“Cold Case – a Hot Topic”

“Cold Case – a Hot Topic”

Dr. Bridget Freyne Infectious Disease Fellow

Royal Children’s Hospital Melbourne

Melbourne Infectious Disease Group Meeting 28/04/2015

Case

20 month old boy

Presentation 3 week history of limp Left knee pain Worse in the mornings Some response to paracetamol and ibuprofen Unwitnessed trauma reported by 4-year old brother Intermittent fevers - 5/6 episodes

Birth history NVD at term

Past Medical History Mild seborrheic dermatitis

Case Immunisations

In line with schedule up to 18 months

Hep A and BCG at 14 months of age for travel to India

Family history No unwell contacts

No history of rheumatological disorders, immune deficiency

Social history Parents from Punjab, non-consanguineous

1 older brother

Suburban Melbourne

No pets

Case Examination

General Height and weight 30th centile

Well nourished

Developmentally appropriate

Well healed BCG scar

Normal tonsillar tissue

No cervical or peripheral adenopathy

No hepatosplenomegaly

Chest clear

Musculoskeletal Mobilising but limping

Tender and swollen over left proximal tibial tuberosity

Investigations Hb 121, Plts 321

WCC 10.8 (N 4.32, L 6.05)

U&E / LFTs -> Normal

CRP < 5 ESR 18

BC no growth

Case Differential diagnosis

Sub-acute bacterial osteomyelitis

TB osteomyelitis

(Septic arthritis)

Plan Imaging +/- surgical washout

Start Flucloxacillin 50 mg/kg iv 6H post op

QFT-IT

TST (Mantoux)

Requested ZN stain, mycobacterial culture and TB PCR on operative samples

There is an aggressive looking lesion involving the proximal metaphysis and epiphysis of the tibia. It is predominantly lucent with internal trabeculations noted in the epiphyseal component. The metaphyseal component is poorly defined with a wide zone of transition.

There is laminar periosteal reaction extending down the tibia. The features of this lesion are more consistent with infection, however malignancy cannot be excluded. MRI is recommended.

12 x 8 x 12 mm dumbbell shaped abscess crossing growth plate

Breach of posterior cortex and non-enhancing area in popliteal fossa ? Extruded sequestrum

Popliteal lymph nodes

Case

Intraoperative findings Osteotomy to anterior tibia

No pus under pressure

Extensive curettage of pale bony fragments

No need for prolonged drainage

Do these imaging & intraoperative findings suggest anything in particular?

Cold abscess pathology and imaging

Cold abscess -> purulent collection with no associated heat or erythema

• Formed by collection of products of liquefaction and reactive exudation

• Migrates in multiple directions along path of least resistance

Tuberculosis Actinomycosis Sporotrichosis Blastomycosis Coccidomycoses Chromomycoses Trichophyton rubrum Protothecosis Bacterial abscess in 1ry immunodeficiency

Case

QFT-IT

TB specific antigens (ESAT-6, FCP10 & TB7.7) 0.01 IU/mL

Mitogen control (PHA) 1.91 IU/mL

Nil control 0.02 IU/mL

Case

QFT-IT

TB specific antigens (ESAT-6, FCP10 & TB7.7) 0.01 IU/mL

Mitogen control (PHA) 1.91 IU/mL

Nil control 0.02 IU/mL

(Mantoux TST cancelled)

Histopathology

Necrotising and suppurative granulomatous osteomyelitis

AFB not seen on Ziehl-Neelsen stain

Gene-Xpert TB PCR positive

Case

Commenced therapy 3 drugs

Isoniazid 10 mg/kg od Rifampicin 15 mg/kg od Pyrazinamide 35 mg/kg od

Major problems with adherence

Personal telephone support hotline Close OPD follow up Admitted for DOTS Play therapy for patient Psychology referral for mother

Case

Development of sinus tract

Draining creamy fluid

Fever

Case



Fever

Case



Fever

Discussion

1. Molecular diagnosis of TB in children

2. BCG vaccination in Australia

3. BCG osteomyelitis & osteitis

4. BCG-itis and immuodeficiency

TB PCR diagnostics – the MTB complex

• Distinguish by fixed deletions and SNPs • Distribution of deletions provides

insights into evolution of MTB • Primarily animal adapted species

identified by deletion of RD9 • Primary hosts vs Maintenance hosts

vs Spillover hosts

Smith et al. Nature Reviews Immunology 2009 “Myths and Misconceptions on the evolution of MTB”

8 species of mycobacterium 99.95% sequence similarity at nucleotide level

15 studies 4768 respiratory samples in in 3640 children <15y Reference standards

Culture positive (12%, n=420) Clinical diagnosis and commencement on TB medication (44% of culture negative patients, n=688)

Population

Mixed HIV status Africa, China, Vietnam, Bangladesh, Spain & Italy Induced sputum, gastric lavage and NPA Cross-sectional, cohort and RCT

Analysis

Bivariate random effects model

Compared to culture Variety in sensitivities by specimen type

Induced Sputum

Specificity 93-100% Sensitivity 25-100%


Gastric fluid



Nasopharyngeal aspirate


DNA PCR (Nested or Multiplex)

Reverse Transcriptase PCR

RT-PCR

mRNA Viable bacteria

Quantification Automation

Non-viable bacteria

Proposed gold standards • Culture on solid and liquid media • BACTEC • Histopathology • Response to TB therapy

Gene Target

IS6110 MTBC, M. smegmatis

65kDa (Rv0440) MTBC

MPB-64/MBT-64 (Rv1980c) MTBC

38kDa (Rv0934) MTBC, M. leprae

Conserved repetitive element (TRC4) MTBC

Guanine-cytosine rich repetitive sequence (GCRS) MTBC, MAC

devR (Rv3133c) MTBC, M. leprae, M. canetti

HupB (Rv2986c) Candidate for differentiation

Gene Target

IS6110 MTBC, M. smegmatis

65kDa (Rv0440) MTBC

MPB-64/MBT-64 (Rv1980c) MTBC

38kDa (Rv0934) MTBC, M. leprae

Conserved repetitive element (TRC4) MTBC

Guanine-cytosine rich repetitive sequence (GCRS) MTBC, MAC

devR (Rv3133c) MTBC, M. leprae, M. canetti

HupB (Rv2986c) Candidate for differentiation

Commercially available

Roche Amplicor (16srRNA) Genprobe MTB direct (16srRNA)

Roche COBAS Taqman (RT-PCR IS6110/MB64) Gene/Xpert (Nested PCR for rpoB gene)

Discussion





BCG Down Under

BCG vaccine is only recommended for:

• all ATSI born in parts of QLD/NT

• parents have TB or leprosy (or who have been treated for leprosy in the past)

• <5 yr old travellers to countries where TB is common

• <16 yr old who live with, or are exposed to, someone with TB, or from a country where TB is present

• healthcare workers who may come into contact with people with TB

172/195 (88%)

Proposed causes of variable efficacy Host genetic factors

Environmental factors Age and route of administration

Vaccine strain

Prior BCG vaccination does not out rule diagnosis of TB

Calmette & Guerin passaged BCG for 13 years on potato slices imbibed with glycerol On loss of virulence -> dissemination -> individual laboratory passages -> 1960s “seed lots” Comparative genomics of vaccine strains Regions of difference (RDs) SNPs Tandem duplications (DU I-IV) in response to selective pressure -> enzymes of glycerol metabolism

BCG Denmark (10 million) BCG Japan (20 million) BCG Russia (85 million)

Effectiveness Clinical Culture BCG-Japan 69% 92% BCG-Serbia 43% 82% BCG-Russia 22% 51% No BCG

Osteitis & osteomyelitis

Rare but most frequent in Scandinavia & Eastern Europe Typically associated with changes to BCG strain

1998 Czechoslovakia switch from Prague -> Russia (35/1,000,000) 1971 Sweden switch from Pasteur -> Gothenburg (1/3,000) Decline after change to Denmark 1331 2012 WHO report “Infrequent” (1/3,333 – 1/108)

General coplication from Jim’s paper

Discussion





Not relevant (n=62)

Post BCG vaccine disease (n=53)

Adults post intra-vesical (n=11)

English language (n=25)

Reviews (n=3)

Case series (n=6)

Case Reports (n=16)

Non-English (n=28) Czech Polish

Turkish French

Portuguese Japanese Russian

BCG AND oste* N=126

Onset 3 months – 5 years post vaccine (average 18 months)

Location Long bone metaphysis Femur 27%, Tibia 19% Humerus 11% Sternum 15% Ribs 11% Case reports: skull, foot and clavicle

Minimal systemic symptoms

Diagnostic Criteria 1971: Foucard & Hjelmsted

BCG vaccination in the neonatal period

Radiology consistent with osteitis No known TB contact

At least 1 of compatible histopathology, ZN stain pos or culture

QFT-IT negative and PPD positive (89%)

ESR (mean 34)

Histopathology Granulomatous inflammation with epitheiloid cells

Caseous necrosis (92%)

AFB positive (45%)

Culture positive (56%)

Management Issues with medical treatment

Pyrazinamide resistance (PTNA deletion)

Variable isoniazid resistance (0.1 ug/L vs 0.4 ug/L)

Role of surgery

Duration

Underlying immune deficiency

Clinical and Radiological resoultion

Report (cases) Regimen Duration Outcome

Finland (222) 1960-1988

Streptomycin 1/12 + INH + Ethionamide Streptomycin 1/12 + INH + Rifampicin INH + Ethionamide + Rifampicin

12 months 97% resolution 4 cases growth disturbance

Denmark (18) 1961-74

Streptomycin 1/12 + INH + Rifampicin

6 months Full resolution

Phone a friend Ethambutol Moxifloxacin Isoniazid Rifampicin Isoniazid Rifampicin

2 months

10 months

Discussion





Conditions increasing susceptibility to BCG infection

Mendelian susceptibility to mycobacterial sisease Primary immune deficiency Acquired

HIV Post BMT

SCID CGD

EDA-ID XR CD40L deficiency AR STAT1 deficiency AR IRF8 deficiency AR TYK2 deficiency

IFN-gR deficiencies AD STAT1 deficiency

XR gp91 phox deficiency AD IRF8 deficiency

XR Nemo deficiency IL-12 & IL12R deficiencies

AR IS GI5 deficiency

Non – infectious phenotype Ectodermal dysplasia (EDA-ID) Family history Consanguinity Infectious phenotype Disseminated NTM disease Systemic features History of past infections Mucocutaneous candidiasis FBE characteristics Total lymphocyte counts Blood dyscrasias Baseline immunological investigations HIV 1/2 antibody Immune globulins T cell subsets Oxidative burst test CD62L (L-selectin shedding)


Mendelian susceptibility to mycobacterial sisease Primary immune deficiency Acquired

HIV Post BMT

SCID CGD




XR Nemo deficiency IL-12 & IL12R deficiencies


Following history, examination, FBE and baseline immunology

tests


Mendelian Susceptibility to Mycobacterial Disease Primary Immune deficiency Acquired

HIV Post BMT

SCID CGD




XR Nemo deificiency IL-12 & IL12R deficiencies



tests


Mendelian Susceptibility to Mycobacterial Disease Primary Immune deficiency Acquired

HIV Post BMT

SCID CGD







tests

MSMD First reported in Maltese kindred 1996

Genetic defects in the interferon gamma (IFN-γ) - (IL-12) pathway

AD / AR / XR

Clinical presentation can be non-specific -> life-threatening

Unifocal NTM OM

IFNgR deficiencies

AD STAT1 deficiency

IL12 & IL12R deficiencies

Evaluation of IFN-gamma – IL12 pathway (RCH)

Royal Children’s Hospital Melbourne

Whole blood & PBMC

Set up on a Tuesday with blood from a healthy control

Method Incubate with PHA +/- IL-12 and the level measure IFN-gamma (Day 3)

Incubate with LPS +/- IFN-gamma and measure TNF-alpha & IL-12p70 (Day 1)

Westmead Immunology Laboratory

Flow cytometry IFN-gamma receptor, IL-12 receptor, Phospho-Stat 1 and Phospho-Stat 4

Gene sequencing for confirmation of suspected defects

Slides and information adapted from Sheree Poulton and Rashelle Farah, RCH Immunology Scientists


Mendelian susceptibility to mycobacterial disease Primary immune deficiency Acquired

HIV Post BMT

SCID CGD






Following IFN-gamma / IL12 pathway assay

TB PCR results need to be interpreted as MTB Complex positivity

Variable sensitivity depending on age, disease location and smear

BCG vaccine is safe and effective

Complication of osteomyelitis/osteitis is rare

Epiphyseal disease associated with worse outcome and warrants conservative approach to treatment duration

Evaluate for HIV, Primary immunodeficiency and MSMD

Conclusions

“Cold Case – a Hot Topic”

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