Vertebral Osteomyelitis Caused by Helicobacter cinaedi Identified Using Broad-range Polymerase Chain Reaction with Sequencing of the Biopsied Specimen.

A 65-year-old man presented with gradually exacerbating low back pain. Magnetic resonance imaging revealed vertebral osteomyelitis in the Th11-L2 vertebral bodies and discs. The patient showed negative findings on conventional cultures. Direct broad-range polymerase chain reaction (PCR) with sequencing of the biopsied specimen had the highest similarity to the 16S rRNA gene of Helicobacter cinaedi. This case suggests that direct broad-range PCR with sequencing should be considered when conventional cultures cannot identify the causative organism of vertebral osteomyelitis, and that this method may be particularly useful when the pathogen is a fastidious organism, such as H. cinaedi.

Introduction

Both blood and biopsy specimen cultures are useful diagnostic tests for confirming the causative pathogen of vertebral osteomyelitis. However, the positivity rates of both tests have been reported to be limited to 58% and 77%, respectively (1). Recently, broad-range 16S rRNA gene polymerase chain reaction combined with sequencing [broad-range polymerase chain reaction (PCR) with sequencing] has been used to identify the causative pathogen in cases where conventional cultures are negative (2). Using this new technique, the causative pathogen is accurately identified by gene amplification and sequencing of a broad-range gene target for bacteria (3).

We herein report the case of a 65-year-old man with vertebral osteomyelitis caused by Helicobacter cinaedi confirmed using broad-range PCR with sequencing that had not been identified by either blood or biopsy specimen cultures.

Case Report

The patient was a 65-year-old man without any significant medical history. He presented with gradually exacerbating low back pain for the past year. He denied a fever or any associated neurological symptoms. He did not have an immunocompromised background.

On admission, his vital signs were as follows: blood pressure, 141/73 mmHg; heart rate, 77 beats/min; body temperature, 36.3℃; respiratory rate, 20 breaths/min; and oxygen saturation on room air, 95%. A physical examination revealed knock pain at the L1 level but no abnormal neurological findings. Laboratory tests showed an elevated C-reactive protein (CRP) level (3.36 mg/dL; normal, <0.3 mg/dL) and an elevated erythrocyte sedimentation rate (41 mm/h; normal, <14 mm/h). His white blood cell count, hemoglobin level, platelet count, serum electrolyte level, plasma glucose level, liver function test results, and creatine phosphokinase level were all normal. Magnetic resonance imaging revealed findings of vertebral osteomyelitis in the Th11-L2 vertebral bodies and disks (Figure).

Figure.

Magnetic resonance imaging. The image in A was taken with TI-weighted imaging, showing a low signal in the Th11-L2 vertebral bodies and disks. The image in B was taken with T2-weighted short-tau inversion recovery imaging, showing a high signal in the Th11-L2 vertebral bodies and disks.

Two sets of blood culture were performed; however, no organism grew on culture. A percutaneous biopsy of the lumbar disk was performed. Bacteria were not observed on Gram staining. In addition, no growth was found on blood agar or chocolate II agar after 72 h of incubation at 35℃ under 5% CO2, nor in thioglycollate broth after 14 days at 35℃.

Direct broad-range PCR with sequencing of the biopsied specimen was performed. The consensus sequence had the highest similarity (1,433/1,441 bp 99.41% match in GenBank, Accession No. NR_025941, 1,431/1,438 bp 99.51% match in EzTaxon, Accession No. ABQT01000054) to the 16S rRNA gene of H. cinaedi.

We started the patient on ceftriaxone, and his low back pain and elevated CRP level gradually improved. We switched ceftriaxone to oral minocycline after six weeks, and he was discharged. Treatment with minocycline was continued for approximately six months, and there have been no signs of relapse during three months of follow-up.

Discussion

The course of the present case emphasizes two important clinical points. First, H. cinaedi should be suspected as a pathogen in vertebral osteomyelitis, even when conventional cultures are negative. Second, broad-range PCR with sequencing of biopsy specimens is particularly useful when conventional cultures cannot identify the causative organism of vertebral osteomyelitis.

H. cinaedi should be suspected as a pathogen in vertebral osteomyelitis even when conventional cultures are negative. Six cases of vertebral osteomyelitis with H. cinaedi have been reported, and two of these cultures were negative, including our case (Table) (4-8). H. cinaedi was detected on blood culture in four cases. A biopsy was performed in four cases, and the biopsy specimen culture was positive in only one case. In two cases, including our present case, broad-range PCR with sequencing directly performed using the biopsy specimen confirmed the presence of H. cinaedi, although the culture was negative. Several reasons for the inability to detect the causative organism have been suggested. First, prior antimicrobial exposure decreases the sensitivity of the conventional culture. However, in our case, no antibiotic had been used prior to performing culture. Second, fastidious organisms, including H. cinaedi, are difficult to culture. The detection of H. cinaedi is known to be particularly difficult because it rarely grows on traditional culture media (9). Nonselective media incubated in a microaerobic condition (5%O2), ideally with hydrogen gas (5-10%), or selective media such as Skirrow or Butzler Blaser were reported to be suitable for the isolation of H. cinaedi (9,10). In our case, we used neither a microaerobic condition nor selective media because we did not suspect H. cinaedi to be the causative organism. In addition, H. cinaedi grows slowly under microaerobic conditions; therefore, a long incubation period is recommended for its detection. Four to ten days are considered necessary for a positive result with a culture bottle of an automatic blood culture system (10). Given the findings of this previous report, we prolonged the incubation time of blood culture to 10 days; however, we did not observe any organism growth.

Table.

A Summary of Five Cases of Vertebral Osteomyelitis Caused by Helicobacter cinaedi Reported in the English Literature along with the Present Case.

Case	Age, years/sex	Underlying conditions	Site of infection	Blood culture	Culture of biopsied specimen	Direct PCR of biopsied specimen	Treatment	Outcome	Reference
1	75 M	Hypertention Coronary artery disease	L5-S1	Positive	NA	NA	Meropenem 1w Ertapenem 5w	Cure	(4)
2	64 M	None	L5	Positive	Negative	NA	(Cefazolin+ Fosfomycin) 8w	Cure	(5)
3	56 M	Bronchial asthma	C6-7	Positive	NA	NA	Ceftriaxone 6w	Cure	(6)
4	66 M	Diabetes, Liver cirrhosis	L1-2	Positive	Positive	Positive	Ampicillin 1w Oral Doxycycline 11w	Cure	(7)
5	55 M	Hypertention	L4-5	Negative	Negative	Positive	Ceftriaxone 8w	Cure	(8)
Present case	65 M	Epilepsy	Th11-L2	Negative	Negative	Positive	Ceftriaxone 6w Oral Minocycline 24w	Cure	Our case

M: male, L: lumbar vertebra, C: cervical vertebra, Th: thoracic vertebra, NA: not applicable, w: week(s)

The optimum antibiotic selection and duration for vertebral osteomyelitis with H. cinaedi have yet to be determined. Indeed, the selection of antimicrobials has varied in previous case reports (Table). We selected ceftriaxone and oral minocycline based on a previous report of successful treatment with these drugs for H. cinaedi bacteremia (11). A treatment duration of more than six weeks in all cases achieved favorable outcomes.

The present case suggests that broad-range PCR with sequencing of biopsy specimens is particularly useful when the causative organism of vertebral osteomyelitis cannot be identified using conventional cultures. Choi et al. reported that broad-range PCR with sequencing is more sensitive than routine culture for the etiological diagnosis of vertebral osteomyelitis (12). In their study, broad-range PCR with sequencing confirmed the causative organism in 16 cases of vertebral osteomyelitis in which biopsy specimen culture had been negative. Given that three-quarters of the patients had been treated with antimicrobial agents, the authors concluded that broad-range PCR would be particularly useful for patients with a history of antimicrobial therapy. The findings of our case further imply that this method might be useful when the pathogen is a fastidious organism, such as H. cinaedi.

In conclusion, we encountered a rare case of vertebral osteomyelitis caused by H. cinaedi identified using broad-range PCR with sequencing of a biopsy specimen that had not been detected with conventional culture. The findings of our case suggest that performing broad-range PCR with sequencing should be considered for the detection of the causative organism when blood or biopsy specimen culture fails.

The authors state that they have no Conflict of Interest (COI).