PATIENTS

We retrospectively examined 32 cases of SIDS and eight controls. The diagnosis of SIDS was based on an adequate negative postmortem examination to exclude evidence of other causes of death. Ages of SIDS cases ranged from 2 to 28 weeks. Controls were defined by death within 1 year of age, and with a known cause of death:Streptococcus pyogenes meningitis, ileal perforation, Escherichia coli septicaemia, pneumonia, necrotising enterocolitis, prematurity (n = 2),S pneumoniae septicaemia, and suffocation. Ages of controls ranged from 3 to 44 weeks.

TISSUE PREPARATION AND HISTOLOGY

Formalin fixed, paraffin embedded specimens of stomach, trachea, and lung were examined from SIDS cases and controls. Tissue sections were prepared aseptically; 5 μm tissue sections were prepared on coated slides for haematoxylin and eosin (H&E) staining, and 10 μm sections were prepared in sterile Eppendorf tubes for subsequent PCR. H&E sections were examined for the presence of bacteria using both low (×40) and high (×100) power light microscopy.

SPECIFIC PRIMERS FOR H PYLORI

For the ureC region, first round primers were 5′-AAG CTT TTA GGG GTG TTA GGG GTT T-3′ corresponding to positions 784–808 and 5′-AAG CTT ACT TTC TAA CAC TAA CGC-3′ corresponding to positions 1054–1085, generating a 301 bp fragment; second round primers were 5′-CTT TCT TCT CAA GCA ATT GTC-3′ corresponding to positions 829–849 and 5′-CAA GCC ATC GCC GGT TTT AGC-3′ corresponding to positions 1012–1032, generating a 252 bp fragment.37

For the cagA gene, oligonucleotide primers were designed using the nucleotide sequence of H pylori 26695.39 First round primers were 5′-CAC CAA CGC CTC CAA GAG TCC TGA T-3′ corresponding to positions 1539–1563 and 5′-TGT TGC CGT TTG GTC TCC AAT TTT-3′ corresponding to positions 1905–1930, generating a 391 bp fragment; second round primers were 5′-AAG AGT CCT GAT AAG GTG GTA GGC-3′ corresponding to positions 1552–1575 and 5′-CCA CTT CTT TCT CTA AAT GCT CTC-3′ corresponding to positions 1877–1900, generating a 348 bp fragment.

DNA EXTRACTION AND AMPLIFICATION

Paraffin was removed from the 10 μm sections usingn-octane, and the DNA extracted in 2 ml of lysis buffer (20 mM Tris/HCl pH 8.3, 2 mM EDTA, 1% Triton-X, 0.5% sodium dodecyl sulphate, 0.5 mg/l proteinase K), and precipated in ethanol and dried under vacuum. Amplifications were performed in a final volume of 50 μl containing 0.4 mM of each primer, 0.2 mM each of dATP, dCTP, dGTP, and dTTP in PCR reaction buffer (10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris/HCl pH 8.8, 2 mM MgSO₄, 0.1% vol/volTriton X-100, 15 mM MgCl₂). Template DNA (10 μl) was added to the mixture and denatured at 94°C for 10 minutes, cooled on ice, and 2.5 U taq DNA polymerase (Perkin Elmer) added prior to 40 amplification cycles (94°C for one minute; 55°C for one minute; 72°C for one minute), a 10 minute final elongation step (72°C), and holding at 4°C. For the second round reaction in each case digoxigenin labelled dNTPs were used (Boehringer-Mannheim). DNA extracted from H pylori NCTC 11637 (positive control), and sterile distilled water (negative control) were incorporated in each run. DNA extracted from a specimen of human ureter was also used as a negative control. Samples were run in duplicate and amplifications were performed on a 9600 thermal cycler (Perkin Elmer). Contamination was minimised by utilisation of separate laboratory areas and pipettes for pre-PCR, PCR, and post-PCR stages of the procedure, use of sterile bunged pipette tips, and inoculation of the positive control as a last step in the pre-PCR preparation. Tissues were processed in a randomised and blinded fashion.

DETECTION BY ELISA

Amplicons were then detected semiquantitatively on the solid phase by ELISA using probes specific for the ureCand cagA genes. For theureC gene, the probe was 5′-AGA ATT GAA GCA TTG CGC GAT TGG GGA TAA GTT TGT GAG CGA AT-3′ corresponding to positions 904–946.37 For thecagA gene, the probe was 5′-ACA AGA AAG CTA ATA AGC TTA TCA AAG ATT TTT TCA GCA GC-3′ corresponding to positions 1722–1755.

The ELISA was performed by adding 35 μl of amplified PCR mixture to 40 μl of NaOH for 10 minutes at room temperature to denature the double stranded DNA. This was then mixed with 425 μl hybridisation solution containing the respective biotinylated probe at a concentration of 7.5 pmol/ml and the solution vortexed. This mixture was then transferred in duplicate to a streptavidin coated microtitre plate (200 μl/well) and incubated at 37°C with shaking for three hours. The wells were then washed five times, 200 μl of antidigoxigenin monoclonal antibody–horseradish peroxidase (HRP) solution added to each well, and the plate incubated at 37°C for 30 minutes. The wells were again washed, and 200 μl of a solution of 2′,2′-azino-di-[3-ethylbenzthiazoline sulphonate-6] diammonium (ABTS) substrate solution added to each well, and the optical density (OD) read on a microplate reader (Titertek Multiskan Plus, Labsystems, Finland) at 405 nm. Positive results on PCR–ELISA were defined as those giving an optical density of greater than or equal to the mean OD plus two times the standard deviation of a range of negative controls (table 1).

DETERMINATION OF THE SENSITIVITY OF THE PCR–ELISAS

Sensitivity of the two PCR–ELISAs were evaluated using tenfold dilutions of an overnight broth culture of H pylori NCTC 11637. Viable counts were performed using the method of Miles and Misra.40 A 100 μl aliquot of each dilution was plated onto Columbia agar with 5% (vol/vol) horse blood (Oxoid, Basingstoke, Hants, UK) and incubated at 37°C in a microaerophilic atmosphere. The DNA was then extracted from each dilution using guanidium thiocyanate, using the method of Pitcheret al.41 Bacterial strains were suspended in 10 ml sterile water and the cells lysed by the addition of 0.5 ml of GES reagent (5 M guanidium thiocyanate (Sigma), 100 mM EDTA, and 0.5% vol/vol sarkosyl). Then 25 ml 7.5 M ammonium acetate was added with mixing, the mixture held on ice for 10 minutes, and 50 ml chloroform/2-pentanol (24/1) was added. The mixture was centrifuged for 10 minutes, the supernatant removed, and 50 ml 2-propanol added. The tube was centrifuged for 20 seconds to pellet the DNA, which was then washed twice with 70% ethanol. DNAs were redissolved overnight at 4°C in sterile water and used as template in the above PCR–ELISA tests for H pylori ureC andcagA genes. The highest dilution giving a positive PCR–ELISA test was used to calculate the sensitivity of the PCR–ELISA reactions. Positive (DNA extract from a plate culture ofH pylori NCTC 11637) and negative (sterile water) controls were incorporated in each PCR run. DNA extracted from a specimen of human ureter was also used as a negative control.

PCR FOR TWO REGIONS OF THE HUMAN β GLOBIN GENE

To determine the presence of inhibitory substances, all clinical specimens were also examined for the presence of human DNA by two separate PCRs directed against the β globin gene. Primers GH20 (5′-GAA GAG CCA AGG ACA GGT AC-3′) and PC04 (5′-CAA CTT CAT CCA CGT TCA CC-3′) produce a product of 268 bp; and primers KM29 (5′-GGT TGG CCA ATC TAC TCC CAG G-3′) and RS42 (5′-GCT CAC TCA GTG TGG CAA AG-3′) produce a product of 536 bp. Each of these reactions was performed using 200 μM of each primer and 1 U of Taq polymerase in a mixture of 67 mM Tris/HCl, 16 mM ammonium sulphate, 2 mM MgCl₂, 0.02% gelatin, pH 8.4 and 200 μM deoxynucleoside triphosphate; incubation at 95°C for seven minutes was followed by 40 one minute cycles at 92°C, 55°C, and 72°C. Product detection was by 1.5% agarose gel electrophoresis.

STATISTICAL ANALYSIS

Differences in incidence of H pyloriDNA positivity in tissues of SIDS versus controls were analysed with Fisher's exact probability test; p < 0.05 was considered significant.

HISTOLOGY

The histological examination of each tissue section from SIDS cases and controls confirmed the typical histological appearance of each respective organ. The prime purpose of this examination was to determine the presence of visible bacteria. Bacteria were not observed in any section of stomach, trachea, or lung from the cases of SIDS and controls.

PCR FOR HUMAN DNA

All tissues tested positive for both regions of the human β globin gene.

PCR–ELISA FOR H PYLORI DNA

Table 1 shows optical densities of PCR products from amplification reactions for H pylori ureC and cagAgenes using template DNA extracted from stomach, trachea, and lung sections from cases of SIDS and controls. Results were calculated using a cut off of the mean optical density plus two times the standard deviation of the negative controls. For theureC region, 25 SIDS cases were positive in one or more tissues compared with one of the controls (Yates's corrected χ² = 9.40; p = 0.0022). For thecagA gene, 25 SIDS cases were positive in one or more tissues compared with one of the controls (Yates's corrected χ² = 9.40; p = 0.0022). Considering both gene sequences together, 28 SIDS cases were positive in one or more tissues compared with one of the controls (Yates's corrected χ² = 14.49; p = 0.0001). Results were also calculated using a cut off of the mean optical density plus three times the standard deviation of the negative controls. For both theureC and cagAgenes, 19 SIDS cases were positive in one or more tissues compared with none of the controls (Yates's corrected χ² = 6.82; p = 0.009).

SENSITIVITY OF THE H PYLORIPCR–ELISAS

For both ureC andcagA assays, after nested PCR, the sensitivity of detection was 150 pg H pyloriDNA which corresponds to 40 genome equivalents. In each case, the sensitivity of detection of the PCR–ELISA was 1–5 pgH pylori DNA which corresponds to approximately four genome equivalents.