Diagnosing childhood TB

Brian Eley

Paediatric Infectious Diseases Unit

Red Cross War Memorial Children’s Hospital

Department of Paediatrics and Child Health

University of Cape Town

I. The microorganism must be found in abundance in all

organisms suffering from the disease, but should not be

found in healthy organisms.

II. The microorganism must be isolated from a diseased

organism and grown in pure culture.

III. The cultured microorganism should cause disease

when introduced into a healthy organism.

IV. The microorganism must be re-isolated from the

inoculated, diseased experimental host and identified as

being identical to the original specific causative agent.

Koch postulates

Koch R. Über tuberkulose, 24 March 1882, Berlin Physiological Society

TB diagnosis

• Careful history (including history of TB contact &

symptoms consistent with TB)

• Clinical examination (including growth

assessment)

• Tuberculin skin testing (TST)

• Bacteriological confirmation whenever possible

• Investigations relevant for suspected (1)

pulmonary TB, and (2) extrapulmonary TB

• HIV testing (in high HIV prevalence areas)

WHO, WHO/HTM/TB/2006.361, 2006

WHO, Int J Tuberc Lung Dis 2006;10(10): 1091-7

History Close contact: living in the same household or in frequent contact with a source

case (e.g. caregiver) with sputum smear-positive TB.

- Children < 5 years in close contact with a smear-positive TB should be

screened for TB

- After TB is diagnosed in a child or adolescent, an effort should be made to

detect the adult source cases

- If a child presents with TB, then other childhood contacts should be sought and

screened for active TB, especially undiagnosed household contacts

- After TB is diagnosed in a child who is resident of a long-term facility an

outbreak investigation should be initiated in order to detect adult source cases

and cases of TB among the other child residents

Symptoms: Most children with symptomatic disease develop chronic symptoms

- Unremitting, persistent cough for > 2 weeks

- Fever (>38°C) for 14 days after common causes e.g. pneumonia have been

excluded

- Weight loss (>5%) or failure to thrive (growth faltering in the last 3 months, or

WAZ / WHZ ≤ -2 in the absence of information about recent growth trajectory)

- Persistent unexplained lethargy / reduced playfulness

WHO, Int J Tuberc Lung Dis 2006;10(10): 1091-7

Graham SM, et al. J Infect Dis 2012;205:S199-208)

Outbreak: Children’s home, Khayalitsha

• Index case: 8 year old, CP, culture-confirmed TB

• 38 screened: 32 children, 6 adults

• 46% (n=26): Mantoux ≥ 10mm, 4 commenced on anti-TB RX (3/4

culture-confirmed disease)

• Index strain and 2 contact strains related on genotypying

Spoligotyping W-Beijing Strain

Index Sat 1 Sat 2

David Moore, 2010

Physical examination Signs of hypersensitivity

Phlyctenular conjunctivitis, erythema nodosum, polyarthritis (Poncet arthritis)

PTB: no specific signs

Signs highly suggestive of EPTB

Non-painful enlarged cervical adenopathy – matted ± fistula formation

Gibbus, especially recent onset

Signs requiring investigation to exclude EPTB

Non-painful enlarged lymph nodes without fistula formation

Pleural effusion

Pericardial effusion

Unexplained hepatomegaly, splenomegaly, hepatosplenomegaly

Distended abdomen with ascites

Papable abdominal lymphadenopathy

Meningitis not responding to antibiotic treatment, with sub-acute onset or

raised intracranial pressure

non-painful monarthritis

Cutaneous manifestations e.g. Papulonecrotic-type TB

Adapted from: WHO, Int J Tuberc Lung Dis 2006;10(10): 1091-7

Tuberculin skin test (TST) 5 TU of PPD or 2 TU of PPD RT23 (0.1ml)

Intradermal administration

Position: left forearm, palm-side up

Read between 48-72 hours after administration

Measure horizontal diameter of induration using

a clear flexible ruler

Young child, 20mm induration; courtesy Dr Candyce Levin, August 2013

TU=tuberculin units; PPD=purified protein derivative

TST interpretation Positive TST

induration ≥ 5 mm in HIV and severely malnourished children; ≥ 10 mmin all other

children

Infection vs disease

Negative TST

Never rules out a diagnosis of TB

False-positive TST

Incorrect interpretation of test

BCG vaccination

Infection with nontuberculous mycobacteria

False-negative TST

Incorrect administration or interpretation

HIV infection

Improper storage of tuberculin

Severe TB

Viral infection (e.g. measles, varicella)

Vaccinated with liove virus vaccines (within 6 weks)

Malnutrition

Immunosuppresives (e.g. glucocorticosteroids)

Neonatal period

Primary immunodeficeincy diseases

Low protein states

Diseases of lymphoid tissue (e.g. Hodgkin disease, lymphoma, leukemia, sarcoidosis)

Bacterial confirmation

• Sputum:

– Spontaneously expectorated

– Induced after 3% NaCl nebulisation

• Gastric aspirate / gastric lavage aspirate

• Fine needle aspirate

• Lymph node biopsy

• Other extrapulmonary specimens

Smear microscopy

Fluorescence microscopy (FM)

Detects 10% more TB cases than LM

FM requires 25% of the time taken to

read a ZN stained smear

Bactec MGIT 960 culture system

Liquid culture versus solid culture

Average time to growth detection: 10-14

days (LC) versus 4-6 weeks (SC)

LC 20% more sensitive than SC

Yield from smear & culture

Recent diagnostic study

452 children with at least 1 induced sputum specimen

evaluated

Children screened for suspected TB if cough present for

>14 days plus one of the following: (1) a household contact

infected with M. tuberculosis within last 3 months, (2) LOW

or failure to gain weight in last 3 months, (3) a positive TST,

or (4) a chest radiograph suggestive of PTB

27 (6%) had a positive smear result

70 (16%) had a positive culture result

Nicol MP, et al. Lancet Infect Dis 2011;11(11):819-824

Investigation of EPTB

Infection site Diagnostic approach

Peripheral lymph nodes

(especially cervical)

Fine needle aspiration (FNA) or lymph node biopsy

TB meningitis Lumber puncture(opening pressure, biochemical

analysis, microscopy & culture), CT scan, air

encephalogram to determine whether

hydrocephalus is communicating

Miliary TB Chest radiograph, lumber puncture, eye examination

for choroidal tubercles

Pleural effusion Chest radiograph, pleural tap for biochemical

analysis, microscopy & culture

Abdominal TB Abdominal ultrasound, ascitic tap for biochemical

analysis, microscopy & culture, laparoscopy and

peritoneal biopsy, other histological tissue

Osteoarticular TB Radiographs, joint tap, and/or synovial biopsy

Pericardial TB Echocardiogram, pericardial tap for biochemical

analysis, microscopy & culture

Chronic ear discharge (especially

in HIV-infected children)

Pus swab for microscopy & TB culture

Key features suggestive of TB

Presence of three or more of the following is

strongly suggestive of TB:

Chronic symptoms suggestive of TB

Physical signs highly suggestive of TB

A positive TST

Chest radiograph suggestive of TB

WHO, Int J Tuberc Lung Dis 2006;10(10): 1091-7

Diagnostic certainty: intrathoracic TB

Confirmed TB: (1) at least one symptom or sign of TB plus (2) isolation of M.

tuberculosis on culture of sputum, gastric washings, or fluid/tissue from a site

that is normally sterile

Probable TB: (1) at least one symptom or sign of TB, and (2) chest

radiography consistent with TB, and (3) at least 1 of the following: (a) a positive

response to anti-TB treatment, (b) documented exposure to M. tuberculosis

infection, or (c) immunological evidence of M. tuberculosis infection

Possible TB: At least one symptom or sign of TB, and either (1) one of the

following: (a) a positive response to anti-TB treatment, (b) documented

exposure to M. tuberculosis infection, or (c) immunological evidence of M.

tuberculosis infection, or (2) chest radiography consistent with TB

Graham SM, et al. J Infect Dis 2012;205:S199-208

Impact of HIV on TB diagnosis

History including contact history Important because of poor sensitivity of

TST

Symptoms consistent with TB Lower specificity due to overlap between

symptoms of TB & HIV

High proportion of patients with short

duration of symptoms

Examination including growth Lower specificity because malnutrition

common in TB & HIV

Tuberculin skin testing Lower sensitivity; TST positivity with

immunosuppression

Chest radiograph findings Lower specificity: overlap with HIV-

related disease

Bacteriological confirmation Important but beyond capabilities of

many clinicians, Lacks sensitivity

Investigations relevant for

suspected PTB and EPTB

Wide range of diagnostic possibilities

because of other HIV-related disease

Adapted from: WHO & IUATBLD, Guidance for national tuberculosis & HIV programmes, 2010 (near-final draft)

Newer diagnostic assays

Interferon-gamma release assays: ELISPOT assay (T-SPOT), ELISA

(QuantiFERON-Gold)

Nucleic acid amplification tests (NAAT): loop-medicated isothermal

amplification (TB-LAMP), Line probe assays (e.g. GenoType

MTBDRplus assay, GenoType MTBDRsl), Xpert MTB/RIF

Antigen detection assays: MPB64 (e.g. Capilia TB), urinary

liporarbinomannan (LAM)

Biosignatures: host proteomic & gene expression signatures,

serological response to TB protein arrays

Perkins MD, et al. J Infect Dis 2007;196(Supp 1):S15-S27

Pai M, et al. Sem Respir Crit Care med 2008;29:560-568

Swaminthan S et al. Clin Infect Dis 2010;50(S3):S184-S194

Wallis RS, et al. Lancet 2010;375:1920-1937

Lawn , et al. AIDS 2009;23:1875-1880

Perez-Velez CM, et al. N Engl J Med 2012;367:348-61

Berry MPR, et al. Nature 2010;466:973-79

Interferon-gamma release assays (IGRAs)

Mandakalas, AM, et al (2011), n=31

TST & IGRAs have similar accuracy

Heterogenous methodology limited comparability of studies

Neither TST nor IGRAs perform sufficiently to ‘rule in’ or ‘rule out’ active TB as

a single test

Machingaidze S, et al. (2011), n=20

No clear evidence that IGRAs should replace TST for detecting LTBI in

children

Sensitivity of IGRAs for TB was no different from TST, ranging from 53% to

94%

Sensitivity of IGRA was significantly reduced in high-burden settings

compared to low-burden settings [pooled sensitivity: 55% (CI: 37-73%) vs 70%

(CI 53-84%)]

Diagnostic usefulness of a combination of TST & QFT should be explored

Mandalakas AM, et al. Int J Tuberc Lung Dis 2011;15:1018-1032

Machingaidze A, et al. Pediatr Infect Dis J 2011;30:694-700

IGRAs: WHO policy statement, 2011

Insufficient data on the performance of IGRAs in low- and middle-income

countries

IGRAs cannot accurately predict the risk of infected individuals developing

active TB disease

IGRAs should not be used for the diagnosis of active TB disease

IGRAs are more costly and technically complex to do than TST. Replacing TST

by IGRAs as a public intervention in resource-constrained settings is not

recommended

IGRAs should not replace TST in low- and middle-income countries for

the diagnosis of latent TB infection in children, nor for the diagnostic

work-up of children (irrespective of HIV status) suspected of active TB in

these settings.

WHO, 2011: http://whqlibdoc.who.int/publications/2011/9789241502672_eng.pdf

Liebeschuetz S, et al. Lancet 2004;364:2196

Davies M, et al. AIDS 2009;23:961-969

Boehme CC, et al. N Engl J Med 2010;363:1005-15

Xpert MTB/RIF: WHO policy statement, 2011

Xpert MTB/RIF should be used as the initial test in

individuals with suspected MDR-TB or HIV-associated TB

(strong recommendation)

Xpert MTB/RIF may be considered as follow-on test to

microscopy in settings where MDR-TB or HIV is lesser

concern, especially for further testing of smear-

negative disease (conditional recommendation)

These recommendations support the use of one sputum

specimen for diagnostic testing and apply to adults &

children

WHO: http://whqlibdoc.who.int/publications/2011/9789241501545_eng.pdf

Xpert MTB/RIF in children

Nicol MP, et

al. (2011)1

Prospective, descriptive study

452 children, median age: 19.4 months with ≥ 1 induced sputum

specimen; 108/452 (24%) had HIV infection

Key findings Overall: 27 (6%) smear+; 70 (16%) culture+, 58 (13%) Xpert+

Two independent specimens obtained in 385 children: 58 had culture-

confirmed TB; Xpert MTB/RIF detected 34/58 (58.7%) on 1st sputum

and 44/58 (75.9%) on 2 specimens; specificity=98.8% when both

specimens evaluated.

Xpert MTB/RIF detected all 22 smear-positive cases and 22/36

(61.1%) smear-negative cases

Xpert MTB/RIF done on 2 induced sputum specimens detected twice

as many TB cases as did smear microscopy (75.9% vs 37.9%)

Sensitivity of Xpert MTB/RIF higher in HIV-infected children: 14/14

(100%) vs 30/44 (68.2%)

Median time to results: Xpert MTB/RIF=1 day, TB culture=12 days

Conclusion Two independent specimens increases the diagnostic yield

1Nicol MP, et al. Lancet Infect Dis 2011;11(11):819-824

Xpert MTB/RIF in children (2) Study n Median

age

Sample

type

Key findings

Zar HJ,

et al.

(2012)1

535 19 mo NPA/IS 535 children had at least 1 IS & I NPA specimen; 392 had 2 paired specimens

Smear+, Xpert+, Culture+ in 30(5.6%), 81 (15.1%) & 87 (16.3%) children

Culture yield: IS, 84/87 (96.6%) vs NPA, 61/87 (70.1%), p<0.001

Among children with 2 paired specimens there were 63+ culture cases:

- Yield higher for IS, 60 (95.2%) vs NPA 48 (76.2%), p=0.002

- Sensitivity of 2 Xpert results similar: IS, 45 (71%) vs NPA, 41 (65%), p=0.44

- Lower smear sensitivity: IS, 21 (33%) vs NPA, 16 (25%)

Incremental yield from 2nd specimen:

- IS: 9 cases (17.6%) by culture vs 9 cases (25%) by Xpert

- NPA: 10 cases (26.3%) by culture vs 11 cases (36.7%) by Xpert

Rachow

A, et al.

(2012)2

164 5.8 yr Sputum 28(17.1%) culture-confirmed TB - Xpert detected 100% smear+ / 66%

smear- cases

Xpert detected 21/28 (75%) culture positive cases

Xpert also detected 4/47 (8.5%) of cases of highly probable TB

Xpert detected 3X more confirmed TB than smear microscopy

Increasing sensitivity of Xpert & culture with serial sampling

- 46.4% vs 75% (1st specimen)

- 60.7% vs 96.4% (2nd specimen)

- 75% vs 100% (3rd specimen)

Bates M,

et al.

(2013)3

930 24 mo GLA Performance of Xpert on gastric lavage aspirate and sputum was similar

1Zar HJ, et al. Clin Infect Dis 2012; Aug 3 (Epub ahead of print) 2Rachow A, et al. 2012;54:1388-96 3Bates M, et al. Lancet Infect Dis 2013;13:36-42

Xpert MTB/RIF: extra-pulmonary specimens

Study Country TB Gld

std dx (n)

TB not

dx (n)

Main sample types

testing positive for TB

Xpert

sensitivity %

(95% CI)

Xpert

specificity%

(95% CI)

Armand1 France 32 NA LN (16), pl/fluid (7), bone (5) 53.1 (34.7-70.9) NA

Causse2 Spain 41 299 Tissue (18), CSF (6), g/aspirate

(8), pl/fluid (4) purulent exudates

(5)

95.1 (83.5-99.4) 100 (98.8-100)

Friedrich3 S Africa 20 5 Pleural fluid (25) 25.0 (8.7-49.1) 100 (47.8-100)

Hillerman4 Germany 45 476 Tissue (30), g/aspirates (8), urine

(5) 77.3 (60.5-87.1) 98.2 (96.0-98.9)

Ligthelm5 S Africa 30 18 FNA 96.6 (86.6-100) 88.9 (69.6-100)

Moure6 Spain 108 41 All smear-negative; pl/fluid 926),

LN (34), abscess aspirate (17),

tissue (120

58.3 (48.5-67.8) 100 (91.4-100)

Vadwai7 India 283 250 Tissue (105), pus (98), body fluids

(24) 80.6 (75.5-85.0) 99.6 997.8-100)

Tortoli8 Italy 268 1206 Tissue/FNA (94), pl/fluid (18)

g/aspirates (61), pus (55), CSF

(14), urine (16), cavitary fluid (10)

81.3 (76.2-85.8) 99.8 999.4-100)

1Armand S, et al. J Clin Microbiol 2011;49:1772-6 2Causse M, et al. J clin Microbiol 2011;49:3065-7 3Frierich SO, et al. J Clin Microbiol 2011;49:4341-2 4Hillerman D, et al. J Clin Microbiol 2011;49:1202-5

5Ligthelm LJ, et al. J Clin Microbiol 2011;49:3967-70 6Moure R, et al. J Clin Microbiol 2011;49:1137-9 7Vadwai V, et al. J Clin Microbiol 2011;49:2540-5 8Tortoli E, et al. Eur Respir J 2012;40:442-7

Xpert: extra-pulmonary specimens in children

Samples n Gold standard = positive culture

or clinical diagnosis of TB

Gold standard = positive culture

Sensitivity % (CI) Specificity % Sensitivity % (CI) Specificity %

Biopsy

specimens‡

101 100 (100-100) 100 100 (100-100) 95.6

Pleural fluid 38 100 (100-100) 100 100 (100-100) 100

Gastric

washings

174 81 (70-91) 100 80 (69-91) 97.6

Pus 109 100 (100-100) 100 100 (100-100) 100

CSF 47 75 (45-105) 97.4 75 (45-105) 97.4

Urine 18 66.7 (13-120) 100 66.7 (13-120) 100

Cavitary fluid• 7 50 (0-119) 100 50 (0-119) 100

Total 494 86.9 (80-93) 99.7 86.3 (80-93) 98.2

Tortoli E, et al. Eur Respir J 2012;40:442-7

‡included fine needle aspirates; •includes peritoneal, synovial and pericardial fluid specimens

Xpert MTB/RIF in paediatric practice

• Limitations of Xpert MTB/RIF in paediatric practice

include: lower sensitivity than culture, (2) Xpert MTB/RIF

is not a M. tuberculosis specific test but identifies all

mycobacteria within the M. tuberculosis complex (3)

Xpert MTB/RIF is unable to identify INH-monoresistent

TB isolates

• Xpert MTB/RIF should be used in conjunction with TB

culture

• WHO (provisional): one Xpert MTB/RIF test in place of

smear microscopy

• Xpert MTB/RIF appears useful on selective extra-

pulmonary specimens

• Policy for academic hospitals awaits provincial approval

Line probe assays: GenoType MTBDRplus ® V2

GenoType MTBDRplus ® V2 vs Xpert MTB/RIF

Evaluation: 282 consecutive specimens

Overall sensitivities were 73.1% and 71.2%

respectively

Smear/culture positive specimens:

- Xpert detected 19/21 (90.5%); LPAv2.0 detected

21/21 (100%)

- Smear negative/culture positive specimens

-Xpert detected 18/31 (58%) & LPAv2.0 17/31

(56.6%)

Advantages of LPAv2.0:

- enhanced sensitivity compared to LPAv1.0

- ability to determine INH susceptibility

Disadvantages of LPAv2.0:

- prolonged time to diagnosis

- requires skilled personnel & additional lab space

Barnard M, et al. J Clin Microbiol 12 September 2012

Are alternative diagnostic strategies

required?

• Unresolved challenges: – Technical challenges associated with routine culture

– Low yield from culture & Xpert MTB/RIF in paediatric respiratory

specimens

– Limited usefulness of Xpert MTB/RIF in possible & probable TB

– Absence of tests with optimal diagnostic specifications

• Can improved diagnostic assays be developed? – No clear answer at this juncture

– Role of disease-related proteomic, transcriptomic or

metabolomic biosignatures under investigation

Mistry R, et al. J Infect Dis 2007;195:357-65

Jacobsen M, et al. J Mol Med 2007;85:613-621

Berry MPR, et al. Nature 2010; 466:973-977

Lesho E, et al. Tuberculosis 2011;91:390-9

Lu C, et al. Plos One 2011;6(8):e24290

Maertzdorf J, et al. PNAS 2012;109:7853-8

MPR Berry et al. Nature 2010; 466, 973-977

Whole-blood 86-gene signature of active TB

is distinct from other diseases

Conclusions

• We remain dependent of existing TB diagnostic

methods

• The role of Xpert MTB/RIF has been defined but

awaits widespread implementation

• The role of complex biosignatures requires

exploration