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Background: The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility., Methodology/principal Findings: Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay's reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent., Significance: The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.

"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S-23S rRNA intergenic spacer region (16S-23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S-23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S-23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S-23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S-23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNA(Ala) gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S-23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination.

The emergence of a novel pandemic human strain of influenza A (H1N1/09) virus in April 2009 has demonstrated the need for well-validated diagnostic tests that are broadly applicable, rapid, sensitive, and specific. The analytical performance and clinical validity of results generated with the novel Roche RealTime Ready Influenza A/H1N1 Detection Set using the LightCycler 2.0 instrument were characterized. Analytical performance was assessed by processing respiratory samples spiked with H1N1/09 and seasonal influenza A virus, a set of seasonal influenza A virus subtypes, and samples containing common viral and bacterial respiratory pathogens. The clinical validity of results was assessed in comparison to other assays by analyzing 359 specimens at three clinical sites and one reference laboratory. Direct sequencing was used to resolve samples with discrepant results. The assay detected virus concentrations down to <50 RNA copies per reverse transcription (RT)-quantitative PCR (qPCR). Various influenza A virus subtypes were covered. The analytical specificity was 100%. High clinical validity was demonstrated by the 99% positive agreement between seasonal influenza A viruses, 98% positive agreement between H1N1/09 viruses, and 88% agreement between negative results. The analytical sensitivity was compared to those of three other RT-qPCR assays and was found to be equivalent. The novel Roche RealTime Ready Influenza A/H1N1 Detection Set can be utilized on the widely used LightCycler platform. We demonstrate its usefulness for the rapid detection and surveillance of pandemic H1N1/09 influenza A virus infections.

The aim of the present study was the quantification of Streptococcus mutans adhesion on ten widely used pit and fissure sealant materials and the correlation of these findings to surface roughness (R(a)) and surface free energy (SFE). Additionally, changes in streptococcal adhesion and surface parameters after water immersion and artificial aging have been investigated. Circular specimens of ten fissure sealants (seven resin-based composites, two glass ionomers, and one compomer) were made and polished. Surface roughness was determined by perthometer and SFE by goniometer measurements. Sealant materials were incubated with S. mutans suspension (2.5 h, 37 degrees C), and adhering bacteria were quantified by using a biofluorescence assay in combination with an automated plate reader. Surface properties and S. mutans adhesion were measured prior to and after water immersion after 1 and 6 months and after additional thermocycling (5,000 cycles; 5 degrees C/55 degrees C). The tested sealants showed significant differences in S. mutans adhesion prior to and after the applied aging procedures. Aging resulted in slight increases (mostly <0.2 microm) in surface roughness, as well as in significant decreases in SFE and in significantly lower quantities of adhering bacteria. Ketac Bond and UltraSeal XT plus revealed the lowest adhesion potential after artificial aging. In general, the amount of adhering S. mutans was reduced after aging, which may be related to the decline in SFEs.

Background: The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods., Methods: We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)--a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes--specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin., Results: Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100-1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent., Conclusions: Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.

Impurities in complex biological samples that persist through nucleic acid preparation can inhibit PCR or reduce the sensitivity and efficiency of PCR amplification and thus affect reliable results in PCR diagnostics. To obtain information on the incidence of inhibition events and on the magnitude of the loss of sensitivity, respectively, we devised a relative inhibition assay and examined 100 samples from patients with respiratory tract infections. As a reference, samples were spiked with Legionella (L.) pneumophila. Detection was by standard nucleic acid purification and subsequent real-time PCR. By comparing the crossing points of the fluorescent curves to those of an L. pneumophila standard dilution series, we were able to quantify the respective degrees of inhibition into several categories. We found complete inhibition in 2% of the samples. 12% were not reliably detected. 65% of the tested samples showed moderate to strong inhibition, but were still reliably detected, whereas in 21% of the samples no inhibition was observed. Except for a significantly higher inhibition in tracheal aspirates than in BAL samples, the degree of inhibition did not correlate with the physical properties of the respective sample. The relative inhibition assay established an unexpectedly broad distribution of the inhibition-degrees in inflammatory respiratory materials.

Background: Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC). We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis., Methods: Blood samples from patients with presumed sepsis were cultured with the Bactec 9240 system (Becton Dickinson, Heidelberg, Germany) and aliquots subjected to analysis with the LightCycler SeptiFast (SF) Test (Roche Diagnostics, Mannheim, Germany) at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made., Results: Of 101 blood samples from 77 patients, 63 (62%) yielded concordant negative results, 14 (13%) concordant positive and 9 (9%) were BC positive only. In 14 (13%) samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients). In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%)., Conclusion: The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.

Objective: This study aims at ranking various luting systems according to their susceptibility to adhering Streptococcus mutans and at evaluating the influence of incongruent mixing ratios on adhesion quantities., Material and Methods: Circular specimens measuring 8 mm in diameter were made of nine widely used dental cements -- three of them mixed in different ratios -- and then incubated with S. mutans. Adhering streptococci were quantified using a biofluorometric assay in combination with an automated plate reader for cell quantification. Surface roughness (R(a)) was determined by perthometer measurements., Results: Meron plus revealed the highest R(a) (0.90 microm) and glass the lowest R(a) (<0.01 microm). In regular cement mixtures, the highest mean fluorescence intensities indicated the presence of many viable bacteria [Meron Plus (35,533 relative fluorescence units (rfu)), Maxcem (13,374 rfu), and Panavia F 2.0 (11,701 rfu)]. Moderate fluorescence intensities were found in Harvard (4,171 rfu), Ketac cem (3,766 rfu), Durelon (3,276 rfu), Calibra (3,259), Rely X Unicem (4,358 rfu), and Bifix SE (3,102 rfu). A medium correlation between R(a) and S. mutans adhesion was found. Changes in regular cement proportions (powder/liquid and base/catalyst, respectively) had a significant influence on relative fluorescence intensities, which linearly increased with a higher proportion of liquid in Harvard and with a higher proportion of catalyst in Calibra and Maxcem., Conclusions: Various luting systems revealed considerable differences in their potential to adhere S. mutans. Variations from recommended cement proportions led to significant changes in the amount of adhering streptococci.

In this work, a two dimensional (2D) finite element foot model was established from magnetic resonance imaging (MRI) of a male subject. The model comprises first medial planar cross-section through the foot, representing the foot in standing posture. For specified external load, the stress and strain distribution field under foot structure are determined. The material characterization of foot structure components are stronger related to diabetic phenomena. The new material model for soft tissue based on mixture theory is proposed. The linear finite element model replaced by nonlinear counterpart with segment-to-segment contact element.

Purpose: To investigate and compare the use of molecular biology with the use of traditional Gram stain and organism culture for the laboratory diagnosis of postoperative endophthalmitis., Setting: Twenty-four ophthalmology units together with 9 microbiology laboratories and 2 European reference molecular biology laboratories., Methods: A prospective randomized partially masked multicenter cataract surgery study recruited 16 603 patients. This resulted in 29 cases of presumed postoperative endophthalmitis. Gram stain and culture were performed in the local laboratory according to agreed protocols. Samples of aqueous and/or vitreous were transported to the first referenced molecular biology laboratory (Regensburg, Germany) for polymerase chain reaction (PCR) testing, and an extracted aliquot of DNA was then referred to the second laboratory (Alicante, Spain) for PCR., Results: Of the 29 who presented with presumed postoperative endophthalmitis, 20 were classified as proven infective endophthalmitis with positive Gram stain, culture, or PCR. Fourteen patients were culture-positive; all but 1 of these was also positive by PCR. Six patients were positive by PCR but negative by Gram stain or culture. Nine patients were negative by both microbiology and PCR testing., Conclusions: Use of molecular biology technique increased the laboratory rate of identifying the pathogen by 20%, confirming the technique is very useful for the endophthalmitis specimen. Samples of both aqueous and vitreous should be collected and stored at -20 degrees C for PCR at the time of the diagnostic taps.

Foot print patterns of the bound feet of a 90-year-old Chinese female were made to obtain insight into the ergonomic consequences of a Chinese custom that caused significant disabilities for many women throughout history. Pressure patterns were evaluated using the techniques applied to standard thumb print analsyis. A digital summary of the pressure patterns were compared to the patterns obtained from a normal subject. The outcomes indicated that the bound foot produced greater plantar tissue pressures than the non-bound foot. These observations help explain the discomfort, gait abnormalities, and disabilities exhibited by many older women with bound feet living in China today. Although foot-binding is no longer practiced, this study offers an ergonomic perspective on a custom practiced in China for centuries.

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5x MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

We report on a Mycobacterium monacense infection associated with a pulmonary tumor in a Chinese patient. To our knowledge, this is the first case of M. monacense described in a non-European patient with a tuberculosis-like disease. Further evaluation of the human pathogenic potential of M. monacense is needed.

Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis., Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values., Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results., Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.

Extranodal marginal zone B-cell lymphomas of MALT type (MALT lymphomas) show site-dependent variations in their morphologic, phenotypic, and/or cytogenetic findings. This report describes a comprehensive analysis of 34 ocular adnexa MALT lymphomas, including interphase fluorescence in situ hybridization for MALT lymphoma-associated cytogenetic abnormalities and polymerase chain reaction for Chlamydia psittaci, which has recently been suggested to be associated with ocular adnexa lymphomas. A typical morphologic pattern was identified in 79% of cases, while overtly monocytoid cytology (12%), predominantly plasmacytic features (9%), and lymphoepithelial lesions (3%) were uncommon. Aberrant CD43 or CD5 expression was also uncommon (12% and 3%, respectively). Plasmacytic differentiation (41%) was associated with stage IV disease (P=0.036) and gains of chromosomes 3 and/or 18q (P=0.021) (79%). +3 was more frequent in the orbit than in lacrimal gland or conjunctiva (P=0.005). Each of 31 cases was negative for MALT1 translocations. IGH translocations were identified in 3 cases (10%), although the translocation partner gene could not be identified. Polymerase chain reaction assays targeting species-specific regions within the C. psittaci omp1 and omp2 genes were negative in each of 30 cases. This study identifies the characteristic morphologic, phenotypic, and cytogenetic findings in ocular adnexa MALT lymphoma, including a subset differing from those arising at other anatomic sites. The frequent presence of +3 and/or +18q suggests that these abnormalities may contribute to lymphomagenesis. The lack of C. psittaci in this series, in contrast to some prior reports, indicates that there may also be geographic heterogeneity in the pathogenesis of ocular adnexa MALT lymphoma.

Although Medicare currently reimburses for telemedicine services, advocates are struggling to increase state Medicaid reimbursement. This study provides data from a national study of Medicaid telemedicine reimbursement policies and examines Idaho as a case study for developing telemedicine reimbursement policies. Idahoans have actively advocated for Medicaid telemedicine reimbursement by forming a statewide network. Working with policymakers, Idaho Medicaid and telemedicine advocates established interpersonal connections, providing policymakers information and support. With developing academic, private, and legislative interest, a window of opportunity opened to allow for positive, albeit minimal, movement. To establish protocols for Idaho's use of telemedicine, a national electronic policy survey was conducted to evaluate the direction of telemedicine policy in state Medicaid agencies. Surveys to explore Medicaid reimbursement status were sent to states that were both participating and non-participating in telemedicine. Responses were received from 10 of the 25 states providing Medicaid telemedicine reimbursement and 17 of the 25 states and one U.S. territory not providing reimbursement. Issues common among participating states included provider and reimbursement complications, allowable services, and modification of reimbursement codes. Nonparticipating states indicated an interest in reimbursing for telemedicine and a need to enhance advocate and state Medicaid agency relationships. In addition, the survey results demonstrated the need to provide cost-benefit analysis on the viability of Medicaid reimbursement for telemedicine. Research outcomes were used to develop Idaho's Interactive Video Telemedicine Protocols. These address identified barriers and fears regarding Medicare reimbursement and state budgetary concerns--the additional major issue identified for state Medicaid agencies.

Four bacterial strains were isolated from independent clinical specimens in different countries and their genotypic and phenotypic characters support their classification in a novel species within the genus Mycobacterium. One strain was clearly responsible for a severe, post-traumatic wound infection in a healthy boy. The novel species, for which the name Mycobacterium monacense sp. nov. is proposed, is yellow-pigmented, non-photochromogenic and grows in less than a week on solid medium. Based on phenotypic investigations alone, distinction of these four strains from known scotochromogenic rapidly growing strains is problematic. However, the novel strains differ from any other mycobacterium in each of the molecular species markers investigated: the 16S rRNA gene, the 16S-23S rRNA gene internal transcribed spacer and the hsp65 gene. Of the strains investigated, two different sequevars were detected for the hsp65 region. Phylogenetic analysis revealed that these four strains were most closely related to Mycobacterium doricum. The type strain of Mycobacterium monacense sp. nov. is B9-21-178T (=DSM 44395T=CIP 109237T).

The aims of this cross-sectional study were to determine the prevalence of and risk factors for carriage of Panton-Valentine leukocidin-producing methicillin-resistant Staphylococcus aureus (PVL-MRSA) in residents and personnel of a nursing home in Germany. In this study, PVL-MRSA carriage status among nursing home residents was associated with risk factors reflecting their dependence on nursing care. No specific risk factors were detected among staff.

The results for a real-time PCR assay, using the LightCycler Strep B analyte-specific reagents (Roche Diagnostics Corporation, Indianapolis, Ind.), were compared to a direct plate method combined with a broth enrichment culture method for detection of group B streptococcus colonization in pregnant women. Two separate evaluations were conducted using two different automated nucleic extraction instruments, the MagNA Pure LC instrument (Roche Diagnostics Corporation) and the lower-capacity MagNA Pure Compact instrument (Roche Diagnostics Corporation). The sensitivities, specificities, and positive and negative predictive values for the different evaluation methods were as follow: for the LightCycler Strep B assay with MagNA Pure LC, 100, 97, 90, and 100%, respectively; for the LightCycler Strep B assay with MagNA Pure Compact, 92.5, 99, 97, and 97.5%, respectively. The LightCycler Strep B assay combined with either MagNA Pure LC or MagNA Pure Compact extraction is a suitable method for detecting group B streptococcus colonization in pregnant women. An advantage of the LightCycler assay over culture is the considerably reduced turnaround time for results.

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

Introduction: According to foreign literature Mycobacterium haemophilum causes diseases of the skin, subcutis and lymph nodes in immunocompetent individuals, while in AIDS patients and in subjects after kidney transplantations it is responsible for osteomyelitis and disseminated infections., Material and Methods: The authors tested the possibility of using a BioFM medium with the X-factor for the detection of Mycobacterium haemophilum in clinical samples and Middlebrook's 7H9 medium with ADC and the X-factor to establish the sensitivity of strains to antimicrobials using the MIC method., Conclusions: Given the favourable results of preliminary tests the use of the BioFM medium was included among the routine methods applied at the department. In one year Mycobacterium haemophilum was detected with this method in 3 patients presenting extrapulmonary mycobacteriosis.

In response to several isolations of methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leucocidin gene (PVL-MRSA), the present study was conducted to document the spread of infection in a small region of southeastern Germany. During a 9-month period, two healthcare-associated outbreaks with PVL-MRSA occurred, affecting 83 patients, personnel and contacts of personnel, and 34 additional cases were detected in the community. The clinical spectrum ranged from colonization to skin infection and necrotizing pneumonia. The findings represent the largest number of PVL-MRSA cases detected in Germany so far, and demonstrate the potential of this emerging pathogen to spread within the community and in healthcare institutions.

We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.

With the prevalence of diagnosed autism on the rise, increased efforts are needed to support surveillance, research, and case management. Challenges to collect, analyze and share typical and unique patient information and observations are magnified by expanding provider caseloads, delays in treatment and patient office visits, and lack of sharable data. This paper outlines recommended principles and approaches for utilizing state-of-the-art information systems technology and population-based registries to facilitate collection, analysis, and reporting of autism patient data. Such a platform will increase treatment options and registry information to facilitate diagnosis, treatment and research of this disorder.

The efficiencies of five commercially available nucleic acid extraction methods were evaluated for the recovery of a standardized inoculum of Legionella pneumophila in respiratory specimens (sputum and bronchoalveolar lavage [BAL] specimens). The concentrations of Legionella DNA recovered from sputa with the automated MagNA Pure (526,200 CFU/ml) and NucliSens (171,800 CFU/ml) extractors were greater than those recovered with the manual methods (i.e., Roche High Pure kit [133,900 CFU/ml], QIAamp DNA Mini kit [46,380 CFU/ml], and ViralXpress kit [13,635 CFU/ml]). The rank order was the same for extracts from BAL specimens, except that for this specimen type the QIAamp DNA Mini kit recovered more than the Roche High Pure kit.

We describe a sensitive and reliable method for detection and differentiation of the five relevant European Borrelia burgdorferi sensu lato species ( B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae), based on a heminested ospA-PCR followed by restriction enzyme analysis. Sensitivity was one borrelia per PCR except for B. afzelii, where it was five per PCR. None of seven relapsing fever borreliae, eight Leptospira serovars or two Treponema species were amplified. Except B. garinii, each of the five B. burgdorferi s.l. species is represented by one or two characteristic restriction fragment length polymorphism (RFLP) patterns. Analysis of the heterogeneous group of B. garinii resulted in five different RFLP patterns, corresponding to the OspA types 3-7 associated with this species. In a pilot study on 529 Ixodes ricinus ticks from three different regions in Southern Germany, all species and OspA types were found except B. lusitaniae and B. garinii OspA type 7, arguing for a broad distribution of almost all OspA types. A further notable finding was the focal prevalence of OspA type 4, which has rarely been detected in ticks previously. Thus, the developed method provides a fast and simple tool for epidemiological studies on the heterogeneity of species and OspA types in Europe which has important implications for the development of vaccines and (microbiological) test systems for Europe.

We report the first case of a postoperative wound infection caused by Vibrio metschnikovii on the lower right leg of a patient after saphenectomy. Compared to the healing of an uninfected site, that of the right leg was delayed, and a cure was achieved by intensified wound care. Several swabs taken from the infected site grew a gram-negative rod in pure culture that was identified as V. metschnikovii by the VITEK 2 system. The source of the infection was not detected; however, the absence of putative risk factors (exposure to water or shellfish or an episode of diarrhea), the profession of the patient (butcher), and the isolation of V. metschnikovii in a variety of farm animals (chicken, cattle, swine, and horses) suggest that infections caused by V. metschnikovii may be regarded as zoonotic.

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

Toxoplasma reactivation is a serious complication in patients receiving allogenic stem cell transplantation. Real-time PCR assays allow a rapid diagnosis of toxoplasma infection; however, no comparative data are available on the performance of real-time PCR protocols under routine conditions. Therefore, the aim of this study was to amplify Toxoplasma gondii DNA from routine samples of allogenic stem cell recipients using two real-time PCR assays on a LightCycler, and using conventional nested PCR. Conventional nested PCR revealed T. gondii DNA in 16 samples. Only 12 of the 16 samples yielded a positive result in both real-time PCRs. The accuracy of the conventional PCR results was demonstrated by direct sequencing. Amplification and detection of the amplicon was completed in only 1 h using the real-time PCR assays. Thus, real-time PCR substantially accelerates the detection of T. gondii DNA in the majority of positive specimens; however, conventional nested PCR is required for detection of T. gondii DNA in some samples.

Anaplasma phagocytophilum is an obligate intracellular bacterium that is related to rickettsial organisms and replicates in the hostile environment of neutrophils. Previous studies with SCID mice suggested that T and/or B cells are required for its control in vivo. Here, we used mice deficient for Toll-like receptor (TLR)2 and TLR4, MyD88, tumor necrosis factor, inducible nitric oxide synthase, or phagocyte NADPH oxidase (gp91(phox-/-)) to define the pathways that are critical for the recognition and the killing of this pathogen. Whereas SCID mice developed a 60-fold higher bacterial load in the blood compared to wild-type mice and succumbed to infection, all other gene-deficient mouse strains were fully capable in overcoming a systemic infection with A. phagocytophilum. From these data we conclude that effector mechanisms that are crucial to the defense against numerous other intracellular pathogens are dispensable for the control of A. phagocytophilum.

We developed real-time polymerase chain reaction (PCR) assays for rapid detection of the most common and clinically relevant bacteria in positive blood culture bottles, including Staphylococcus spp., S. epidermidis, S. aureus, Enterococcus spp. (including differentiation of E. faecalis and E. faecium), Streptococcus spp., Streptococcus agalactiae, Enterobacteriaceae, E. coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp., Bacteroides spp., Haemophilus influenzae, and Neisseria meningitidis. A total of 507 positive blood cultures were investigated according to a specific PCR algorithm based on the microscopic result of the blood culture, and the PCR results were compared to the culture results. Apart from-cross reactions between E. coli and Chryseomonas luteola and Enterococcus faecium and E. durans, the PCR assay correctly identified all bacteria in the blood cultures and did not show any false-positive results. Regarding blood cultures positive with a single species of bacteria (n = 474), 98.3% of all bacteria were correctly detected by the PCR algorithm within a few hours. However, in mixed infections, the sensitivity was lower. The PCR algorithm is well suited for rapid identification of the most common bacteria in positive blood cultures.

A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.

This chapter describes a straightforward protein purification strategy for the specific separation of insoluble recombinant proteins (so-called "inclusion bodies") located in the E. coli cytoplasm and their subsequent recovery in form of soluble recombinant proteins. Optimization of this technique can overcome in some cases the application of tedious and yield-reducing standard protein purification procedures. Due to the different behavior of individual recombinant proteins during separation, solubilization, and purification, subsequent purification steps may be required to obtain recombinant proteins in such a purity, that they can be used as antigen components of immunological test systems.

Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

While culture for Bordetella species is highly specific, sensitivity is extremely variable due to patient age, immunization status, antibiotic treatment, and specimen transport conditions. We evaluated a real-time multiplex PCR assay as an alternative to culture for the detection and differentiation of Bordetella pertussis and Bordetella parapertussis. The PCR conditions allowed the simultaneous detection of one B. pertussis organism and five B. parapertussis organisms per reaction. An inhibition control was incorporated into the assay. Of 163 total samples evaluated, 37 of 38 samples positive by either culture or direct fluorescent antibody testing (DFA) were also positive by PCR (97% sensitivity). Of 125 culture- or DFA-negative samples, 101 were also negative by PCR (81% specificity). The described multiplex assay is a rapid, sensitive, contamination-limiting, real-time PCR assay that controls for inhibition. The assay performs well using liquid or swab samples and from dried material on slides.

Background: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid., Methods: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity., Results: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii., Conclusion: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

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.

In order to meet the need of many microbiological laboratories for a standardized system for detecting Chlamydia pneumoniae in respiratory specimens, a hybridization probe-based LightCycler (Roche Diagnostics, Germany) PCR assay was developed. The assay's analytical sensitivity and specificity were evaluated according to the recommendations of the Centers for Disease Control and Prevention (USA) and Laboratory Centre for Disease Control (Canada). Seventy-four bacterial species other than Chlamydia pneumoniae, including strains of Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pecorum, tested negative. Six of six representative Chlamydia pneumoniae strains tested positive. An analytical sensitivity of 1 inclusion forming unit per ml of bronchoalveolar lavage fluid, corresponding to 0.02 inclusion forming units per PCR reaction, was observed. The assay showed 100% specificity and sensitivity for Chlamydia pneumoniae when testing DNA preparations from 12 specimens of patients with known pulmonary Chlamydia pneumoniae infection and from 78 specimens of patients with respiratory tract disease of other origin. The newly developed LightCycler assay may contribute to the urgently needed standardization of laboratory diagnosis of Chlamydia pneumoniae.

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum.

The performance of a commercial line probe assay (LiPA) (Inno-LiPA Mycobacteria; Innogenetics, Belgium) for the detection and identification of Mycobacterium species from liquid and solid culture was evaluated at five routine clinical laboratories. The LiPA method is based on the reverse hybridization principle, in which the mycobacterial 16S-23S ribosomal RNA (rRNA) spacer region is amplified by polymerase chain reaction (PCR). Amplicons are subsequently hybridized with oligonucleotide probes arranged on a membrane strip and detected by a colorimetric system. The test detects the presence of Mycobacterium species and specifically identifies Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium chelonae - Mycobacterium abscessus complex. The results of LiPA were compared with the results obtained using traditional biochemical and molecular tests (DNA probe-based techniques, PCR restriction enzyme analysis of the 65 kDa heat-shock protein gene, and sequencing of the 16S rDNA). A total of 669 isolates, 642 of which were identified as Mycobacterium species and 27 as non- Mycobacterium species, were tested by LiPA. After analysis of 14 initially discordant results and exclusion of one isolate, concordant results were obtained for 636 of 641 Mycobacterium isolates (99.2% accuracy). All Mycobacterium species reacted with the MYC ( Mycobacterium species) probe (100% sensitivity), and all non- Mycobacterium species were identified as such (100% specificity).