Subclinical Propionibacterium Acnes Infection Estimation in the Intervertebral Disc (SPInE-ID)
Condition: Infection; Intervertebral Disc Herniation; Propionibacterium Infection; Spinal Diseases; Discitis
MeSH terms: Infections; Communicable Diseases; Discitis; Intervertebral Disc Displacement; Spinal Diseases
Study type: Observational

Summary: 
      Subclinical infection of the intervertebral disc after lumbar disc herniation surgery has
      been correlated to chronic low back pain and vertebral endplate changes. The most commonly
      reported agent is Propionibacterium acnes. However, the real incidence is unclear, as it has
      been reported in some series ranging from 3.7% to 46%. Recently, a systematic review
      concluded that there is a relationship between P. acnes and endplate changes, but, there are
      so far no studies to verify whether the reported presence of that pathogen in the
      intervertebral discs is due to local infection or whether intraoperative contamination
      occurred during the collection of samples.

      Thus, the main objective of this study is to estimate the incidence of subclinical infection
      in patients surgically treated for lumbar disc herniation.

      To this end, a prospective cohort study will be conducted with a minimum of 95 patients
      between 18 and 65 years of age who have been submitted to surgery after failure of
      conservative treatment. The extruded disc will be removed and cultured for bacterial
      identification. As controls, the ligamentum flavum and the multifidus muscle, taken
      respectively before and after removal of the herniated fragment will also be cultured.
      Patients will be followed-up for a year and MRI will be done at the end of this period.
    

Detailed description: 
      INTRODUCTION Low back pain is a frequent condition in the population, as well as vertebral
      endplates abnormalities, described by Modic et al.(1,2), that affect up to 6% of the general
      population, and, up to 46% of patients with low back pain(3). Modic type I changes are
      described as vertebral bone marrow edema related to acute low back pain(4). When Modic
      changes are detected, chances of one presenting unspecific low back pain are 4.5 times
      higher(1,2).

      Subclinical infection caused by low-virulence pathogen can possibly lead to vertebral
      endplate abnormalities, detected in magnetic resonance imaging (MRI) studies, and
      differentiation between infection and Modic changes may be difficult(5,6). Subclinical
      infection can also be associated with increasing low back pain(7). Albert et al(8) reported
      61 patients who had undergone surgical treatment for lumbar disc herniation where 46% of
      cases had a positive culture. The same authors also reported that 80% of patients with a
      positive culture for anaerobic pathogens presented Modic type I changes at the adjacent
      vertebra after a two-year follow-up, against 44% of patients with negative culture. Some
      studies demonstrated the presence of low-virulence pathogens in intervertebral disc tissue
      cultures(6-10), most commonly reported to Propionibacterium acnes.

      Chronic low back pain and Modic type I changes have been treated with antibiotics for up to
      100 days with superior outcomes compared to sham treatment(7). Patients were treated with
      amoxicillin/clavulanate (500mg/125mg)(7) based on the study where sciatica is associated with
      Propionibacterium acnes(8).

      However, Carricajo et al(11) suggest that the presence of P. acnes in the intervertebral
      discs is due to either external surgical or laboratory contamination. These authors detected
      positive disc culture in only 3.7% of cases out of 54 patients. Furthermore, same authors
      demonstrated that samples of spinal muscle and ligamentum flavum collected intraoperatively
      at the end of procedure had positive cultures in 14.8% of cases with a negative disc culture.

      A systematic review performed by Urquhart et al(12) concluded, that there is moderate
      evidence that a relationship between positive culture with Modic type I changes and low back
      pain exists, although there was low evidence for relationship of cause. For that, authors
      concluded that new studies should be made to determine whether pathogens in the disc are
      originated from external contamination or if they are truly involved in the development of
      chronic back pain.

      HYPOTHESIS Lumbar disc herniation is related to subclinical infection of the intervertebral
      disc Null hypothesis: incidence of subclinical infection is the same as incidence of cases
      without infection in patients with lumbar disc herniation treated with surgery.

      OBJECTIVES Main purpose of this study is to identify if the presence of a infection pathogen
      in the intervertebral disc is real or if it is intraoperative contamination.

      Secondary objectives are to analyze clinical prognostic factors in patients and diagnosis of
      infection.The study also proposes to analyze the relationship between radiological changes
      (Modic I and II) and infection.

      METHODS

      Study design:

      An open prospective cohort study will be performed at a single center, (Hospital Israelita
      Albert Einstein - HIAE) taking 1 year for recruiting, and ending 1 year after inclusion of
      last patient. Patients' data will be collected with a specific form created for this study.
      Patients will be summoned for a new magnetic resonance image of the lumbar spine one year
      after their surgical procedure.

      All included patients will go through further treatment of ten sessions of postoperative
      physical therapy.

      Population:

      Patients who have failed conservative treatment for lumbar disc herniation undergoing lumbar
      decompression open surgery (microdiscectomy) will be consecutively included in the study.

      Patient enrollment in the study:

      Patients accepting their participation will date and sign the Informed Consent Form (ICF).
      After ICF is properly signed, patient will undergo an interview to complete initial
      demographic data and pretreatment forms.

      Patient recruitment will be carried out for 24 months, when 95 patients shall be included
      (details of estimated n reported at sample size determination).

      Patient allocation:

      Patients will undergo surgery according to surgeons' preference. Attending surgeons will
      determine chosen operative technique according to their experience and preference.

      Blinding:

      Patients will not have access to the results of tissue cultures for pathogens, as well as the
      attending physician.

      The radiologist that will analyze imaging studies of performed magnetic resonance will also
      be blinded to patients' data or laboratory culture results. A blinded investigator will
      analyze pain and function scores.

      Early stopping of participation in the study:

      Patients will be excluded from the study when:

        -  Withdrawn of ICF

        -  Diseased

        -  Patient selection flaw - incompatible eligibility criteria

        -  Lost to follow-up

        -  If patient presents clinical symptoms of infection such as severe lumbar or radicular
           pain, fever with no other detected foci, abnormal ESR, CRP, leucogram, and, altered
           imaging studies that leads to interruption of blinding of the results of culture exams.

      Study stages

      Sample collection:

      Included patients will undergo standard fashion general anesthesia and prepped with
      clorhexidine solution. Intravenous antibiotics prophylaxis will be administered within first
      hour before skin incision, according to standard protocol of HIAE Infection Control Committee
      published at the hospital Pharmaceutics Manual.

      Preoperative blood sample will be collected for leucogram, Erythrocyte Sedimentation Rate
      (ESR), and C-Reactive Protein (CRP). Same laboratory will be repeated at 1, 6 and 12 months
      time-point.

      The excised herniated disc fragment will be immediately sent to microbiology laboratory
      analysis in a universal sterile container (screw cap tube) in no more than 30 minutes to be
      processed as follows. Same process will be applied to samples of deep muscle and ligamentum
      flavum at the end of the surgical procedure. Three cultures of the intervertebral disc will
      be done, as well as three of the ligamentum flavum and three of the multifidus muscle for
      each patient.

      Search for pathogens:

      Herniated intervertebral disc will be split in three equally sized fragments of over 2x2x5 mm
      and squashed in a laminar flow cabinet until homogeneous material is achieved. Same process
      will be carried out for the ligamentum flavum and multifidus muscle samples, although split
      in three fragments. Tissues will be cultured in specific growth medium and incubated
      according to the respective culture:

      Similar criteria used by the Infectious Diseases Society of America (IDSA) to detect joint
      replacement infection will be adopted, which is the recommendation on at least two positive
      tissue cultures by the same pathogen to confirm diagnosis of infection(13).

        -  A - Aerobic culture For aerobic cultures, samples will be cultured in 5% sheep blood
           agar, chocolate agar and MacConkey agar plate, and will be placed in a 35°C incubator
           (CO2 atmosphere) for 5 days. If a positive bacteria culture is detected in the plate,
           the colony will be identified by MALDI TOF (Matrix Assisted Laser Desorption
           Ionization-Time of Flight) Microflex LT (Bruker Daltonics/BD).

        -  B - Anaerobic culture For anaerobic culture, tissue will be cultured in a Thioglycolate
           tube and incubated in a 35°C incubator for up to 21 days. If turbidity occurs at the
           Thioglycolate medium, material will be cultured in anaerobic blood agar and incubation
           at 35°C will be done in an anaerobic atmosphere. After growing of colonies,
           identification will be done by MALDI TOF.

        -  C - Histological analysis Anatomic pathology analysis of the other fragment of the
           herniated disc (2x2x5 mm) will be done. Sample will be transported in a universal
           container with tamponaded formalin (10%), followed by dehydration in alcohol diafanized
           in xylol and inclusion in paraffin (60-65°C), which will be stained in hematoxylin-eosin
           (HE) and GRAM staining.

             -  HE: Histological cuts of 4μm will be performed, followed by clearing with xylol for
                10 minutes twice, embedding with alcohol under increasing concentrations and
                stained with hematoxylin for 5 minutes, running water for 5 minutes, eosin for 1
                minute and running water for 2 minutes followed by assembly of glass microscope
                slide with Entellan®.

             -  GRAM: Another slide will be embedded with crystal violet for 1 minute, running
                water for 1 minute followed by lugol for another 1 minute and additional wash with
                running water. Unstaining of the slide will be done with alcohol 95% for 10 seconds
                followed by running water and then, stain with fuchsine for 30 seconds, running
                water wash again, drying and slide assembly with Entellan®.

        -  D - Molecular analysis of pathogens Positive cultures that present aerobic or anaerobic
           pathogens culturing, will be isolated and stowed refrigerated in -80°C freezer for
           posterior molecular analysis.

      Molecular typing will be performed through Pulsed Field Gel Electrophoresis technique of
      isolated samples according to the protocol described by Oprica et al.(14) using Spe-I
      restriction enzyme and Bionumerics software for analysis of results.

      Questionnaires See outcome measures

      Imaging studies Magnetic resonance imaging studies will be performed in Siemens or General
      Electric 1.5T devices.

      Lumbar disc herniation will be diagnosed through MRI in eligible patients. Included patients
      will be submitted to new MRI 12 months after surgery.

      Two radiologists with expertise in musculoskeletal MRI will independently classify and
      perform measurements.

      Confounding variables Data on the following confounding variables will be collected: age,
      gender, alcohol intake, smoking, body mass index (BMI), spinal injections with corticoid
      within 6 months before surgery, usage of oral corticoids up to 3 months before surgery,
      diabetes.

      Since this information may change over time, data will be considered at time of last
      assessment before surgery.

      Statistical planning Rate of subclinical infection (or Modic change) will be obtained by the
      ratio between number of positive cultures from surgical samples and total number of patients,
      and estimates will follow 95% confidence intervals. After infection cases are identified, we
      will investigate if there is an association between detected infection and patient outcomes
      by logistic regression models for Modic changes, ordinal logistic regression for Modic volume
      and size, and linear regression or general linear models for numeric outcomes, such as: low
      back pain, quality of life and function. All models will consider confounding variables such
      as: smoking and alcohol intake, diabetes, corticosteroids injection, BMI, gender, and age.
      Results will be presented as effects estimates such as odds ratio or mean ratio, 95%
      confidence intervals and p values. Study dropout cases, for any reason, will be considered
      for final analysis.

      Sample size calculation Sample size was calculated to estimate of incidence of subclinical
      infection in patients with lumbar disc herniation. Considering that the rate of infection
      lies around 46%(3), we need to observe a minimum of 95 patients to achieve a 95% confidence
      interval with 10% absolute accuracy.

      The necessary sample size to analyze secondary endpoints will depend on the observed rate of
      cases with subclinical infection in our study sample. If the observed rate is too small, an
      increase in the number of included patients will be needed. To better evaluate this, the
      sample size calculation will be revisited by the time we have reached half of initially
      planned sample size (48 patients).
    

Intervention name(s): Microdiscectomy
Intervention type(s): Other
Intervention description(s): It will be an observational study without interventions. Subjects with diagnose of lumbar disc herniation undergoing open decompression surgery (microdiscectomy) will be included and analyzed. There will be no direct intervention to the patient proposed by the study.
Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Patients with lumbar disc herniation who failed conservative treatment undergoing surgical
        treatment open decompression surgery (discectomy).
      
Biospecimen description: 
      Lumbar Intervertebral Disc, Flavum ligament, Multifidus muscle
    
Keywords: infection; intervertebral disc; Propionibacterium acnes; spine