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A polymerase chain reaction (PCR) assay for the detection of Legionella spp. in clinical bronchial fluid samples was constructed. The assay could detect a 375 bp fragment of the 16S RNA gene, equivalent to 10 cfu in simulated clinical bronchial fluid and blood samples. Seven clinically relevant Legionella spp. involving 12 serogroups produced a positive signal, whereas three Legionella spp. and none of a panel of 26 bacterial strains produced a signal in the constructed assay. Investigation of bronchial fluid from 51 patients with clinically suspected Legionnaire's disease revealed the presence of Legionella DNA by PCR in seven patients. The results were verified by Southern blot analysis of the amplified DNA fragment. None of 37 control patients produced a positive signal. Only one of the seven bronchial fluid samples positive for Legionella by PCR was positive by conventional culture, thus indicating the increased sensitivity of PCR compared to culture. [ABSTRACT FROM AUTHOR]

Background: Pandemic preparedness is critical to respond effectively to existing and emerging/new viral pathogens. Important lessons have been learned during the last pandemic at various levels. This revision discusses some of the major challenges and potential ways to address them in the likely event of future pandemics., Objectives: To identify critical points of readiness that may help us accelerate the response to future pandemics from a clinical microbiology laboratory perspective with a focus on viral diagnostics and genomic sequencing. The potential areas of improvement identified are discussed from the sample collection to information reporting., Sources: Microbiologists and researchers from five countries reflect on challenges encountered during the COVID-19 pandemic, review published literature on prior and current pandemics, and suggest potential solutions in preparation for future outbreaks., Content: Major challenges identified in the pre-analytic and post-analytic phases from sample collection to result reporting are discussed. From the perspective of clinical microbiology laboratories, the preparedness for a new pandemic should focus on zoonotic viruses. Laboratory readiness for scalability is critical and should include elements related to material procurement, training personnel, specific funding programmes, and regulatory issues to rapidly implement "in-house" tests. Laboratories across various countries should establish (or re-use) operational networks to communicate to respond effectively, ensuring the presence of agile circuits with full traceability of samples., Implications: Laboratory preparedness is paramount to respond effectively to emerging and re-emerging viral infections and to limit the clinical and societal impact of new potential pandemics. Agile and fully traceable methods for sample collection to report are the cornerstone of a successful response. Expert group communication and early involvement of information technology personnel are critical for preparedness. A specific budget for pandemic preparedness should be ring-fenced and added to the national health budgets., Competing Interests: Transparency declaration The authors declare that they have no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Syndromic testing for lower respiratory tract infections with BioFire® FilmArray® Pneumonia Panel Plus (BF) detects 27 pathogens with a turn-around-time of one hour. We compared the performance of BF with culture. Samples from 298 hospitalized patients with suspected pneumonia routinely sent for culture were also analyzed using BF. Retrospectively, patients were clinically categorized as having "pneumonia" or "no pneumonia." BF and culture were compared by analytical performance, which was evaluated by pathogen concordance, and by clinical performance by comparing pathogen detections in patients with and without pneumonia. The BF results for viruses and atypical bacteria were not included in the performance analysis. In 298 patient samples, BF and culture detected 285 and 142 potential pathogens, respectively. Positive percent agreement (PPA) was 88% (125/142). In patients with community-acquired pneumonia (CAP), clinical sensitivity was 70% and 51%, and specificity was 43% and 71% for BF and culture, respectively. In patients with hospital-acquired pneumonia, the corresponding numbers were 55% and 23%, and 47% and 68%. There was no significant improvement of performance, when only high-quality sputum samples were considered. Efficacy of both BF and culture was low. Both tests are best used in CAP patients for whom the diagnosis has already been clinically established. Indiscriminate use may be clinically misleading and a cause of improper use of antibiotics., (© 2022 Scandinavian Societies for Medical Microbiology and Pathology.)

Background: Stratification by clinical scores of patients suspected of infection can be used to support decisions on treatment and diagnostic workup. Seven clinical scores, SepsisFinder (SF), National Early Warning Score (NEWS), Sequential Orgen Failure Assessment (SOFA), Mortality in Emergency Department Sepsis (MEDS), quick SOFA (qSOFA), Shapiro Decision Rule (SDR) and Systemic Inflammatory Response Syndrome (SIRS), were evaluated for their ability to predict 30-day mortality and bacteraemia and for their ability to identify a low risk group, where blood culture may not be cost-effective and a high risk group where direct-from-blood PCR (dfbPCR) may be cost effective., Methods: Retrospective data from two Danish and an Israeli hospital with a total of 1816 patients were used to calculate the seven scores., Results: SF had higher Area Under the Receiver Operating curve than the clinical scores for prediction of mortality and bacteraemia, significantly so for MEDS, qSOFA and SIRS. For mortality predictions SF also had significantly higher area under the curve than SDR. In a low risk group identified by SF, consisting of 33% of the patients only 1.7% had bacteraemia and mortality was 4.2%, giving a cost of € 1976 for one positive result by blood culture. This was higher than the cost of € 502 of one positive dfbPCR from a high risk group consisting of 10% of the patients, where 25.3% had bacteraemia and mortality was 24.2%., Conclusion: This may motivate a health economic study of whether resources spent on low risk blood cultures might be better spent on high risk dfbPCR., (© 2021. The Author(s).)

Human orthopneumovirus, formerly known as respiratory syncytial virus (RSV), is a frequent cause of hospitalization among infants due to respiratory tract infection. Fast, reliable, and easy to perform tests are needed to optimize treatment and to identify children that should be contact isolated to avoid nosocomial outbreaks. We prospectively tested 200 respiratory samples with a new assay (ImmuView RSV Antigen Test, SSI Diagnostica) and compared the results to the Alere BinaxNOW RSV Card by using our laboratory-developed real-time reverse transcription polymerase chain reaction (PCR) as reference. In addition, 300 retrospectively collected respiratory samples were included in the study. The sensitivities of both antigen kits were very low (<50%). Sensitivities were higher when samples came from children less than 6 years, when samples came from nasopharynx or lower respiratory airways, or when samples were positive for RSV serotype A compared to when samples came from adults, samples were throat swabs, or samples were positive for RSV serotype B. In conclusion, the ImmuView RSV antigen kit did not perform well and may at the most be used as a quick guidance for clinical decision. Thus, it cannot stand alone without reverse transcription PCR confirmation of negative results., (© 2020 Wiley Periodicals LLC.)

Sensitive, probe-based detection of multiple DNA targets is limited by the competitive reannealing of the antiparallel duplex DNA helix with the complementary DNA strand. To address this, we developed Cliffhanger primers, which create single-stranded DNA overhangs on PCR amplicons while simultaneously increasing the multiplex PCR efficacy and allowing PCR amplification using crude lysates of human faecal samples. A multiplex PCR that targeted eight genes from diarrhoeagenic Escherichia coli plus an internal control was performed and compared to a routine method that consisted of culture followed by multiplex PCR with fragment length separation. A total of 2515 clinical faecal samples from patients with diarrhoea were tested using both methods, and there was a significant increase in clinical sensitivity and negative predictive value with the Cliffhanger method for seven out of eight genes. All Cliffhanger-only positive samples were confirmed by Sanger sequencing of the PCR amplicon. Notably, the Cliffhanger method reduced the total sample turn-around time in the laboratory from 20 hours to 6 hours. Hence, use of Cliffhanger primers increased assay robustness, decreased turn-around time and increased PCR efficacy. This increased the overall clinical sensitivity without the loss of specificity for a heavily multiplexed PCR assay., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: UVS financial competing interests: Current ownership of stocks, previous paid employment and previous consulting for Anapa Biotech A/S. Inventor of WO 2011/137911 “Method for generating a double stranded nucleic acid with a single stranded overhang” which is fully entrusted to Anapa Biotech A/S. Non-financial competing interests: None. NDM financial competing interests: Previous paid employment for Anapa Biotech A/S. Non-financial competing interests: None. FS financial and non-financial competing interests: None. AF-M financial and non-financial competing interests: None. GL financial competing interests: Previous paid employment and previous consulting for Anapa Biotech A/S. Inventor of WO 2009/112032 “Target amplification and sequencing with primers comprising triplex forming monomer units” and WO 2011/137911, which are fully entrusted to Anapa Biotech A/S. Non-financial competing interests: None. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (T(m)) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that Delta T(m) of base mismatches on PT is remarkably high (between 7.4 and 15.2 degrees C) compared to antiparallel duplexes (between 3.8 and 9.4 degrees C). The specificity of PT by Delta T(m) increases when shorter TFOs and higher pH are chosen. To increase Delta Tms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3' (preferable) or 5' of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays.

Background: Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.Hoogsteen based parallel triplex helix formation requires protonation of cytosines in the triplex forming strand. Therefore, nucleic acid triplexes show strong pH dependence and are stable only at acidic pH. This led us to establish a new pH independent fluorophore based measuring system on the LightCycler for thermal stability studies of parallel triplexes., Results: A novel LightCycler FRET pair labelled with ATTO495 and ATTO647N was established for parallel triplex detection with antiparallel duplex as a control for the general applicability of these fluorophores for Tm determination. The ATTO fluorophores were pH stable from pH 4.5 to 7.5. Melting of triplex and duplex structures were accompanied by a large decrease in fluorescence intensity leading to well defined Tm and high reproducibility. Validation of Tm showed low intra- and inter-assay coefficient of variation; 0.11% and 0.14% for parallel triplex and 0.19% and 0.12% for antiparallel duplex. Measurements of Tm and fluorescence intensity over time and multiple runs showed great time and light stability of the ATTO fluorophores. The variance on Tm determinations was significant lower on the LightCycler platform compared to UV absorbance measurements, which enable discrimination of DNA structures with very similar Tm. Labelling of DNA probes with ATTO fluorophore increased Tm of antiparallel duplexes significantly, but not Tm of parallel triplexes., Conclusions: We have established a novel pH independent FRET pair with high fluorescence signals on the LightCycler platform for both antiparallel duplex and parallel triplex formation. The method has been thoroughly validated, and is characterized by an excellent accuracy and reproducibility. This FRET pair is especially suitable for DeltaTm and Tm determinations of pH dependent parallel triplex formation.

Objectives: To evaluate a decision support system (TREAT) for guidance of empirical antimicrobial therapy in an environment with a low prevalence of resistant pathogens., Methods: A retrospective trial of TREAT has been performed at Copenhagen University, Hvidovre Hospital. The cohort of patients included adults with systemic inflammation and suspicion of community-acquired bacterial infection. The empirical antimicrobial treatment recommended by TREAT was compared with the empirical antimicrobial treatment prescribed by the first attending clinical physician., Results: Out of 171 patients recruited, 161 (65 with microbiologically documented infections) fulfilled the inclusion criteria of TREAT. Coverage achieved by TREAT was significantly higher than that by clinical practice (86% versus 66%, P = 0.007). There was no significant difference in the cost of future resistance between treatments chosen by TREAT and those by physicians. The direct expenses for antimicrobials were higher in TREAT when including patients without antimicrobial treatment, while there was no significant difference otherwise. The cost of side effects was significantly lower using TREAT., Conclusions: The results of the study suggest that TREAT can improve the appropriateness of antimicrobial therapy and reduce the cost of side effects in regions with a low prevalence of resistant pathogens, however, at the expense of increased use of antibiotics.

Unlabelled: Streptococcus pyogenes (SP) is a common bacterial pathogen. For the past two decades, several studies have reported an increase in the severity and the incidence of SP infections., Case: a 60-year-old female admitted to the hospital with tonsillitis acuta verified by strep-A test was initially treated with V-penicillin, but developed septic shock and disseminated intravascular coagulation (DIC), and treatment was changed to cefuroxime and clindamycin. She recovered fully. This case combined with other studies illustrates that SP infections have become more severe. Clindamycin should be added to penicillin for the treatment of invasive SP infections.

Background & Aims: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide., Methods: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies., Results: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids., Conclusions: We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics.

Background: Histological similarity between sarcoidosis and tuberculosis, with epithelioid cell granulomas as the typical finding, suggests that sarcoidosis could be a specific manifestation of infection with mycobacteria. However, convincing evidence for a causal relationship between mycobacterial infection and sarcoidosis has not been produced., Objective: To examine whether mycobacteria could be cultured from mediastinal lymph nodes from patients with newly diagnosed pulmonary sarcoidosis using a prolonged culture technique., Methods: The series comprised 15 patients (10 men) with a median age of 45 years (range 25-61) and chest X-ray showing mediastinal lymphadenopathy. The diagnosis of sarcoidosis was based on biopsy specimens of lymph nodes obtained by mediastinoscopy. Lymph node tissue was processed in a prolonged (12 month duration) culture for mycobacteria. Bronchial washings were obtained by bronchoscopy and examined for mycobacteria using fluorescence microscopy, polymerase chain reaction (PCR) for Mycobacterium tuberculosis (M. tuberculosis) DNA and standard culture (7-week duration)., Results: In all lymph node specimens, prolonged culture for mycobacteria was negative. In all bronchial washings, microscopy for mycobacteria, analysis for M. tuberculosis DNA, and standard culture for mycobacteria was negative., Conclusion: Our study with prolonged culture failed to demonstrate the presence of mycobacteria in sarcoid tissue, especially M. tuberculosis and M. paratuberculosis, and does not suggest that mycobacterial infection could play a direct pathogenic role in patients with pulmonary sarcoidosis.

The culture of viable microorganisms from the blood or from cardiac tissue is currently the most important test for diagnosis of IE. This is followed by phenotypic identification methods used for taxonomic positioning of isolates. However, in those cases where the invading microorganism is difficult or impossible to culture (including instances of prior antimicrobial treatment), molecular methods provide the best means for detection. Molecular identification methods, either nucleic acid target or signal amplification alone or in combination with sequence analysis can offer a more specific and in some cases a more rapid alternative to the phenotypic methods. We propose revised Duke criteria of IE, including positive identification of an organism by molecular biology methods.

Brain tissue from 25 patients with clinically definite multiple sclerosis (MS) and as controls brain tissue from 36 patients without neurological disease was tested for the presence of human coronaviral RNA. Four PCR assays with primers specific for N-protein of human coronavirus strain 229E and three PCR assays with primers specific for the nucleocapsid protein of human coronavirus strain OC43 were performed. Sporadic positive PCR assays were observed in both patients and controls in some of the PCR assays. However, these results were not reproducible and there was no difference in the proportion of positive signals from the MS patients compared to controls. Evidence for a chronic infection with the human coronaviruses strain 229E or OC43 in brain tissue from patients with MS or controls has not been found in this study.

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.

The use of nucleic acid amplification methods in routine clinical microbiology laboratories is becoming increasingly widespread. The theory of polymerase chain reaction is described, including discussion of suitable microbal targets, extraction of nucleic acid from clinical samples, choice of primers, optimization of the process, laboratory design, contamination, and other problems as well as quality control. Other nucleic acid amplification methods such as ligase chain reaction, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification are described and the choice of technology is discussed.

The aim of this study was to compare a commercially available PCR kit (Amplicor, Roche) with our present routine analysis (SYVA EIA) for the detection of genital C. trachomatis infection in females. Furthermore, we wished to investigate the possibility of pooling samples for PCR analysis. Two hundred and sixty-eight consecutive female patients attending two gynecology clinics in Copenhagen, Denmark were included in this study. Compared to the number of samples regarded as true positives, PCR had a sensitivity of 100% (18/18) and EIA a sensitivity of 83.3% (15/18). The specificity of the PCR analysis was 99.2% (248/250) compared to 100% (250/250) for the EIA. By pooling patient samples (five patient samples in each pooled sample), a 39% reduction in reagent costs was obtained without affecting the sensitivity. In conclusion, the implementation of a standardized commercially available C. trachomatis PCR kit leads to a marked increase in analytical sensitivity compared to EIA without affecting the specificity. When pooled samples were analyzed, the cost per patient sample was reduced, but further large-scale studies are needed to rule out the possibility of a reduced sensitivity due to the dilution of individual patient samples.

The aim of this study was to apply a polymerase chain reaction for diagnosis of CMV infection and determine its clinical value in renal transplant recipients. We have applied the PCR to urine and blood specimens collected from 27 renal transplant recipients as well as blood from 49 normal blood donors. Ten of twenty-seven patients, compared to 3 of 49 normal blood donors, had CMV-DNA present in one or more samples. Six of the ten CMV-DNA-positive patients had positive CMV serology, and 3 of the 10 had severe clinical symptoms of active CMV infection. In four additional patients with positive CMV serology - but without clinical signs of active CMV infection - no CMV-DNA could be detected by the PCR. In the three patients with severe symptoms, PCR could detect CMV-DNA before serology became positive, and in one of these three patients, serology remained negative despite the patient's death from clinically obvious CMV pneumonia. PCR thus appears capable of detecting active CMV infection at a time when CMV serology is inconclusive, but cannot substitute for serology as the only routine analysis since a transient viraemia might be missed. However, in immunosuppressed patients with severe CMV infection, PCR could provide an early diagnosis, enabling the clinician to implement early specific treatment.

The etiology of sarcoidosis has not yet been established, but several mycobacterial species have been implicated in the pathogenesis of this disease. We have used a nested primer polymerase chain reaction (PCR) specific for the IS900 Mycobacterium paratuberculosis insertion element to analyse paraffin-embedded mediastinal lymphoid tissue from 18 patients with pulmonary sarcoidosis verified by mediastinoscopy. Only the positive control contained M. paratuberculosis-specific DNA. The present study does not support a role for M. paratuberculosis in the pathogenesis of sarcoidosis. However, further studies are needed in order to clarify the possible role of other mycobacteria in this disease.

The discovery of the polymerase chain reaction (PCR) enables the detection of just a few target gene copies present in almost every kind of tissue. The PCR technique uses two separated specific DNA-sequences for identification of a desired genetic sequence. This identification is followed by an almost unlimited production of the specific target gene sequence by a heat stable DNA-polymerase. The PCR technique will revolutionize several diagnostic areas, and especially the identification of virus, fungi and slowly growing bacteria will benefit from this new genetic technology. The impressive sensitivity is, however, the greatest pitfall of the technique, as just a few contaminating DNA fragments can initiate a false positive result. Great care is therefore needed when designing a PCR laboratory, as well as high demands upon the motivation and technical skills of the personnel involved. PCR is so far limited to research, but is expected to be released for routine diagnostic purposes in 1993.