A Large, Refractory Nosocomial Outbreak of Klebsiella pneumoniae Carbapenemase-Producing Escherichia coli Demonstrates Carbapenemase Gene Outbreaks Involving Sink Sites Require Novel Approaches to Infection Control.

Carbapenem-resistant Enterobacteriaceae (CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs. We investigated a large Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli outbreak and wider CRE incidence trends in the Central Manchester University Hospital NHS Foundation Trust (CMFT) (United Kingdom) over 8 years, to determine the impact of infection prevention and control measures. Bacteriology and patient administration data (2009 to 2017) were linked, and a subset of CMFT or regional hospital KPC-producing E. coli isolates (n = 268) were sequenced. Control interventions followed international guidelines and included cohorting, rectal screening (n = 184,539 screens), environmental sampling, enhanced cleaning, and ward closure and plumbing replacement. Segmented regression of time trends for CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis (n = 268 isolates) identified the spread of a KPC-producing E. coli outbreak clone (strain A, sequence type 216 [ST216]; n = 125) among patients and in the environment, particularly on 2 cardiac wards (wards 3 and 4), despite control measures. ST216 strain A had caused an antecedent outbreak and shared its KPC plasmids with other E. coli lineages and Enterobacteriaceae species. CRE acquisition incidence declined after closure of wards 3 and 4 and plumbing replacement, suggesting an environmental contribution. However, ward 3/ward 4 wastewater sites were rapidly recolonized with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-producing E. coli outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and the persistence of blaKPC in E. coli, including pathogenic lineages, are of concern.

Comparison of Binax NOW urine antigen test and pneumococcal DNA assay using qPCR before and after nasopharyngeal swabbing in healthy Malawian children.

Diagnosis of invasive pneumococcal disease is challenging. We compared Binax NOW pneumococcal urinary antigen test with blood pneumococcal PCR in healthy Malawian children with and without pneumococcal carriage, and we found a high false-positive rate with Binax NOW. Blood pneumococcal PCR positivity was 66/88 (75%) compared to 5/27 (18%) when nasopharyngeal swabbing was performed first compared to after blood sampling for pneumococcal blood PCR. We speculate that nasopharyngeal swabbing may be causing a breach of mucosal integrity, leading to invasion into the bloodstream. These findings need to be confirmed with autolysin-based PCR assays.

MBL2 deficiency is associated with higher genomic bacterial loads during meningococcemia in young children.

Mannose binding lectin (MBL2) is a soluble pattern recognition receptor that is key to generating innate immune responses to invasive infection, including against the cardinal Gram-negative bacterium Neisseria meningitidis. Individuals homozygous or heterozygous for any of three variant alleles of MBL2 (O/O or A/O genotypes) have deficient concentrations of MBL2 in circulating blood, but previous studies linking MBL deficiency to susceptibility to meningococcal disease have not revealed a consistent association. We genotyped 741 patients with microbiologically-proven meningococcal disease and correlated MBL2 genotype with plasma bacterial load of N. meningitidis with blood samples taken during hospital admission. We show that individuals with genotypes compatible with MBL2 deficiency have higher measurable levels of bacterial plasma genomic load with the greatest effect seen in children <2 years of age. However, the overall impact of this is minor, because there was no evidence that such genotypes are more common in children with meningococcal disease compared with uninfected cohorts. The findings suggest that MBL2 supports innate immune defence against meningococcal disease in the early months of life, before acquired immunity is sufficiently robust for effective natural protection.

Detection of herpes viruses in the cerebrospinal fluid of adults with suspected viral meningitis in Malawi.

PURPOSE: We looked for herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA in Malawian adults with clinically suspected meningitis. METHODS: We collected cerebrospinal fluid (CSF) from consecutive adults admitted with clinically suspected meningitis to Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi, for a period of 3 months. Those with proven bacterial or fungal meningitis were excluded. Real-time polymerase chain reaction (PCR) was performed on the CSF for HSV-1 and HSV-2, VZV, EBV and CMV DNA. RESULTS: A total of 183 patients presented with clinically suspected meningitis. Of these, 59 (32 %) had proven meningitis (bacterial, tuberculous or cryptococcal), 39 (21 %) had normal CSF and 14 (8 %) had aseptic meningitis. For the latter group, a herpes virus was detected in 9 (64 %): 7 (50 %) had EBV and 2 (14 %) had CMV, all were human immunodeficiency virus (HIV)-positive. HSV-2 and VZV were not detected. Amongst those with a normal CSF, 8 (21 %) had a detectable herpes virus, of which 7 (88 %) were HIV-positive. CONCLUSIONS: The spectrum of causes of herpes viral meningitis in this African population is different to that in Western industrialised settings, with EBV being frequently detected in the CSF. The significance of this needs further investigation.

Chronic norovirus infection in pediatric hematopoietic stem cell transplant recipients: a cause of prolonged intestinal failure requiring intensive nutritional support.

Norovirus infection is a major cause of nonbacterial gastroenteritis. In immunocompetent individuals the illness caused by norovirus is mostly self limiting. Excretion of norovirus has been reported to be prolonged in the immunocompromised including adult HSCT recipients. We report a case series of 13 children who received HSCT and required prolonged parenteral and enteral nutrition due to severe gut dysfunction accompanying protracted norovirus excretion that was monitored by RT-PCR. The median duration of viral excretion was 150 days (range 60-380) and the eventual clearance of norovirus from feces was closely associated with donor T cell recovery in the peripheral blood. There was no disease manifestation beyond the gut but the severity and length of norovirus associated illness suggests that HSCT should be delayed where possible in patients excreting the virus prior to conditioning therapy.

Continued emergence and changing epidemiology of oseltamivir-resistant influenza A(H1N1)2009 virus, United Kingdom, winter 2010/11.

During the winter period 2010/11 27 epidemiologically unlinked, confirmed cases of oseltamivir-resistant influenza A(H1N1)2009 virus infection have been detected in multiple, geographically dispersed settings. Three of these cases were in community settings, with no known exposure to oseltamivir. This suggests possible onward transmission of resistant strains and could be an indication of a possibility of changing epidemiology of oseltamivir-resistant influenza A(H1N1)2009 virus.

Active CMV disease does not always correlate with viral load detection.

The use of quantitative cytomegalovirus (CMV) real-time polymerase chain reaction (RT-PCR) and preemptive ganciclovir therapy is replacing prophylaxis as the management of choice in high-risk patients undergoing stem cell transplantation (SCT). However, there are limited data defining its role in this setting. In the current retrospective single-centre study, quantitative RT-PCR was used to determine CMV in 577 consecutive patients undergoing SCT (172 allogeneic and 405 autologous) over a 5-year period. CMV RT-PCR was performed weekly until cessation of immunosuppression (allogeneic) or for 30 days post-SCT (autologous). Treatment was commenced after two consecutive positive results or a high copy on the first occasion (> 1000 copies/ml, > 3 log). The overall CMV reactivation rate in patients undergoing allogeneic SCT was 30%, with reactivation observed in 72% of high-risk patients (recipient positive patients). CMV end-organ disease was observed in eight patients (1%); of these, four were CMV RT-PCR negative at the time of diagnosis of end-organ CMV disease, with three remaining negative throughout the course of the disease. CMV-related mortality was recorded in three patients. The current data support a preemptive treatment strategy-based CMV RT-PCR, but indicate that in symptomatic patients, a negative CMV PCR result does not exclude CMV end-organ disease.

Pulmonary aspergillosis in a patient with chronic granulomatous disease: confirmation by polymerase chain reaction and serological tests, and successful treatment with voriconazole.

A 25-year-old male with chronic granulomatous disease developed severe bilateral pulmonary aspergillosis confirmed by a positive result of polymerase chain reaction for Aspergillus species from sputum and positive serological tests for Aspergillus. He was successfully treated with voriconazole. The diagnostic difficulties and the new therapeutic options in the era of new antifungal agents are discussed.

Diagnosis of invasive pneumococcal infection by PCR amplification of Streptococcus pneumoniae genomic fragments in blood: a multi-centre comparative study.

The increasing interest in the prevention of pneumococcal disease by immunisation necessitates improved organism-specific surveillance. This is particularly the case with regard to the contribution of Streptococcus pneumoniae infection to community-acquired pneumonia where blood cultures are often negative and sputum culture results ambiguous. Examination by PCR of blood samples taken at hospital admission offers one possibility for such improvement. The sensitivity, specificity and convenience of three pneumolysin gene PCR assays were compared in a large study, using EDTA blood from 175 patients (95 with proven pneumococcal bacteraemia, 80 with bacteraemia due to other organisms). The assays used were a PCR-enzyme immunoassay, a hybridisation probe assay run on the Roche LightCycler and a hydrolysis probe (TaqMan) assay run on an ABI 7700. Overall samples from only 57% of patients with bacteraemic pneumococcal infection yielded a positive result in at least one assay. Individual sensitivities ranged from 45% (TaqMan/ABI) through 35% (PCR-EIA) to 21% (Hybridisation/LightCycler). Specificity (PCR negative in the 80 control patients) ranged from 97-100%. The TaqMan/ABI assay was run in two centres and concordance between results was 91.4%, discrepancies being associated with very weakly positive samples. Overall, the TaqMan/ABI was the most sensitive and convenient assay; however, this method does not appear to offer any significant improvement over conventional blood cultures and is unlikely to be sufficiently sensitive to confirm a pneumococcal aetiology for non-bacteraemic pneumococcal pneumonia. For the present, therefore, blood culture is the preferred option.

Improved case confirmation in meningococcal disease with whole blood Taqman PCR.

BACKGROUND: The clinical diagnosis of meningococcal disease (MCD) can be difficult. Non-culture methods like the previous ELISA meningococcal PCR improved case confirmation rates, but were not ideal. A Taqman meningococcal PCR, using DNA extracted from serum (S-Taqman), which has an improved sensitivity compared to the ELISA method in vitro, was introduced into clinical practice in July 1997. A new whole blood DNA extraction method for Taqman (WB-Taqman) was introduced in September 1999. AIMS: To determine the degree of improvement in the confirmation rate in clinically diagnosed MCD, following the introduction of WB-Taqman. METHODS: A total of 192 patients (WB-Taqman) with possible or probable MCD, including those admitted to our paediatric intensive care unit, were studied. Admission EDTA samples obtained were sent for bacterial DNA detection at the Meningococcal Reference Unit (MRU), Manchester. These patients were compared to 319 patients with possible and probable MCD, seen at the same hospital prior to the introduction of WB-Taqman. RESULTS: Following the introduction of WB-Taqman, 82 of the 95 probable cases (88%) had a positive meningococcal PCR result. This gives a diagnostic sensitivity and specificity for WB-Taqman of 87% and 100% respectively. Following WB-Taqman all blood culture positive patients were also PCR positive. Confirmation of cases by PCR rose from 47% (S-Taqman, n = 166) to 88% (WB-Taqman). When all confirmatory tests were included, case confirmation increased from 72% (S-Taqman) to 94% (WB-Taqman). CONCLUSION: The sensitivity of PCR in confirming clinical MCD has improved significantly with this new method. The gold standard for confirming cases of MCD is now the WB-Taqman PCR.

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay.

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.

Meningococcal bacterial DNA load at presentation correlates with disease severity.

AIMS: To determine bacterial loads in meningococcal disease (MCD), their relation with disease severity, and the factors which determine bacterial load. METHODS: Meningococcal DNA quantification was performed by the Taqman PCR method on admission and sequential blood samples from patients with MCD. Disease severity was assessed using the Glasgow Septicaemia Prognostic Score (GMSPS, range 0-15, severe disease > or =8). RESULTS: Median admission bacterial load was 1.6 x 10(6) DNA copies/ml of blood (range 2.2 x 10(4) to 1.6 x 10(8)). Bacterial load was significantly higher in patients with severe (8.4 x 10(6)) compared to milder disease (1.1 x 10(6), p = 0.018). This difference was greater in septicaemic patients (median 1.6 x 10(7) versus 9.2 x 10(5), p < 0.001). Bacterial loads were significantly higher in patients that died (p = 0.017). Admission bacterial load was independent of the duration of clinical symptoms prior to admission, with no difference between the duration of symptoms in mild or severe cases (median, 10.5 and 11 hours respectively). Bacterial loads were independent of DNA elimination rates following treatment. CONCLUSION: Patients with MCD have higher bacterial loads than previously determined with quantitative culture methods. Admission bacterial load is significantly higher in patients with severe disease (GMSPS > or =8) and maximum load is highest in those who die. Bacterial load is independent of the duration of clinical symptoms or the decline in DNA load.

Evaluation of CMV viral load using TaqMan CMV quantitative PCR and comparison with CMV antigenemia in heart and lung transplant recipients.

BACKGROUND: Quantitative assessment of cytomegalovirus (CMV) infection using the antigenemia test has been used to monitor CMV infection in heart and lung transplant patients enabling a preemptive treatment strategy. However, the method is labour intensive, samples have to be processed within a few hours and requires skilled interpretation. A comparative prospective evaluation of a real-time TaqMan CMV quantitative PCR (QPCR) with the CMV antigenemia was undertaken. METHODS: A real-time quantitative TaqMan CMV PCR from EDTA bloods was developed. In this study 25 heart transplant and single-lung transplant patients were monitored posttransplantation by antigenemia and TaqMan CMV QPCR. CMV DNA extracted from EDTA blood was amplified by TaqMan QPCR using primers and probe designed from the CMV glycoprotein B (gB) gene. Quantification of the genome copies is extrapolated from a standard curve generated from amplification of quantified standards. RESULTS: Antigenaemia levels and TaqMan CMV QPCR genome copies showed a linear correlation between the two assays (R=0.843, P=0.001). A clinically significant threshold of 50 CMV pp65 antigen positive polymorphonuclear leucocytes (PMNLs) per 200 000 cells previously reported was used to extrapolate an equivalent value of 40 000 (log 4.6) genome copies per ml of blood for the TaqMan CMV QPCR. CONCLUSIONS: The TaqMan system enables a rapid high-throughput of samples. The TaqMan CMV QPCR can be used as an accurate and robust alternative to the antigenemia test to predict CMV disease and to monitor effectiveness of treatment.

Rapid identification of candida species by TaqMan PCR.

AIM: To develop and evaluate a TaqMan(TM) polymerase chain reaction (PCR) for the rapid identification and speciation of candida species. METHODS: Species specific primer and probe sets were designed for the identification of Candida albicans, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, and C. glabrata from clinical isolates in a 5' exonuclease (TaqMan(TM)) assay. The probes were labelled with three fluorescent dyes to enable differentiation between species when three primer and probe sets were combined in two multiplexes. The specificity of these assays was evaluated against a range of National Collection of Pathogenic Fungi strains, clinical isolates of yeast, bacterial and viral pathogens. RESULTS: The primer and probe sets have been shown to be 100% specific for their respective species; there was no crossreaction between any set and human DNA, or extracts from other candida species, fungal, bacterial, or viral pathogens tested. Extracts from two clinical isolates, originally identified as C albicans on the basis of germ tube formation, were not amplified by any of the primer and probe sets. These isolates have been putatively re-identified as C dubliniensis after sequencing of the variable internal transcribed spacer region ITS2 and lack of growth at 45 degrees C. CONCLUSION: This TaqMan assay provides a rapid alternative to conventional culture based techniques for the identification and speciation of the most frequently isolated candida species. The simple extraction method followed by TaqMan PCR can identify the six species mentioned in four hours.

Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus.

A novel real-time quantitative PCR (QPCR) assay is described for monitoring CMV DNA load in clinical specimens using the LightCycler. The assay is rapid (< 40 min), easy to carry out, robust, reliable and is capable of detecting from 10 to over 2 x 10(5) CMV DNA copies with a wide linear range. Amplification and detection occur simultaneously, avoiding the need for post-PCR analysis and thereby minimising the risk of carryover contamination. The assay proved to be accurate, specific and reproducible when evaluated in three different laboratories. In addition, LightCycler results were comparable with those of TaqMan, an independent real-time QPCR assay.

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.

A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

Screening for cytomegalovirus (CMV) infection in allogeneic bone marrow transplantation using a quantitative whole blood polymerase chain reaction (PCR) method: analysis of potential risk factors for CMV infection.

Potential risk factors for CMV infection and the use of quantitative CMV PCR screening to guide pre-emptive anti-CMV therapy were reviewed retrospectively in 32 allogeneic bone marrow transplant patients accrued over a 2-year period. Significant CMV PCR positivity (an indicator of CMV infection) developed in 34% of patients. When analysed by recipient CMV IgG serostatus, 69% of seropositive recipients developed significant CMV PCR positivity while none of the seronegative recipients did so (P = 0.00007). Considering only the seropositive recipients, 100% of those who received the low intensity campath-1H/fludarabine/melphalan 'mini-allograft' conditioning regimen developed significant CMV PCR positivity, while only 44% of those who had received cyclophosphamide/TBI did so (P = 0.0337). The mean time to first episode of significant CMV PCR positivity for those who had received campath/fludarabine/melphalan was 25 days while for those who had received cyclophosphamide/TBI, this was 66 days (P = 0.0372). For the first episode of significant CMV PCR positivity, the mean index and peak CMV PCR counts for those who had received campath/fludarabine/melphalan were 4.54 and 5.22 log copies/ml respectively, while for cyclophosphamide/TBI, the corresponding figures were 3.85 and 4.12 log copies/ml respectively (P = 0.2986 and P = 0.0472 for index and peak values). 85% of those who had significant CMV PCR positivity with the campath/fludarabine/melphalan regimen developed more than one such episode, while 50% of those receiving cyclophosphamide/TBI regimen did so (P = 0.491). Significant CMV PCR positivity was associated with symptoms in a proportion of patients (pyrexia 45%, cough 18%, rise in AST 72%). No patient developed overt CMV disease. CMV PCR is useful for guiding pre-emptive anti-CMV therapy and for monitoring response.

PCR diagnosis.

Nonculture diagnosis is of increasing importance in maximizing case ascertainment of disease owing to Neisseria meningitidis (1). In the United Kingdom (UK), greater use of pre-admission antibiotics has lead to a steady decline in the total number of cases confirmed by culture, compared to the number reported to the Office for National Statistics (ONS). In addition, since the introduction of serogroup C oligosaccharide-protein conjugate vaccine (2) in the UK and its imminent introduction elsewhere, it is necessary to maximize case ascertainment to determine the true level of disease in the population and establish the impact of vaccination programs. Although serodiagnosis is available for confirmation, results are retrospective and often inconclusive (1). Amplification by polymerase chain reaction (PCR) provides a rapid, highly sensitive, and specific method for detecting meningococcal DNA from clinical samples. A number of assays have been described, some of which provide additional information about serological markers such as serogroup, serotype, and serosubtype (3-10). The introduction of PCR at the UK Public Health Laboratory Service (PHLS) Meningococcal Reference Unit (MRU) has resulted in a dramatic increase in the number of confirmed cases of meningococcal disease (11). In 1998 an additional 56% of cases were confirmed by PCR alone compared to those confirmed by culture only.

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.