Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis.

Severe sepsis is increasingly a cause of death. Rapid and correct initial antimicrobial treatment reduces mortality. The aetiological agent(s) cannot always be found in blood cultures (BCs). A novel multiplex PCR test (SeptiFast (alpha version)) that allows identification of 20 bacterial and fungal species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms were isolated with BC, and 186 microorganisms were identified with SeptiFast; 231 microorganisms were found by combining the two tests. Of the 96 isolates identified with BC, 22 isolates were considered to be contaminants. Of the remaining 74 non-contaminant BC isolates available for comparison with SeptiFast, 50 were identified as a species identical to the species identified with SeptiFast in the paired sample. Of the remaining 24 BC isolates for which the species, identified in the BC, could not be detected in the paired SeptiFast sample, 18 BC isolates were identified as a species included in the SeptiFast master list, and six BC isolates were identified as a species not included in the SeptiFast master list. With SeptiFast, 186 microorganisms were identified, 12 of which were considered to be contaminants. Of the 174 clinically relevant microorganisms identified with SeptiFast, 50 (29%) were detected by BC. More than half of the remaining microorganisms identified with SeptiFast (but not isolated after BC) were also found in routine cultures of other relevant samples taken from the patients. Future clinical studies should assess whether the use of SeptiFast is of significant advantage in the detection of bloodstream pathogens.

Coronaviruses in brain tissue from patients with multiple sclerosis.

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.

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls.

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.

A prospective, controlled, randomized study of the effect of a slow-release silver device on the frequency of urinary tract infection in newly catheterized patients.

OBJECTIVE: To test the effect on urinary tract infections (UTIs) in patients needing continuous indwelling catheterization, of a newly designed urine-collecting system containing an antibacterial device which slowly releases silver ions onto the inner surface of the system. PATIENTS AND METHODS: The study comprised a prospective controlled randomized trial; 213 patients fulfilled the inclusion criteria. They were randomized to a urine drainage system (comprising a Unometer 400 metering system or PP 2000N closed urine-bag system, both from Maersk Medical, Denmark) either with or without the antibacterial device. The efficacy was assessed as the number of UTIs and the time to infection in the 170 patients eligible for analysis. RESULTS: There were fewer UTIs in those using the system containing the antibacterial device (19% vs 24%), but the difference was not statistically significant (P < 0.05). CONCLUSION: The potential importance of different infection routes were highlighted, suggesting that modifications to Foley catheters and urine-collecting systems attempting to prevent UTIs should focus not only on the intraluminal pathway, but on the internal and external pathways of infection.

Application of nucleic acid amplification in clinical microbiology.

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.

Coronaviruses in spinal fluid of patients with acute monosymptomatic optic neuritis.

UNLABELLED: Acute monosymptomatic optic neuritis (AMON) may be an initial symptom of multiple sclerosis (MS). Coronaviruses have been implicated in the etiology of MS. The objective of the present study was to look for coronaviral RNA in AMON, which could be present in the initial stages of the development of MS. MATERIAL AND METHODS: Spinal fluids from 37 patients with AMON and 15 surgical control patients with protrusion of the intervertebral disk were assayed with a nested multiplex polymerase chain reaction with primers specific for human coronaviruses strain (HCV) 229E and OC43. RESULTS: Four patients and 1 control were positive for HCV-229E. No evidence of HCV-OC43 was found. The frequency of positive samples was low and there was no statistical difference between AMON and controls. CONCLUSION: This study does not provide evidence for an etiological role of human coronaviruses in acute monosymptomatic optic neuritis.

Random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing in the analysis of a hospital outbreak of Salmonella enteritidis.

Isolates of Salmonella Enteritidis from 81 patients from Herlev Hospital or from Copenhagen County were analysed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing. Fourteen polymorphic markers from five decamer primers unambiguously placed all isolates into six RAPD groups: 65 isolates of phagetype 6 (PFGE type I) were resolved into three RAPD groups constituting 86, 12, and 2%, respectively. A fourth RAPD group of 10 isolates was coincident with phage type 8 (PFGE type II) and two isolates, one phage-type 1, the other phage-type 4 (both PFGE type I) formed the fifth group. The sixth group of four isolates was not phage typeable and was PFGE type III. Forty outbreak-related isolates of phage-type 6 were resolved into three strains. No diversity of phage-type 6 was found among isolates unrelated to the outbreak. It is concluded that RAPD is useful as a tool in investigations of microbial outbreaks in its own right, or to supplement phage-typing and PFGE of Salmonella Enteritidis.

Three allelic forms of the human endogenous retrovirus, ERV3, and their frequencies in multiple sclerosis patients and healthy individuals.

A possible association between the endogenous retrovirus, ERV3, and multiple sclerosis (MS) was examined. Samples of DNA from 74 MS patients and 159 healthy blood donors were subjected to enzymatic amplification followed by single strand conformational analysis to detect polymorphisms in the long terminal repeats of ERV3. Using this approach we detected six single base pair variations and a drop-out of a nucleotide. The linkage pattern of these base pair variations enabled us to define three allelic forms of ERV3. Polymorphisms exclusively present in the group of patients were not found and the distribution of the three allelic forms did not differ significantly between the group of controls and the MS group. Neither was there a significant difference in the distribution of the three alleles between MS patients with the progressive form and patients with relapsing/remitting MS. Our results are not in support of an association between ERV3 and MS.

Mycobacterium paratuberculosis in intestinal tissue from patients with Crohn's disease demonstrated by a nested primer polymerase chain reaction.

BACKGROUND: The etiology of Crohn's disease remains unknown, but current research has concentrated on autoimmunity and/or mycobacterial infection. The polymerase chain reaction (PCR) enables the detection of genetic material even when very few microorganisms are present. METHODS: A nested primer PCR for detection of a multi-copy insertional element (IS900) specific for Mycobacterium paratuberculosis was applied to DNA extracted from fresh and from paraffin-embedded intestinal tissue obtained from patients undergoing surgery. RESULTS: In fresh intestinal tissue from 11 of 24 patients with Crohn's disease, from 2 of 10 patients with ulcerative colitis, and from 3 of 28 patients with other colonic disorders, specific M. paratuberculosis DNA was found. In paraffin-embedded Crohn's disease tissue the presence of specific M. paratuberculosis DNA was also increased. CONCLUSIONS: Whether the presence of M. paratuberculosis is connected to the inflammatory bowel disease or is a mere coincidence cannot be stated. We find this presence interesting and encouraging for further investigations.

Detection of Chlamydia trachomatis in individual and pooled endocervical and urethral scrapes by a commercially available polymerase chain reaction.

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.

Polymerase chain reaction as a rapid diagnostic assay for cytomegalovirus infection in renal transplant patients.

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.

Construction of a DNA amplification assay for detection of Legionella species in clinical samples.

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.

Search for an HTLV-I-like retrovirus in patients with MS by enzymatic DNA amplification.

A retroviral etiology to MS has been sought for some time, so far reports of retroviral presence have not been confirmed by other groups. DNA was isolated from peripheral blood mononuclear cells from 67 patients with MS and brain capillaries from six patients with MS. Enzymatic amplification by the polymerase chain reaction was conducted with ten primer sets homologous to highly conserved HTLV-I/HTLV-II genetic sequences at stringent and non-stringent annealing conditions. No HTLV I/II related DNA fragments were seen judging from hybridization to an HTLV-I probe, even at relaxed conditions. The present study does not support a role for a HTLV-I-like virus in MS.

Search for Mycobacterium paratuberculosis DNA in tissue from patients with sarcoidosis by enzymatic gene amplification.

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.