Diagnosis of Tuberculosis

Diagnosis of TB

• This presentation is about diagnosing Tuberculosis (TB)
• The presenter is Dr Marianne Black
• The presenter works at the Mycobacteriology Referral Laboratory in Braamfontein, Johannesburg
• She works at the Department of Clinical Microbiology and Infectious Diseases at the University of the Witwatersrand
• The date of presentation is July 2019

Lecture Objectives

• List specimen types suitable for TB investigations
• List advantages and limitations of direct microscopy for TB diagnosis
• List advantages and limitations of culture as a diagnostic method for TB
• List advantages and limitations of phenotypic drug resistance determination in TB
• List advantages and limitations of genotypic drug resistance determination in TB
• Compare Xpert MTB/RIF and Hain Line Probe assays
• Grade TB diagnostic platforms based on increasing sensitivity
• Explain the principle of latent TB diagnosis and list available platforms
• List limitations of existing tests for latent TB diagnosis
• Describe the principle of urine lipoarabinomannan assay and WHO recommendations

Specimen Collection and Transport

• Specimen Collection:
  • Safety for healthcare workers (HCWs)
  • Clear labeling
  • Good quality, representative specimens (early morning preferred, but not always feasible)
• Specimen Transport:
  • Safe packaging to minimize leakage/contamination
  • Delivery to the lab within 4 hours
  • Maintain cold chain, out of direct sunlight
  • Important to avoid overgrowth of other bacteria
  • High temperatures can kill bacilli (important for culture of M. tuberculosis)
  • Transportation should be on ice if necessary
• Specimen Types (for Culture):
  • Pulmonary TB: Sputum, induced sputum, gastric aspiration, bronchial washings, bronchoalveolar lavage (BAL)
  • Disseminated TB/Isolated Organ TB: Blood, cerebrospinal fluid (CSF), urine, lymph node aspirate, tissue, other body fluids, bone marrow, skin samples

Specimen Collection and Transport (Specific Details)

• Sputum: Sterile, wide-mouthed containers, induced sputum (nebulized hypertonic saline) - strict airborne precautions
• Gastric Aspirates: Early morning aspirate after fasting 4-6 hours
• Urine: First morning, clean catch, midstream urine (minimum 40 ml), 24-hour pooled specimens not acceptable
• Blood Culture: Aseptic collection, inoculated 5 ml at the bed site into broth media, BACTEC MyocoF Lytic
• Cerebrospinal Fluid (CSF): Minimum of 5 ml
• Tissue and Fluids: (e.g., lymph node/pleural fluid aspirate/biopsy), sterile container with no fixative or preservative

Active TB Disease Diagnosis

• Direct Microscopy: Auramine smear
• Culture: Specialized liquid culture media (mycobacteria growth indicator tube - MGIT)
• Drug susceptibility testing:
  • Phenotypic methods
  • Genotypic methods

Direct Microscopy (Advantages and Limitations)

• Advantages: Inexpensive, rapid results, quantitative measure of bacterial load (smear grade correlates with infectiousness - negative, scanty, 1+, 2+, 3+), treatment monitoring
• Limitations: Low sensitivity (25%-65%), visual detection limit is 10,000 bacilli/ml, influenced by specimen quality (sputum vs. saliva, site, HIV status). Cannot differentiate mycobacterial species (MTB complex vs. NTM), Cannot differentiate between viable/nonviable bacilli, Cannot determine drug susceptibility

Culture (Advantages and Limitations)

• Advantages: Gold standard, high sensitivity (10-100 bacilli/ml), precise species identification, allows for (culture-based) phenotypic drug susceptibility testing, useful to monitor treatment response
• Limitations: Mycobacteria divide once/day - lengthy time to result, prone to contamination (bacteria/fungi may outgrow mycobacteria), requires biosafety level 3 laboratory infrastructure

Drug Susceptibility Testing (DST)

• Phenotypic (culture-based) methods: Comparison of growth in the presence and absence of the drug, gold standard
• Genotypic methods: Detect mutations in the DNA sequence that confer resistance
• Gene Xpert MTB/RIF assay (GXP) / Xpert® MTB/RIF Ultra, Hain Lifescience® Line probe assays (LPA)

Gene Xpert MTB/RIF Assay

• Identify MTB complex and provides rifampicin susceptibility result
• Performed on pulmonary and selected extra-pulmonary specimens (e.g., purulent fluid, tissue, CSF)
• Turn-around time of 24 hours

Xpert MTB/RIF Ultra Assay

• Concentrates bacilli and removes inhibitors
• Sample is filtered and washed
• Ultrasonic lysis of filter to release DNA
• DNA mixed with dry PCR reagents
• Semi-nested real-time amplification and detection in integrated reaction tube
• Time-to-result: 1 hour 45 minutes
• Printable test result
• Minimal expertise needed
• Turnaround time (TAT) of 24 hours (laboratory-based)

Drug Susceptibility Testing (Genotypic methods - LPA)

• Hain Line probe assay (LPA) MTBDRplus and MTBDRsl
• Detects MTB complex
• 1st line LPA: rifampicin and isoniazid
• 2nd line LPA: fluoroquinolones and second-line injectable agents
• Performed on clinical specimens and culture
• More complex process, expertise needed, laboratory infrastructure
• TAT: 2-3 days

Latent TB Infection Diagnosis

• Principle: Detect immune response (cell-mediated immunity) to previous infection with M. tuberculosis
• Available platforms:
  • Skin: Induration in response to inflammation (tuberculin skin test [TST] or purified protein derivative [PPD], Mantoux test)
  • Blood: Production of interferon-gamma (IFN-γ) by white blood cells (interferon gamma release assays [IGRAs])

Latent TB Infection Diagnosis - Limitations

• Cannot differentiate between latent TB infection and active TB disease
• May be positive in both disease and infection
• Requires further microbiological testing
• False negatives may occur in immunocompromised or advanced TB disease
• Limited use in highly endemic countries
• TST: Time-consuming, subjective, less specific than IGRA
• IGRA: Expensive, but more specific than TST (injected antigens specific to M. tuberculosis)

Lateral Flow Urine Lipoarabinomannan Assay (Urine LAM Test)

• Based on detection of mycobacterial lipoarabinomannan (LAM) antigen in urine
• Released from metabolically active or degenerating mycobacterial cells
• Point-of-care test for TB
• LAM only present in people with active TB disease
• Urine is easy to collect and store, and lacks infection control risks associated with sputum collection
• Improved sensitivity in HIV-TB co-infection (lower CD4 counts, especially below 100 cells/µL)
• Limitations: Not good for general screening, negative result does not exclude TB (other diagnostic tests needed), does not differentiate species of mycobacteria

WHO Recommendations for Urine LAM

• Inpatient Settings: WHO strongly recommends using LF-LAM to assist in the diagnosis of active TB in HIV-positive adults, adolescents, and children with signs and symptoms of TB, advanced HIV disease or seriously ill, or with a CD4 count of less than 200 cells/mm³
• Outpatient Settings: WHO suggests using LF-LAM to assist in the diagnosis of active TB in HIV-positive adults, adolescents, and children with signs, symptoms of TB or seriously ill, or with a CD4 count of less than 100 cells/mm³
• WHO recommends against using LF-LAM in outpatient settings without assessing TB symptoms or without TB symptoms and a CD4 count above 200 cells/mm³

Limit of Detection

• Microscopy: 5,000-10,000 bacilli/mL
• MTBDRplus: 160 bacilli/mL
• Xpert MTB/RIF: 130 bacilli/mL
• Culture: 10-100 bacilli/mL
• GXP Ultra: 10-100 bacilli/mL

TB Diagnostic Algorithm

• Refer to presentation for the detailed algorithm

This quiz covers the fundamentals of diagnosing Tuberculosis (TB), presented by Dr. Marianne Black from the University of the Witwatersrand. Participants will explore various diagnostic methods, specimen types, advantages, and limitations involved in TB investigations. Test your knowledge on contemporary TB diagnostic platforms and drug resistance determination techniques.