A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Targeted case finding for hepatitis B using dry blood spot testing in the British-Chinese and South Asian populations of the North-East of England.

Chronic infection with the hepatitis B virus (HBV) is a frequent cause of cirrhosis and liver cancer. Targeted HBV screening is recommended by the Centre for Disease Control (CDC) and Prevention for subjects born in countries with >2% HBV prevalence. However, there are no UK guidelines. Here, we applied the (CDC) recommendations to the British-Chinese and British-South Asian community of North-East (NE) England. British-Chinese and South Asian subjects were invited to attend for HBV education and screening sessions held in community centres. Hepatitis B surface antigen (HBsAg) and hepatitis B core total antibody (HBcAb) were tested with dry blood spot tests. South Asians were also tested for hepatitis C antibody (HCVAb). A total of 1126 subjects (606 Chinese and 520 South Asian) were screened. Sixty-two (5.5%) were HBsAg positive. Ten of these reported a previous diagnosis of HBV. The prevalence of HBsAg positivity was 4.6% when previously diagnosed individuals were excluded. The HBsAg prevalence was significantly higher in the Chinese subjects compared with South Asians (8.7% VS. 1.7% P < 0.001). In Chinese subjects, HBsAg positivity was highest in subjects born in Vietnam (17.4%), followed by China (11%), Hong Kong (7.8%) and the UK (6.7%). Subjects from Pakistan had the highest HBsAg and HCV Ab prevalence in the South Asians (3.1% and 1.8%, respectively). Ten percentage of HBsAg positive patients who had follow-up assessment had active disease requiring antiviral treatment. Undiagnosed HBV infection was above the 2% threshold for screening suggested by the CDC in the British-Chinese and Pakistani community of NE England, which provides evidence for a UK HBV-targeted screening programme.

Clinical features, aetiology and outcome of empyema in children in the north east of England.

BACKGROUND: The incidence of empyema in children in the UK is increasing. The reason for this is unclear. A prospective study was undertaken to investigate the clinical features, aetiology, and outcome of cases of empyema and parapneumonic effusion presenting to a tertiary paediatric respiratory centre between February 1997 and August 2001. METHOD: Routine bacterial culture of blood and pleural fluid was performed for 47 cases. Forty three pleural fluid specimens, culture negative for pneumococcus, were analysed for pneumococccal DNA by real time polymerase chain reaction (PCR). Penicillin susceptibility was determined for DNA positive specimens using complementary PCR assay. Capsular serotype specific antigen detection was by enzyme immunoassay (EIA) using monoclonal antibodies to serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Clinical data were obtained from patient notes, supplemented by a postal questionnaire. RESULTS: The median (range) age of the patients was 5.6 (0.6-16.9) years and 70% were male. The median (range) duration of illness before referral to hospital was 5 (0-25) days. Forty five (96%) had received antibiotics before referral; 32 (68%) required decortication and eight (21%) thoracocentesis. Median postoperative stay was 4 days (2-8). Thirty two (75%) pneumococcal culture negative specimens were pneumococcal DNA positive; 17 (53%) of these were serotype 1. All were penicillin sensitive. CONCLUSIONS: Pneumococcus is the major pathogen in childhood empyema and serotype 1 is the prevalent serotype. This has implications for vaccine development and immunisation strategy as the current 7-valent pneumococcal conjugate vaccine does not protect against serotype 1.

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.