STUDY SUBJECTS

Between September 1996 and March 1998 114 consecutive adult patients with a clinical and radiological picture suggestive of community acquired pneumonia admitted to the emergency room of a 500 bed university hospital in Catalonia (Spain) were enrolled in the study. Informed consent was obtained from all patients and the study was approved by the ethical and scientific committees of our institution. Fifty patients with a wide variety of diseases and without a recent history of pneumococcal infection were used as controls.

SPECIMEN COLLECTION

The following battery of samples was collected from patients with community acquired pneumonia:

(1)

two blood samples for aerobic and anaerobic conventional cultures;

(2)

an additional blood sample for study by nested PCR was obtained and stored at –70ºC and processed at the end of study; this sample was also collected from controls;

(3)

when present, pleural fluid was biochemically examined and processed for aerobic and anaerobic cultures and, in some cases, for detection of S pneumoniae genome by nested PCR;

(4)

considerable effort was made to obtain a good sputum sample; this was microscopically assessed to confirm its quality and, if satisfactory, it was cultured by conventional methods. Sputum was also stained and cultured for Mycobacteriumspp or opportunistic pathogens only when it was indicated;

(5)

after exclusion of contraindications, transthoracic needle aspiration (TNA) was performed in patients with severe pneumonia by the previously described technique.13 The sample obtained was processed for culture in aerobic and anaerobic media and for nested PCR to detect S pneumoniaegenome;

(6)

based on clinical suspicion, a urine sample was obtained from some patients for detection of Legionella pneumophila antigen;

(7)

a serum sample was obtained for serological investigations and stored at –70ºC. After 4–6 weeks of follow up a second serum sample was collected and processed with the first one to detect antibodies to Mycoplasma pneumoniae(immunofluorescence test), Chlamydia pneumoniae (microimmunofluorescence test),Coxiella burnetii (complement fixation test), and L pneumophila (immunofluorescence test).

PCR TECHNIQUE

200 μl samples of whole blood from patients with community acquired pneumonia and control subjects or 200 μl specimens obtained by TNA or thoracocentesis were processed using the QIAamp Blood Kit for extraction of S pneumoniae DNA. Proteinase K is used in this method for DNA extraction.

The selection of two primer pairs was based on the published pneumolysin gene sequence14; the outer primers Ia (5′-ATTTCTGTAACA GCTACCAACGA-3′) and Ib (GAATTCC CTGTCTTTTCAAAGTC-3) amplified a 348 bp region and the inner primers IIa (5′-CCCACTTCTTCTTGCGGTTGA-3′) and IIb (5′-TGAGCCGTTATTTTTTCAT ACTG-3′) amplified a 208 bp region of the pneumolysin gene.

A mixture was prepared using the following reagents: 10 μl PCR buffer (200 mM Tris-ClH pH 8.4, 500 mM ClK), 3 μl 50 mM Cl₂Mg, 1 μl 10 mM dNTP mixture, 1 μl of the first primer pair (Ia and Ib) (100 pmol for each reaction), 2.5 Taq polymerase units, made up to 50 μl with distilled water. In a 1.5 ml tube 100 μl of mineral oil, 50 μl of mixture 2X, and 50 μl of the study sample were instilled and the following thermocycle programme was performed (Mod IHB2024, Cherlyn Electronics Ltd, Cambridge, UK): one cycle of four minutes at 95ºC; 30 cycles of one minute at 95ºC, one minute at 55ºC, and one minute at 72ºC: and one cycle of 10 minutes at 72ºC. The same operation was repeated with the second pair of primers (IIa and IIb).

The amplified product was analysed by 2% agarose gel electrophoresis and ethidium bromide staining and examined by ultraviolet transillumination. Identification of the size of the band was performed by comparison with standard molecular weights (100 base pairs DNA ladder; Gibco BRL) and the band obtained from positive controls.

To prevent contamination a strict spatial separation of different PCR steps was maintained during the process and the recommendations of Kwok and Higuchi were followed.15 In addition, a positive control formed by a suspension of pneumococci in distilled water and a negative control for each sample consisting of target free distilled water were included in the procedure. Sensitivities of the assay on TNA and blood samples have previously been reported.9 ,10

AETIOLOGICAL CLASSIFICATION OF PATIENTS

According to the microbiological results and excluding PCR results provided by blood samples, the patients were classified into four groups:

(1)

Patients with pneumococcal pneumonia: a definite diagnosis of pneumococcal pneumonia was established whenS pneumoniae was isolated from an uncontaminated sample (blood, pleural fluid, or lung aspirate) or the S pneumoniae genome was detected in pleural fluid or TNA sample. In contrast, a probable diagnosis was made when S pneumoniae was only isolated in a pure culture from the sputum. Mixed infections, includingS pneumoniae in the isolated flora, were also considered as pneumococcal pneumonias.

(2)

Patients with pneumonia due to other aetiologies: a fourfold or greater rise in serological titres, the isolation of a micro-organism from uncontaminated samples (blood, pleural fluid, or TNA) or, for obligate pathogens, from the sputum, as well as the detection of L pneumophila antigen in urine, constituted a definite aetiological diagnosis. Conventional pathogens isolated only from the sputum were considered as a probable microbial diagnosis. Some patients finally diagnosed as having tuberculosis were included in this group.

(3)

Patients with pneumonia of unknown aetiology: all the remaining patients with non- diagnostic microbiological results were classified into this group.

(4)

Patients with other pathologies were used as controls.

STATISTICAL ANALYSIS

The sensitivity and specificity of PCR for S pneumoniae in the blood were calculated by comparison with the results provided by other diagnostic methods.