Malcolm Guiver

Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR

ABSTRACT 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. pneumoniaewere 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrAprimers 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 thebexA primers amplified H. influenzaetypes 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.

Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines

Adoptive transfer of CMV-specific T cells offers the potential for reconstitution of viral immunity after allogeneic transplantation. However, the logistics of producing virus-specific T-cell clones has limited the application of cellular therapies. We treated 16 patients for CMV infection with polyclonal CMV-specific T-cell lines generated by short-term culture. Massive in-vivo expansions of CMV-specific cytotoxic T lymphocytes were observed, resulting in reconstitution of viral immunity. In eight cases antiviral drugs were not required, and subsequent episodes of reactivation occurred in only two patients. Our findings indicate that application of CMV-specific cell lines is both feasible and effective in a clinical environment.

Non-culture diagnosis and serogroup determination of meningococcal B and C infection by a sialyltransferase ( siaD ) PCR ELISA

Rapid, non-culture, serogroup determination of meningococcal infection is important in contact management where vaccination may be possible. The impending availability of polysaccharide-protein conjugate vaccines for serogroup C disease requires maximal case ascertainment, with serogroup determination, at a time when the number of culture confirmed meningococcal infections is decreasing. A polymerase chain reaction assay (PCR), based on a restriction fragment length polymorphism (RFLP) in the meningococcal serogroup B and C sialytransferase (siaD) gene, was developed to combine the non-culture diagnosis of meningococcal infection from CSF, whole blood and serum with serogroup (B and C) identification. The PCR assay was adapted to an ELISA format incorporating hybridization with serogroup-specific B and C oligonucleotide probes. Specificity for CSFs was 100% and sensitivities were respectively 81, 63 and 30% for CSFs, whole blood and sera. The serogroup-specific PCR ELISA is a significant addition to currently available tests for non-culture diagnosis of meningococcal infection and outbreak investigation.

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 ≥8). Results: Median admission bacterial load was 1.6 × 106 DNA copies/ml of blood (range 2.2 × 104 to 1.6 × 108). Bacterial load was significantly higher in patients with severe (8.4 × 106) compared to milder disease (1.1 × 106, p = 0.018). This difference was greater in septicaemic patients (median 1.6 × 107 versus 9.2 × 105, 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 ≥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.

High pneumococcal DNA loads are associated with mortality in malawian children with invasive pneumococcal disease

Background: In bacteremia owing to Streptococcus pneumoniae, high bacterial counts at presentation have been shown to be predictive of the development of serious invasive disease. Using real-time PCR, we aimed to determine pneumococcal DNA loads in blood and CSF, and their relationship to cytokine concentrations, clinical presentation and outcome. Methods: Children with confirmed meningitis (n = 82) or pneumonia (n = 13) were prospectively recruited, and blood and CSF samples taken for pneumococcal bacterial DNA loads and cytokine determination. Results: At the time of admission, the median bacterial load in blood was 1.6 × 103 DNA copies/mL (range 0.00–1.54 × 106) and in CSF it was 5.77 × 107 DNA copies/mL (range 4.42 × 102 to 6.15 × 108). Median blood and CSF bacterial loads (log DNA copies/mL) were significantly higher in nonsurvivors than in survivors; blood (3.80 vs. 2.97, P = 0.003), CSF (8.17 vs. 7.50, P = 0.03). In HIV-infected children (n = 59), blood and CSF loads and plasma tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6 and IL-10 were all significantly higher in nonsurvivors than in survivors, but in HIV-uninfected children (n = 36) this difference was not significant. Blood bacterial loads and plasma cytokine concentrations were significantly associated, and were all significantly higher in children with meningitis than in those with pneumonia. In children with meningitis, median CSF cytokine concentrations were significantly higher than median plasma cytokine concentrations (P < 0.001) and CSF bacterial loads were significantly associated with CSF IL-1β (P = 0.002) and IL-10 (P = 0.001) concentrations. Conclusions: Pneumococcal DNA loads are associated with plasma cytokine concentrations, and are higher in meningitis than in pneumonia. High blood and CSF pneumococcal DNA loads are associated with a fatal outcome.

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.

Invasive meningococcal disease in England and Wales: implications for the introduction of new vaccines.

A number of meningococcal vaccines have either been recently licensed or are in late-phase clinical trials. To inform national vaccination policy, it is important to define the burden of disease and the potential impact of any new vaccine. This study describes the epidemiology of invasive meningococcal disease across all age groups in England and Wales for recent epidemiological years between 2006 and 2010. The Health Protection Agency (HPA) conducts enhanced national meningococcal surveillance through a combination of clinical and laboratory reporting. Between 2006/07 and 2010/11, the average annual incidence of invasive meningococcal disease across all age groups was 2.0/100,000. Capsular group B (MenB) accounted for 87% (4777/5471) cases, with an overall incidence of 1.8/100,000. The highest MenB incidence observed among infants (36.2/100,000) where cases increased from birth to 5 months of age then gradually declined. An annual average of 245 MenB cases occurred in infants (135 in those aged ≤ 6 months) representing 26% (and 14%) of all MenB cases, respectively. After infancy, MenB rates declined until the age of 12 years, rising to a second smaller peak at 18 years. MenB case fatality ratio (CFR) was 5.2% (247/4777 cases) overall and was highest among ≥ 65 year-olds (28/161; 17.4%). The largest number of deaths (n=125), however, occurred among <5 year-olds. Clonal complexes cc269 and cc41/44 each accounted for around a third of cases across the age groups. Other capsular groups rarely caused invasive disease, although capsular group Y (MenY) cases more than doubled from 35 in 2006/07 to 86 in 2010/11. Thus, universal meningococcal vaccination with an effective broad-spectrum formulation has potential to prevent most disease, particularly if the vaccine is immunogenic early in infancy, but, there is currently little justification for routine quadrivalent ACWY conjugate vaccination in the UK, although the increase in MenY disease warrants continued surveillance.

Interlaboratory Comparison of PCR-Based Identification and Genogrouping of Neisseria meningitidis

ABSTRACT Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCR-based nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

Characterization of fHbp, nhba (gna2132), nadA, porA, and sequence type in group B meningococcal case isolates collected in England and Wales during January 2008 and potential coverage of an investigational group B meningococcal vaccine.

ABSTRACT Invasive disease caused by meningococcal capsular groups A, C, W-135, and Y is now preventable by means of glycoconjugate vaccines that target their respective polysaccharide capsules. The capsule of group B meningococci (MenB) is poorly immunogenic and may induce autoimmunity. Vaccines based on the major immunodominant surface porin, PorA, are effective against clonal epidemics but, thus far, have a limited scope of coverage against the wider MenB population at large. In an alternative approach, the first-generation, investigational, recombinant MenB (rMenB) plus outer membrane vesicle (OMV) (rMenB-OMV) vaccine contains a number of relatively conserved surface proteins, fHBP, NHBA (previously GNA2132), and NadA, alongside PorA P1.4-containing OMVs from the New Zealand MeNZB vaccine. MenB currently accounts for approximately 90% of cases of meningococcal disease in England and Wales. To assess potential rMenB-OMV vaccine coverage of pathogenic MenB isolates within this region, all English and Welsh MenB case isolates from January 2008 (n = 87) were genetically characterized with respect to fHBP, NHBA, NadA, and PorA. Alleles for fHbp, nhba, and porA were identified in all of the isolates, of which 22% were also found to harbor nadA alleles. On the basis of genotypic data and predicted immunological cross-reactivity, the potential level of rMenB-OMV vaccine coverage in England and Wales ranges from 66% to 100%.

Severity of Meningococcal Disease Associated with Genomic Bacterial Load

BACKGROUND Diagnostic polymerase chain reaction (PCR) detection of Neisseria meningitidis has enabled accurate quantification of the bacterial load in patients with meningococcal disease. METHODS Quantification of the N. meningitidis DNA level by real time-PCR was conducted on whole-blood samples obtained from patients presenting with meningococcal disease to hospitals throughout England and Wales over a 3-year period. Levels were correlated with clinical outcome, infecting serogroup, and host factors including, interleukin-1 genotype (IL-1). RESULTS Bacterial loads were available for 1045 patients and were not associated with the age of the patient, delay in sample submission, or administration of antibiotics prior to admission. The median log bacterial load was higher in 95 patients who died (5.29 log(10)copies/mL; interquartile range, 4.41-6.30 log(10)copies/mL) than in 950 patients who survived (3.79 log(10)copies/mL; interquartile range, 2.87-4.71 log(10)copies/mL). Logistic regression revealed that age (odds ratio, 1.04 per 1-year increase in age) and bacterial load (odds ratio, 2.04 per log(10)-copies/mL increase) had a statistically significant effect on the risk of death. Infection with N. meningitidis serogroup C was associated with increased risk of death and an increased bacterial load. Also associated with a higher bacterial load were prolonged hospitalization (duration, >10 days); digit, limb, or soft-tissue loss; and requirement of hemodialysis. Carriage of IL-1RN(+2018) was associated with increased mortality (odds ratio, 2.14; P=.07) but not with a higher bacterial load. CONCLUSIONS In meningococcal disease, bacterial load is associated with likelihood of death, development of permanent disease sequelae, and prolonged hospitalization. The bacterial load was relatively higher in patients infected with N. meningitidis serogroup C than in those infected with other serogroups. The effects of age and IL-1 genotype on mortality are independent of a high genomic bacterial load.