100% to detect EMB resistance. According to the molecular methods, mutations conferring resistance to INH, RMP, or EMB were detected in 13.5%, 5.8%, and 3.1% of the isolates, respectively, while mutation conferring MDR was present in 5.0% of the isolates. Of 244 M. tuberculosis isolates, 59.0% were classified as known lineages; Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%) lineage. Interestingly, 31.6% of the isolates were grouped in to four previously undefined phylogenetic lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating high rate of recent transmission. Similarly, 66.7% of MDR strains were grouped in clusters.

Humane Astroviren (HAstV) stellen wichtige Erreger kindlicher Gastroenteritiden dar, die in ihrer Bedeutung lange Zeit unterschätzt wurden. Sie werden in acht Serotypen klassifiziert, die nach dem bisherigen Kenntnisstand mit den Genotypen korrespondieren. Ziel dieser Arbeit war es, Einsicht in die molekulare Epidemiologie der in Deutschland zirkulierenden Astroviren zu vermitteln. Eine HAstV-spezifische RT-PCR bildete die Grundlage für die phylogenetische Analyse eines Genomabschnitts, der für die Kapsidproteine des Virus kodiert. Dazu wurden 16 deutsche Astrovirusisolate aus den Jahren 1997-1999 unter Einbeziehung bereits publizierter Sequenzdaten der Serotypen 1-8 untersucht. In Anlehnung an ein in der vorliegenden Literatur verwendetes Klassifizierungsschema erfolgte die Einteilung der deutschen Isolate in unterschiedliche Genotypen. Hierbei bildete eine Konvergenz von mindestens 85% das Kriterium für die Zuordnung differenter Isolate zum gleichen Genotyp. Es konnte gezeigt werden, dass in Deutschland wenigstens vier HAstV-Genotypen (1-4) kozirkulieren. Über dem betrachteten Genomabschnitt stimmten einige Isolate aus unterschiedlichen geografischen Regionen in ihrer Sequenz überein. Im Rahmen dieser Arbeit wurde erstmals die Totalsequenz eines humanen Astrovirus vom Serotyp 3 ermittelt. Während in der phylogenetischen Analyse des betreffenden Isolats nur ein Genomabschnitt betrachtet wurde, ließ sich an seiner Totalsequenz demonstrieren, dass die Einordnung in den Geno- bzw. Serotyp 3 auch in anderen Genomregionen Gültigkeit besitzt. Analog zu den bisher bekannten Gesamtgenomsequenzen der Serotypen 1, 2 und 8 lassen sich drei überlappende offene Leseraster (ORFs) identifizieren. In den beiden am 5'-Ende gelegenen ORFs 1a und 1b erweisen sich die putativen Motive der Protease und der RNA-Polymerase zwischen den vier Serotypen als hochkonserviert, ebenso wie vier potentielle Transmembrandomänen, ein ribosomales frameshift-Signal und ein nukleäres Lokalisationssignal. In dem am 3'-Ende gelegenen ORF 2 befinden sich drei hochkonservierte potentielle N-Glykosylierungs-Sites sowie ein hochkonserviertes Glykosaminoglykan-Attachment-Site. Ein wesentlicher Befund im Zusammenhang mit der Totalsequenz ist der Nachweis einer 45 Nukleotide umfassenden Deletion im ORF1a im Originalmaterial (Stuhl). Diese wurde bisher nur bei Astroviren gefunden, die in Zellen kultiviert wurden. Von Interesse und weiteren Untersuchungen vorbehalten ist ihre Nähe zur nukleären Lokalisationssequenz, die für die Beeinflussung des Zielzell-Tropismus von Astroviren verantwortlich sein könnte. / Human astroviruses (HastV) are an important cause of infantile gastroenteritis. To date, there are eight recognized serotypes which correlate with genotypes. The aim of this study was to investigate the molecular epidemiology of astroviruses circulating in Germany. Based on a HAstV-specific RT-PCR, phylogenetic analysis of a segment of the capsid protein gene was performed. The examination included sequence data of 16 German astrovirus isolates from the years 1997-1999 as well as published sequence information of the serotypes 1-8. Molecular typing was carried out following published classification strategies. The criterion for classification of isolates into one genotype was sequence identity of at least 85%. Astroviruses of at least four different genotypes (1-4) were found to cocirculate in Germany. The nucleotide sequences of several isolates from different geographical regions were identical. As part of this study, the complete genomic sequence of a type 3 human astrovirus was determined. The classification of the virus as a genotype 3 astrovirus as suggested by phylogenetic analysis over a limited genome section was supported by sequence comparison over two different genomic regions. Similar to the known total sequences of serotypes 1,2 and 8, three overlapping open reading frames (ORFs) were identified. The 5' end ORFs 1a and 1b contain the putative protease and polymerase motifs, which are highly conserved between the four serotypes. A high degree of sequence identity was also found for four potential transmembrane domains, a ribosomal frameshift signal and a nuclear localisation signal. The 3' end ORF 2 encodes three almost totally conserved potential N-glycosylation sites and one highly conserved putative glycosaminoglycan attachment site. As an outstanding feature, the virus, which was isolated and sequenced directly from diarrheal feces, presents a 45-nucleotide deletion in ORF 1 a. This deletion has previously only been found in cell cultured astroviruses. Further studies are needed to determine whether all viral genomes within the quasispecies carry the deletion.

In der vorliegenden Arbeit wurde die Sensitivität des Nachweises von Leishmanien in Giemsa-gefärbten Bioptaten aus Hautulzerationen mittels direkter Mikroskopie mit der Sensitivität der ITS1-PCR verglichen. Bei der ITS1-PCR wurde eine Sensitivität von 87 % mit einem positiven predictive value von 100 %, sowie eine Spezifität von 100 % mit einem negativen predictive value von 85 % nachgewiesen. Weiterhin wurden vier verschiedene Nachweismethoden miteinander verglichen: die in vitro Kultivierung in NNN Medium, die direkte Mikroskopie von Giemsa gefärbten Hautbioptaten, die PCR Amplifizierung der ITS1 Region aus auf Filterpapier aufgetragenen Hautbioptaten (FP) sowie die ITS1-PCR von ungefärbten Hautbioptaten (US). Die PCR der US erwies sich als die sensitivste Methode. Die Verbreitung von Leishmanien Arten in Jericho wurde mittels molekularer Epidemiologie untersucht. Die räumliche (Spatial) Analyse zeigte drei statistisch relevante Cluster innerhalb der kutanen Leishmaniose (CL): ein Cluster mit L. major und zwei L. tropica Cluster. Bei der Raum-Zeit–Analyse wurden vier Cluster von Kutanen Leishmaniose, zwei L. major und drei L. tropica Cluster nachgewiesen. Insgesamt 106 Stämme, die aus verschiedenen endemischen Regionen in Zentralasien, im Nahen Osten und Afrika stammen, wurden mit 10 Mikrosatellitenmarkern untersucht. Die Auswertung erfolgte über zwei Analysemethoden: die Distanz-basierte und die Modell-basierte Methode. Anhand der L. major Genomsequenz wurden PCR-Primer zur Amplifizierung von Mikrosatellitenloci von L. major entwickelt, die auf den Chromosomen 1, 3, 5, 21 und 35 liegen. Sieben unterschiedliche L. major Populationen einschließlich zweier genetisch isolierter Populationen im Nahen Osten wurden mit diesen Markern nachgewiesen. / In this study we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR. ITS1-PCR showed a sensitivity of 87% with positive predictive value of 100% and a specificity of 100% with negative predictive value of 85%. In-vitro cultivation using NNN medium and direct smear microscopy of Giemsa-stained slides, PCR amplifying region 1 of internal transcribed spacer (ITS1) using skin scrapings spotted on filter papers (FP) and unstained tissue smears (US) were compared. PCR using US was more sensitive than all other methods Molecular epidemiology was used to study the distribution of Leishmania species in Jericho. Spatial analysis showed three statistically significant clusters of CL, one cluster for L. major and two clusters for L. tropica. In the case of space-time, four clusters for CL, two for L. major and three for L. tropica were detected. A total of 106 strains isolated in different endemic regions of Central Asia, Middle East and Africa were analysed using 10 pairs of microsatellite markers under two cluster methods: distance and model-based. Markers were designed to amplify microsatellite loci identified in the genome sequence of L. major on chromosomes 1, 3, 5, 21 and 35. Seven discrete populations of L. major including two genetically isolated populations in the Middle East were revealed.