PCR analysis is superior to histology for diagnosis of Whipple's disease mimicking seronegative rheumatic diseases.

OBJECTIVES: The diagnosis of Whipple's disease (WD) is commonly confirmed by histology demonstrating Periodic Acid Schiff (PAS)-positive macrophages in the duodenal mucosa. Analysis of intestinal tissue or other specimens using polymerase chain reaction (PCR) is a more sensitive method. However, the relevance of positive PCR findings is still controversial. Therefore, we evaluated the relevance of histology and PCR findings to establishing the diagnosis of WD in a series of WD patients initially presenting with suspected rheumatic diseases. METHOD: Between 2006 and 2014, 20 patients with seronegative rheumatic diseases tested positive for Tropheryma whipplei (Tw) by PCR and/or histology and were enrolled in a retrospective analysis of the diagnostic value of both procedures. RESULTS: Seven of the 20 cases (35%) were diagnosed with 'classic' WD as indicated by PAS-positive macrophages. In the remaining 13 patients, the presence of Tw was detected by intestinal (n = 10) or synovial PCR analysis (n = 3). Two of the 20 patients (10%) with evidence of Tw did not respond to antibiotic therapy. They were not considered to suffer from WD. Therefore, relying only on histological findings of intestinal biopsies would have missed 11 (61%) of the 18 patients with WD in our cohort. In comparison, PCR of intestinal biopsies detected Tw-DNA in 14 (93%) of the 15 WD patients evaluated. Patients with a positive histology did not differ from PCR-positive patients with regard to sex, age, or duration of disease, but more often presented with gastrointestinal symptoms. CONCLUSIONS: A substantial number of WD patients present without typical intestinal histology findings. Additional PCR analysis of intestinal tissue or synovial fluid increased the sensitivity of the diagnostic evaluation and should be considered particularly in patients presenting with atypical seronegative rheumatic diseases and a high-risk profile for WD.

[PCR-based detection of pathogens in clinical rheumatology]. / Polymerasekettenreaktion-gestützte Erregerdiagnostik in der Rheumatologie.

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.

An assessment of the current Chlamydia trachomatis laboratory practices in Germany.

BACKGROUND AND OBJECTIVES: Currently, no information is available about the number of Chlamydia trachomatis (CT) tests performed, testing facilities available or diagnostic methods used in Germany. This study aimed to map CT diagnostic facilities so that representative laboratories can be recruited for CT sentinel surveillance. METHODS: Using a questionnaire, we collected information about population coverage, the number of tests performed, accreditation and current testing methods and systems for German facilities that potentially offer CT diagnostics. RESULTS: Overall, 725/1,504 (48%) facilities responded; of the respondents, 143 reported that they perform CT diagnostics. Of the laboratories performing diagnostics, 45% were privately owned, and 42% were located in a hospital. Of the laboratories that provided information about their catchment area, 61% received samples from at least one federal state and therefore covered more than their surrounding area. The median length of time that CT diagnostics had been performed was 11.5 years. Over half (54%) of the laboratories that provided information on their accreditation status were accredited, for a median duration of 6 years. In accordance with national guidelines, 77% used nucleic acid amplification tests (NAAT) for acute CT infections. CONCLUSIONS: The long duration since Ct diagnostics have been performed and laboratories have been accredited can be seen as an indication of the high diagnostic quality of German laboratories. Additionally, laboratories mostly serviced doctors and patients from a large region and are not representative for people living in the area where the lab is located. This has to be considered when sampling representative labs for CT sentinel surveillance and further epidemiological studies.

Chlamydia trachomatis prevalence, genotype distribution and identification of the new Swedish variant in Southern Germany.

PURPOSE: In Germany, reliable data about the prevalence of urogenital Chlamydia trachomatis infections, causative genotypes, as well as corresponding clinical, demographic and behavioural information are sparse. We, therefore, performed a prospective prevalence study including 1,003 sexually active volunteers of a Southern German city. METHODS: Study participants completed a standardised questionnaire and provided first void urine samples for analysis. Our screening strategy included the performance of two nucleic acid amplification tests with different target genes, enabling the detection of the new Swedish variant of C. trachomatis (nvCT). Direct genotyping of positive specimens was performed by sequence analysis of the ompA gene. RESULTS AND CONCLUSION: The overall prevalence of C. trachomatis infection was 4.2 % in women and 4.6 % in men. A relatively high prevalence of 8.3 % was found in men older than 25 years. Never using condoms was an independent risk factor for infection. The most common symptom was discharge; however, 64.5 % of infected females and all of the infected men were asymptomatic, supporting the need for screening programmes. The most frequently encountered genotypes were E (46.5 %), F (20.9 %) and K (14.0 %). Since the nvCT was detected in one female student, this is one of the rare studies that reports on the molecular identification of nvCT apart from Sweden.

Fatal anthrax infection in a heroin user from southern Germany, June 2012.

Blood cultures from a heroin user who died in June 2012, a few hours after hospital admission, due to acute septic disease, revealed the presence of Bacillus anthracis. This report describes the extended diagnosis by MALDI-TOF and real-time PCR and rapid confirmation of the anthrax infection through reference laboratories. Physicians and diagnostic laboratories were informed and alerted efficiently through the reporting channels of German public health institutions, which is essential for the prevention of further cases.

Granulomatous pneumonia caused by Mycobacterium genavense in a dwarf rabbit (Oryctolagus cuniculus).

A juvenile dwarf rabbit (Oryctolagus cuniculus) with clinical signs of dyspnea and suspected ascites was submitted for necropsy. The main macroscopic findings were a watery red pleural effusion and some whitish striated foci in the lungs. In addition, there were multifocal scars in the cortex of the kidneys. The histologic examination of the lungs showed a severe granulomatous pneumonia with detection of acid-fast bacilli, in the kidneys, an interstitial chronic lymphoplasmacellular nephritis with interstitial fibrosis, and in the brain, a multifocal granulomatous and partly necrotizing encephalitis with detection of spores, suggestive of encephalitozoonosis. In the lungs, Mycobacterium genavense was verified by polymerase chain reaction and 16S ribosomal RNA gene sequencing. To our knowledge, this is the first report of an M. genavense infection in a rabbit, with the lungs being the only affected organ. Therefore, an aerogen infection seems to be the most contemplable way of infection.

The Swedish new variant of Chlamydia trachomatis (nvCT) remains undetected by many European laboratories as revealed in the recent PCR/NAT ring trial organised by INSTAND e.V., Germany.

The May 2009 round of INSTAND's ring trial "Chlamydia trachomatis detection PCR/NAT" included a sample with high amount of the Swedish new variant of C. trachomatis (nvCT). A spectrum of at least 12 different commercial diagnostic nucleic acid amplification tests (NAATs) and many different in house NAATs were applied by the 128 participating laboratories which reported 152 results. Approximately 80% of the results correctly reported the presence of C. trachomatis in the nvCT specimen. The nvCT sample was mainly missed, as expected, by participants using the Roche COBAS Amplicor CT/NG (15.5% of reported results) but also by several participants using in house NAATs. The trend towards using nvCT-detecting NAATs is obvious and in addition to the new dual-target NAATs from Roche and Abbott, and BD ProbeTec ET, also a number of new CE mark-certified commercial tests from smaller diagnostic companies as well as many different in house NAATs were used. Laboratories using commercial or in house NAATs that do not detect the nvCT are encouraged to carefully monitor their C. trachomatis incidence, participate in appropriate external quality assurance and controls schemes, and consider altering their testing system. The reliable detection of low amounts of the wildtype C. trachomatis strain in other samples of the ring trial set indicates a good diagnostic performance of all applied commercial NAATs while also detecting the nvCT strain.

First sequence-confirmed case of infection with the new influenza A(H1N1) strain in Germany.

Here, we report on the first sequence-confirmed case of infection with the new influenza A(H1N1) virus in Germany. Two direct contacts of the patient were laboratory-confirmed as cases and demonstrate a chain of direct human-to-human transmission.

Single-nucleotide polymorphism in the SCCmec-orfX junction distinguishes between livestock-associated MRSA CC398 and human epidemic MRSA strains.

A number of real-time PCR assays for direct detection of methicillinresistant (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic T(m) value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.

Detection of influenza A(H1N1)v virus by real-time RT-PCR.

Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic.

Is Ureaplasma spp. the leading causative agent of acute chorioamnionitis in women with preterm birth?

OBJECTIVES: Aim of this study was to detect microorganisms in fetal membranes and placental tissue in preterm chorioamnionitis by combining fluorescence in situ hybridization (FISH) with broad range PCR. The combination of the two molecular techniques enables identification and localization of the microorganisms within the tissue, confirming their clinical relevance. METHODS: In a prospective cohort study, we compared 31 women with preterm premature rupture of membranes or preterm labour and preterm delivery by caesarean section with a control group of 26 women undergoing elective caesarean section at term. Fetal membranes and placental tissue were analysed by FISH and broad range 16S rRNA-gene PCR and sequencing. RESULTS: For 20 women in the preterm group, caesarean section was performed because of a clinical diagnosis of chorioamnionitis. Microorganisms were detected in the tissues by both molecular techniques in 11 out of 20 women. Among those, Ureaplasma spp. was most abundant, with five cases that remained culture-negative and would have been missed by routine diagnostic procedures. Other infections were caused by Staphylococcus aureus, Streptococcus mitis or Escherichia coli. FISH and PCR were negative for all women without suspected chorioamnionitis and for the control group. CONCLUSIONS: Combination of FISH with broad-range PCR and sequencing permitted unambiguous identification of the causative microorganisms in chorioamnionitis. The high prevalence of Ureaplasma spp. should lead to a re-evaluation of its clinical significance and possible therapeutic consequences.

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'.

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.

Application of molecular biology-based methods to the diagnosis of infectious diseases.

The basis for effective treatment and cure of a patient is the rapid diagnosis of the disease and its causative agent, which is founded on the analysis of the clinical symptoms coupled with laboratory tests. As we approach the 21st century, clinicians are becoming increasingly able to diagnose and treat diseases at the molecular level. The rapid development of new methods and techniques in the area of molecular biology has gained new insights into the genetic and structural features of a considerable number of human pathogens. These results obtained by intensive basic research are currently leading to improved diagnostic procedures. Basically, there are four different possibilities for laboratory diagnosis of infections: 1. direct detection of the pathogens (e.g., microscopy and/or culture), 2. detection of protein components of the pathogens with the help of specific antibodies (e.g., antigen capture ELISA) 3. IgA-, IgM- and IgG-specific detection of antibodies directed against a given pathogen and changes in their corresponding titer, and as the most sensitive method, 4. specific detection of nucleic acids (e.g., PCR) of the pathogens. Here, the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis are serving as examples to review the recent developments as well as the future perspectives in molecular biology-based laboratory diagnosis.

Strategy for typing human papillomaviruses by RFLP analysis of PCR products and subsequent hybridization with a generic probe.

Genital human papillomaviruses (HPV) were detected by PCR using L1 consensus primers MY09 and MY11. To determine the underlying HPV type(s). PCR products were subsequently analyzed employing a combination of restriction fragment length polymorphism (RFLP) and hybridization with a generic oligonucleotide probe that binds to a conserved region located close to the MY11-binding site within the PCR products. Using computer-assisted sequence analysis, the lengths of the corresponding BamHI, DdeI, HaeIII, HinfI and PstI restriction fragments hybridizing with the generic probe were calculated, revealing distinct patterns for each of the 45 mucosal HPV types. This method is superior to RFLP analysis since it is not impaired by large amounts of restriction fragments resulting from nonspecific PCR products. Moreover, considering clinical specimens containing two or three different HPV types, direct sequencing of PCR products will be inconclusive, and the increased number of restriction fragments will complicate interpretation of RFLP patterns. Subsequent hybridization with the generic probe, however, results in the appearance of, at most, 2 or 3 bands per restriction enzyme digest and thus facilitates identification of the underlying HPV types.

Characterization of an isolate belonging to the newly described species Mycobacterium hassiacum.

The isolation, from a urine sample, of a rapidly growing acid-fast mycobacterium assigned to the thermophilic species Mycobacterium hassiacum led to further insight into present knowledge of this newly described organism. Already known phenotypic traits of M. hassiacum were extended and its susceptibility to additional antimicrobials was investigated. The high-performance liquid chromatography pattern of mycolic acids is, for the first time, presented. So far, no clinical relevance was proved for our isolate; likewise for the one which led to the species' original description.

Expression and purification of an Epstein-Barr virus encoded 23-kDa protein and characterization of its immunological properties.

Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens by immunofluorescence and enzyme-linked immunosorbent assay-based tests, or in part on the detection of heterophile antibodies by the Paul-Bunnell-Davidson heterophile assay. In the past few years, the specificity and the sensitivity of serodiagnostic assay systems has been improved considerably by the use of purified recombinant EBV antigens. Screening of EBV-positive sera for antigenic reactivities by immunoprecipitation with extracts of EBV-positive cells revealed a 23-kDa protein (p23) that was recognized by antibodies from all EBV carriers tested. Open reading frame BLRF2 was identified as the coding region for this protein. After cloning and high-level expression of the BLRF2 open reading frame as DHFR fusion protein in Escherichia coli, the recombinant protein was purified to near homogeneity with the help of continuous elution electrophoresis. Sera from both EBV-positive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23. Since anti-p23 antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera had either IgM and/or IgG antibody responses to this protein, recombinant p23 seems to be a useful diagnostic marker for EBV-infection.

Specific detection of monkeypox virus by polymerase chain reaction.

The open reading frame coding for the A-type inclusion body protein (ATI) of monkeypox virus (MPV) was identified and sequenced for two strains. Nucleotide sequence comparison revealed 72-95.3% homology with the reported open reading frame sequences of the ATIs of other orthopoxvirus species, such as variola, vaccinia, cowpox, ectromelia, and camelpox viruses. Each MPV strain contained an 8-bp deletion, which caused a frameshift that introduced a premature stop in the open reading frame at base 2091 relative to the ATI open reading frame of cowpox virus strain Brighton. The sequences enabled a primer pair to be designed that flanked the deletion and specifically amplified a 601-bp fragment that identified and differentiated 19 MPV strains examined from five other Old World orthopoxvirus species examined. The specificity was confirmed by cleavage of the 19 MPV strain amplicons with BglII, which produced three subfragments of expected sized, based on the determined MPV sequences.

Quantitative PCR. A survey of the present technology.

PCR-based amplification of nucleic acids has had a major impact in almost every field of basic research and has already found extensive applications in the area of clinical diagnosis. For many of these applications, quantitative data are sought to relate the quantity of amplified product to the amount of original target nucleic acid present in the sample. Since the PCR methodology with its exponential nature can be adapted for this purpose, a lot of different strategies have emerged in the last few years for sensitive and specific PCR product detection and quantification. Basic strategies, including the use of external and internal standards, are presented with respect to statistical aspects, and the advantages as well as the limitations of individual protocols are discussed. Furthermore the suitability of conventional laboratory techniques, such as gel systems or HPLC, nonradioactive labeling procedures, and the principles of advanced solid-phase-mediated strategies for the precise determination of amplification products, are outlined with the help of selected examples.

Nonradioactive labeling and high-sensitive detection of PCR products.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.

Nonradioactive labeling of polymerase chain reaction products.

Polymerase chain reaction (PCR) was originally introduced to amplify in vitro particular DNA sequences by the application of temperature cycles (1). In a modification, RNA molecules also may serve as templates by an additional reverse transcription step converting RNA in complementary DNA sequences (2).