Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR

Abstract

Helicobacter cinaedi is the most frequently reported enterohepatic Helicobacter species isolated from humans. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods. Here, we report a nested PCR assay that rapidly detects the cytolethal distending toxin gene (cdt) of H. cinaedi with high specificity and sensitivity. Specificity of the assay was validated by using different species of Helicobacter and Campylobacter, as well as known H. cinaedi-positive and -negative samples. The sensitivity of detection for the cdt gene in the assay was 10(2) CFU/ml urine or 10(2) CFU/10(5) infected RAW 264.7 cells. In an H. cinaedi-infected mouse model, the cdt gene of H. cinaedi was effectively detected via the assay with urine (6/7), stool (2/3), and blood (2/6) samples. Importantly, it detected H. cinaedi in blood, urine, and stool samples from one patient with a suspected H. cinaedi infection and three patients with known infections. The assay was further used clinically to follow up two H. cinaedi-infected patients after antibiotic treatment. Stool samples from these two patients evaluated by nested PCR after antibiotic therapy showed clearance of bacterial DNA. Finally, analysis of stool specimens from healthy volunteers showed occasional positive reactions (4/30) to H. cinaedi DNA, which suggests intestinal colonization by H. cinaedi in healthy subjects. In conclusion, this nested PCR assay may be useful for the rapid diagnosis, antimicrobial treatment evaluation, and epidemiological study of H. cinaedi infection.

Fig 1

Design and specificity of nested PCR. (A) Arrows indicate the locations of PCR primers in the whole genome sequence, and the sizes of the products of the primary and nested amplification reactions are shown. (B) Nested PCR amplification of bacterial DNA. Lane 1, 1-kb marker; lane 2, standard clinical isolate of H. cinaedi; lane 3, master mix without template; lane 4, Milli-Q water control for DNA extraction; lanes 5 to 14, H. cinaedi clinical isolates from 10 patients; lane 15, H. pylori PAGU 151^T (ATCC 43504^T); lane 16, H. hepaticus PAGU 604^T (LMG 16316^T). (C) First PCR amplification of the cdt gene of H. cinaedi. Lane 1, 100-bp DNA ladder; lane 2, H. cinaedi PAGU 597^T (CCUG18818^T); lane 3, H. fennelliae PAGU 601^T (LMG 18294^T); lane 4, H. pametensis PAGU 607^T (LMG 12678^T); lane 5, H. acinonychis PAGU 609^T (LMG 12684^T); lane 6, H. muridarum PAGU 606^T (LMG 13646^T); lane 7, H. hepaticus PAGU 604^T (LMG 16316^T); lane 8, H. bilis PAGU 599^T (LMG 18386^T); lane 9, H. pylori PAGU 151^T (ATCC 43504^T); lane 10, H. canadensis PAGU 600^T (CCUG 47163^T); lane 11, H. canis PAGU 598^T (NCTC 12379^T); lanes 12 to 14, clinical isolates of H. cinaedi; lane 15, C. fetus subsp. fetus PAGU 74^T (ATCC 27374^T). (D) Sensitivity of detection of the H. cinaedi-specific cdt gene by nested PCR. Serial titrations of pure H. cinaedi culture were performed, samples were mixed with 10⁶ RAW 264 cells, and then nested PCR was performed by using the extracted DNA of the mixture of bacteria and cells (left). Tenfold dilutions of pure bacterial culture were spiked into healthy human urine, and nested PCR was performed by using the extracted DNA from contaminated urine (right).

Fig 2

Nested PCR with experimental samples collected from H. cinaedi-infected mice. (A and B) Nested PCR amplification of DNA extracted from blood (A) and urine (B) samples from intraperitoneally infected mice at the indicated time points. (C) Nested PCR amplification of DNA extracted from stool samples from uninfected control and orally infected mice. Stool samples were collected at 7 days p.i. Lanes 1, 2, and 3, DNA extracted from three different mice.

Fig 3

Nested PCR with clinical specimens collected from patients with known or suspected H. cinaedi infections. Patients 1 and 2 had cellulitis, fever, and positive blood tests for growth of anaerobic bacteria that were subsequently confirmed to be H. cinaedi by conventional PCR. (A) Brief clinical history of patient 1. WBC, white blood cells; div, intravenous drip infusion; CRP, C-reactive protein. (B) Nested PCR with DNA from the indicated specimens from patient 1. (C) Left, morphological appearance of bacterial growth on Helicobacter agar plates inoculated with a stool specimen from patient 1. Plates were incubated for 4 days at 37°C in a microaerobic atmosphere containing 10% H₂ and 10% CO₂. Right, bacterial morphology of an isolate from culture of stool from patient 1 (acridine orange staining). (D and E) Nested PCR with DNA from the indicated specimens from patient 2 (D) and patient 3 (E). Patient 3 had cellulitis and fever, but a blood test was negative for anaerobic bacterial growth.

Fig 4

Follow-up of H. cinaedi-infected patients by means of nested PCR of DNA from stool and blood specimens during antibiotic treatment. (A) Patient 4. Various amounts of products from the first PCR were used as templates for the nested PCR. The diffuse signal in the lane of the 5.0-μl template, at 90 days p.o. for the blood DNA, was due to nonspecific PCR products caused by too much template DNA being used. The lower panel gives a brief clinical and therapeutic history. Arrowheads indicate times of collection of stool or blood samples for bacterial culture and DNA amplification. (B) Patient 1. Samples were inoculated onto Helicobacter agar plates, and suspected colonies from the resultant bacterial growth were subcultured to obtain pure H. cinaedi cultures (Fig. 3C). Nested PCR was performed with DNA from both the stool specimen and the stool culture. The lower panel gives a brief clinical and therapeutic history of recurrent infection. Arrowheads indicate times of collection of stool samples. The nucleotide sequences of all PCR products from patients were consistent with that of the H. cinaedi type strain.

Fig 5

Genetic analysis of clinical isolates of H. cinaedi. The 16S rRNA gene was analyzed by means of the FASTA search system. Phylogenetic relationships for various clinical isolates recently obtained, mainly in Japan (the isolation location is given in parentheses), and H. bilis were analyzed on the basis of the 16S rRNA gene sequence (1,430-bp area). The Helicobacter cinaedi strains isolated from patient 1 from the primary and the recurrent infections (indicated in the dashed box; PAGU1625-Kumamoto and PAGU1679-Kumamoto) were found to be almost genetically identical on the basis of the 16S rRNA gene sequence. Numbers at nodes are percent occurrence in 1,000 bootstrapped trees.