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In der Differenzialdiagnostik müssen Rheumatologen häufig neben autoimmunbedingten Erkrankungen auch Infektionen (z. B. eine Lyme-Arthritis) oder reaktive Krankheitsbilder (z. B. eine reaktive Arthritis nach einem urogenitalen bakteriellen Infekt) in Erwägung ziehen. Des Weiteren können Infektionen mit atypischer Präsentation oder mit atypischen Erregern (opportunistische Infektionen) unter der immunsuppressiven Therapie von rheumatischen Erkrankungen auftreten. In der mikrobiologischen Diagnostik kommen hierfür nicht nur konventionelle Kulturverfahren, sondern zunehmend auch PCR (Polymerasekettenreaktion)-basierte Verfahren zum direkten Nachweis von Erregern zum Einsatz. Ziel dieser Übersicht ist es, in der Rheumatologie häufig eingesetzte PCR-Verfahren vorzustellen und ihre Vorteile bzw. Grenzen im Vergleich zu kulturellen Nachweisverfahren aufzuzeigen. In the differential diagnostics of autoimmune-mediated rheumatic diseases, rheumatologists often have to consider infections (e. g. Lyme arthritis) or reactive diseases (e. g. reactive arthritis after urogenital bacterial infections). Furthermore, infections with an atypical presentation or caused by atypical pathogens (opportunistic infections) can complicate the immunosuppressive therapy of autoimmune diseases. For this purpose not only conventional microbiological culture methods but also PCR-based methods are increasingly being applied for the direct detection of pathogens in clinical specimens. The aim of this overview is to present commonly used PCR methods in the clinical practice of rheumatology and to describe their benefits and limitations compared to culture-based detection methods.

In Deutschland ist seit Jahren eine kontinuierliche Zunahme von Methicillin-resistentem Staphylococcus (S.) aureus (MRSA) zu verzeichnen. Um eine weitere Ausbreitung von MRSA zu verhindern, werden Screeninguntersuchungen bei Aufnahme der Patienten ins Krankenhaus empfohlen. In den meisten Fällen ist ein Screening von Risikopatienten mit kulturellen mikrobiologischen Methoden ausreichend. Auf Intensivstationen mit einer hohen MRSA-Inzidenz (>2 Fälle/100 Patienten) und einem hohen Anteil nosokomialer MRSA-Infektionen (>0,3/100 Patienten) wird das Aufnahmescreening bei allen Patienten kosteneffektiv: Bei einer Reduktion nosokomialer MRSA-Infektionen um 50% ist hier eine Kosteneffektivität bis zu EUR 16/Abstrich (einschließlich Folgekosten), bei einer Reduktion nosokomialer MRSA-Infektionen um 75% bis zu EUR 24/Abstrich (einschließlich Folgekosten) gegeben. Das Screening mithilfe der Polymerase-Kettenreaktion (PCR) wird kosteneffektiv bei Risikopatienten mit einer hohen MRSA-Inzidenz und falls die Isolationsmaßnahmen prophylaktisch durchgeführt werden. Hier wurden Verfahren entwickelt, die eine eindeutige Identifikation von MRSA direkt aus Nasenabstrichen ermöglichen.In Deutschland ist seit Jahren eine kontinuierliche Zunahme von Methicillin-resistentem Staphylococcus (S.) aureus (MRSA) zu verzeichnen. Um eine weitere Ausbreitung von MRSA zu verhindern, werden Screeninguntersuchungen bei Aufnahme der Patienten ins Krankenhaus empfohlen. In den meisten Fällen ist ein Screening von Risikopatienten mit kulturellen mikrobiologischen Methoden ausreichend. Auf Intensivstationen mit einer hohen MRSA-Inzidenz (>2 Fälle/100 Patienten) und einem hohen Anteil nosokomialer MRSA-Infektionen (>0,3/100 Patienten) wird das Aufnahmescreening bei allen Patienten kosteneffektiv: Bei einer Reduktion nosokomialer MRSA-Infektionen um 50% ist hier eine Kosteneffektivität bis zu EUR 16/Abstrich (einschließlich Folgekosten), bei einer Reduktion nosokomialer MRSA-Infektionen um 75% bis zu EUR 24/Abstrich (einschließlich Folgekosten) gegeben. Das Screening mithilfe der Polymerase-Kettenreaktion (PCR) wird kosteneffektiv bei Risikopatienten mit einer hohen MRSA-Inzidenz und falls die Isolationsmaßnahmen prophylaktisch durchgeführt werden. Hier wurden Verfahren entwickelt, die eine eindeutige Identifikation von MRSA direkt aus Nasenabstrichen ermöglichen.

We describe the molecular identification of human tuberculosis (TB) from vertebral bone tissue samples from three different populations of ancient Egypt. The specimens were obtained from the predynastic to early dynastic necropolis of Abydos (7 individuals, c. 3500–2650 B.C.), from a Middle Kingdom to Second Intermediate Period tomb of the necropolis of Thebes-West (37, c. 2100–1550 B.C.) and from five further Theban tombs used in the New Kingdom and the Late Period (39, c. 1450–500 B.C.). A total of 18 cases tested positive for the presence of ancient DNA (aDNA) of the M. tuberculosis complex. Out of the 9 cases with typical macromorphological signs of tuberculous spondylitis, 6 were positive for mycobacterial aDNA (66·7%). Of 24 cases with non-specific pathological alterations, 5 provided a positive result (20·8%). In 50 cases of normally appearing vertebral bones 7 tested positive (14·0%). There were only minor differences in the frequencies between the three populations. These data strongly support the notion that tuberculosis was present and prevalent in ancient Egypt since very early periods of this civilization. The unexpectedly high rate of mycobacterial aDNA in normal bone samples is presumably due to a pre- to perimortal systemic spread of the bacteria and indicates a generalized infection by M. tuberculosis.

A pathogenic scotochromogenic Mycobacterium xenopi-like organism was isolated from the lung of an immunocompetent young woman. This pathogen caused severe bilateral cavitary lung disease, making two surgical interventions necessary after years of chronic disease. This case prompted us to characterize this mycobacterium by a polyphasic taxonomic approach. The isolate contained chemotaxonomic markers which were typical for the genus Mycobacterium, i.e., the meso isomer of 2,6-diaminopimelic acid, arabinose, and galactose as diagnostic whole-cell sugars, MK-9(H(2)) as the principal isoprenoid quinone, a mycolic acid pattern of alpha-mycolates, ketomycolates, and wax ester mycolates, unbranched saturated and unsaturated fatty acids plus a significant amount of tuberculostearic acid, and small amounts of a C(20:0) secondary alcohol. On the basis of its unique 16S rRNA and 16S-23S spacer gene sequences, we propose that the isolate should be assigned to a new species, Mycobacterium heckeshornense. This novel species is phylogenetically closely related to M. xenopi. The type strain of M. heckeshornense is strain S369 (DSM 44428(T)). The GenBank accession number of the 16S rRNA gene of M. heckeshornense is AF174290.

A polymerase chain reaction (PCR) assay was applied for the selective amplification of a characteristic sequence within aSalmonella-specific chromosomal fragment. A two-temperature PCR cycle enhanced both the speed and overall sensitivity of the amplification procedure. Twenty-one well-characterizedSalmonella strains and a number of non-Salmonella strains were tested. With the exception of the rarely isolatedSalmonella arizonae strain, the PCR-based approach enabled the specific identification ofSalmonella with a detection limit of 103 organisms. In combination with a nested PCR assay, as few as ten organisms were detectable. Specificity was demonstrated as no distinct amplification products were detectable with any of the testednon-Salmonella strains. With a pre-enrichment step using paramagnetic antiSalmonella beads, an increase in sensitivity was observed in the case of clinical samples while the amplification process was not influenced.

Owing to the persistence of viral DNA in leukocytes after primary CMV infection, detection of CMV DNA in these cells does not necessarily represent active infection. To identify CMV replication more precisely we have analysed immediate-early, early and late CMV transcripts by RNA amplification. The assay seems to be specific for active infection since no RNA-derived PCR products were obtained from healthy seropositive persons. The late UL83 transcript was detected in 80% of the patients with active CMV infections. Diagnosis of CMV replication by amplification of early and immediate-early transcripts was considerably less sensitive. In the case of continual CMV DNA detection in blood leukocytes by PCR without pp65 antigenemia the analysis of defined CMV transcripts would allow differentiation of active and non-active infection. In two cases the RNA assay became negative prior to DNA PCR analysis and pp65 antigen detection upon antiviral treatment, indicating that RNA amplification could be a suitable assay for early detection of the end of viral replication. No strong correlation was found between RNA detection and appearance of clinical symptoms. The development of CMV disease probably depends more on the extent of the functional impairment of the immune system.

Background. The results of excimer laser PRK are promising as more than 80% of eyes with up to −6.0 diopters of attempted correction have refractive results within 1.0 diopter of emmetropia. However, throughout the dioptric range some unexpected results have been observed with individual patients showing an aggressive wound healing response with excessive myopic regression and severe corneal haze. Unfortunately, only limited data are available about the cellular and extracellular responses in human corneas after PRK and this information is important to establish adequate postoperative pharmaceutical treatment.Methods. We made a histopathological and immunohistochemical study on 20 human corneal samples from patients with severe corneal haze and myopic regression. The indirect immunofluorescence method was used for demonstration of collagen types I, III, IV laminin, chondroitin sulphate, dermatan sulphate, and keratin.Results. All corneal specimens showed a hyperplastic epithelium. Histologically, most samples (16/20) showed mainly a loose lamination of extracellular material which could be identified as collagen type IV. The remaining four samples had newly synthesised collagen type III.Conclusions Our histopathological results indicate that corneal wound healing after excimer laser PRK varies among individuals. In some people epithelial basement proteins, such as collagen type IV, are the main wound healing products, whereas in others mainly collagen type III is found postoperatively, which does not effect the synthesis of collagen type IV. This suggests the need for individually-tailored postoperative pharmaceutical treatment regimens.

Immunity after infection with the parvovirus B19 is assumed to be conferred by a humoral immune response with development of neutralizing antibody. In contrast, little is known about the nature of T-cell-mediated responses to parvovirus B19 infection in humans. We used recombinant proteins VP1, VP2, and NS1, as well as a recombinant VP1-specific amino-terminal sequence, to test the proliferative responses of peripheral blood mononuclear cells after infection of otherwise healthy individuals with parvovirus B19. These proteins were used as antigens for the stimulation of freshly isolated cells. The results show that a B19 virus-specific cellular immunity develops that is directed against the capsid proteins VP1 and VP2. We also demonstrate that viral determinants are presented to CD4+ T cells by HLA class II molecules.

Purpose Listeria monocytogenes is a rare cause of endogenous endophthalmitis. To date 15 cases have been published in the literature. All eyes showed similar clinical features and profound visual loss mainly due to delayed diagnosis.Methods An additional case of an otherwise healthy 73 year-old male, who was referred to our hospital because of acute iridocyclitis with secondary glaucoma, is reported. Within a few days the severity of the intraocular infection increased dramatically, resulting in the clinical picture of acute endophthalmitis.Results In contrast to most published cases, early identification of the causative pathogen in the aqueous humor after anterior chamber puncture using polymerase chain reaction (PCR) and the initiation of a specific, systemic antibiotic medication, resulted in complete recovery of visual acuity.Conclusions PCR is very useful for the identification of the pathogen in intraocular infections.

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ABSTRACTDirect sequencing of the 16S rRNA gene (16S rDNA) ofMycobacterium celatumisolates showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded two copies of the gene, which differed by insertion of a thymine at position 214 and by additional mismatches. Restriction fragment length polymorphism analysis confirmed the presence of two copies of 16S rDNA within the bacterial chromosome.

ABSTRACTWe examined 49 Legionellaspecies, 26 L. pneumophilaand 23 non-pneumophila Legionellaspp., using partial 16S rRNA gene sequencing. This approach accurately identified all the L. pneumophilaisolates, characterized all non-pneumophila Legionellaisolates as such within this genus, and classified most (20/23; 87%) of the non-pneumophila Legionellaisolates to the species level.

Cat scratch disease (CSD) is a common infectious disease, however, its association with disciforme keratitis is a previously unreported ocular complication. With the use of the 16S rDNA-PCR technique with subsequent DANN sequencing on corneal material obtained by corneal scrape we were able to identify Bartonella henselae in an unusual form of disciforme keratitis.