Background: In adults, viral causes of communityacquired pneumonia (CAP) are poorly characterised. The aims of this study were to characterise the viral aetiology of CAP in adults by using an extensive array of viral diagnostic tests and to compare the characteristics of viral pneumonia with those of pneumococcal pneumonia. Methods: Adults admitted to Christchurch Hospital over a 1-year period with CAP were included in the study. Microbiological testing methods included blood and sputum cultures, urinary antigen testing for Streptococcus pneumoniae and Legionella pneumophila, antibody detection in paired sera and detection of respiratory viruses in nasopharyngeal swabs by immunofluorescence, culture and PCR.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, but it is undoubtedly underdiagnosed. We used a nested PCR assay (targeting the pneumolysin gene) to detect S. pneumoniae DNA in multiple sample types from 474 adults with community-acquired pneumonia and 183 control patients who did not have pneumonia. Plasma or buffy coat samples were PCR positive in only 6 of the 21 patients with positive blood cultures for S. pneumoniae and in 12 other patients (4 of whom had no other laboratory evidence of S. pneumoniae infection). Buffy coat samples from two control patients (neither having evidence of S. pneumoniae infection), but no control plasma samples, were PCR positive. Although pneumococcal antigen was detected in the urine from 120 of 420 (29%) patients, only 4 of 227 (2%) urine samples tested were PCR positive. Overall, 256 of 318 (81%) patients had PCR-positive sputum samples, including 58 of 59 samples from which S. pneumoniae was cultured. Throat swab samples from 229 of 417 (55%) patients were PCR positive and, in those who produced sputum, 96% also had positive PCR results from sputum. Throat swabs from 73 of 126 (58%) control patients were also PCR positive. We conclude that the pneumolysin PCR assay adds little to existing diagnostic tests for S. pneumoniae and is unable to distinguish colonization from infection when respiratory samples are tested.

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PCR-based methodologies offer increased sensitivity for the detection of most respiratory viruses in young children. The inclusion of PCR into diagnostic testing strategies is needed to broaden our understanding of the natural ecology of respiratory viruses and the significance of multiple infections.

Real-time PCR with melting curve analysis of PCR products is a rapid procedure for detecting and genotyping herpes simplex virus (HSV). When testing mucocutaneous samples for HSV by a real-time PCR assay targeting the DNA polymerase gene, we found that some PCR products had atypical melting curves that did not conform to the expected melting temperatures for HSV type 1 or 2. Sequence analysis showed that these strains had base-pair mismatches over the probe binding sites. An alternative assay is required to type such atypical isolates.The development of real-time PCR assays has provided a rapid alternative to traditional viral cell culture isolation for detection and typing of herpes simplex virus (HSV). (3-6, 8) These assays enable detection of HSV within a few hours in a closed system that reduces the risk of contamination by amplicon carryover. Furthermore, genotyping is possible through analysis of the melting curve of the PCR product, with each genotype being distinguished by a different melting temperature (T m ).While evaluating real-time PCR for detection of HSV in clinical samples, we noticed that some HSV PCR products had atypical melting curves that did not conform to the expected T m for HSV type 1 or HSV type 2. These samples and amplicons were further analyzed to determine the reasons for these findings.For detection of HSV in mucocutaneous samples, we use a modification of the LightCycler PCR assay developed by Olfert Landt (TIB Molbiol, Berlin, Germany) and described by Burrows et al. (3) This assay targets the HSV DNA polymerase gene, and our modification involves the use of 4.0 mM MgCl 2 , a 0.5 M concentration of forward primer, a 0.1 M concentration of reverse primer (asymmetric primers to minimize the hook effect [2]), and a 0.2 M concentration of each probe. All PCRs were carried out by using the LightCyclerFastStart-DNA Master Hybridization Probe kit (Roche Diagnostics).PCR products were analyzed by using the LightCycler melting curve analysis software and designated HSV type 1 or type 2 based on the T m . The melting curve process involves the continuous acquisition of fluorescence as the temperature is steadily increased from 55 to 90°C. The presence of two mismatch base pairs over the LightCycler 640 labeled probe binding site for HSV type 1 causes the probe to separate at a lower temperature (59°C) than for HSV type 2 (72°C), which has a perfect match (Fig. 1A).Of the 745 genital and cutaneous samples that we tested, 266 (36%) were determined to be positive for HSV. Of the positive samples, 124 (46.6%) were found to be positive for HSV type 1, 127 (47.7%) were found to be positive for HSV type 2, and 15 (5.7%) failed to exhibit the expected melt curve for HSV type 1 or HSV type 2. The atypical melting curves from the 15 samples conformed to six different T m patterns (Fig. 1).We sequenced the PCR product from each representative pattern by using Megabase (Amersham Pharmacia Biotech) at the Waikato Sequencing Facility (Waikato University, Hamilton, New Zealand). CLUSTALX (v.1.81) align...

We directly compared a conventional hemi-nested PCR assay with a real-time (LightCycler) PCR assay for the detection of Bordetella pertussis in nasopharyngeal samples. Of the 152 samples tested, 49 (32%) were positive by first-round conventional PCR, 56 (37%) were positive by the hemi-nested PCR assay, and 39 (26%) were positive by the real-time assay. All samples testing positive with the real-time assay were also positive by the hemi-nested assay (both first- and second-round PCR), and all culture-positive samples tested positive by both PCR assays. These results suggest that the hemi-nested assay is more sensitive than the real-time assay for detecting B. pertussis.

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To establish that in Canterbury, New Zealand, women over the age of 40 with biopsy confirmed high grade squamous intraepithelial lesions (HSIL) have human papilloma virus (HPV) detectable by the Hybrid Capture 2 (HC2) test. Fifty-two women with abnormal cytology under going colposcopy had HC2 performed. HPV status, cytology and histology were compared. HC2 was positive in 30/31 with grade 2 cervical intraepithelial neoplasia (CIN2) or worse, 5/6 with HPV changes or CIN1, and 10/15 with no demonstrated abnormality.