ADVANCES in BACTERIOLOGICAL

DIAGNOSIS of TUBERCULOSIS

Assoc. Prof. Alpaslan Alp

Hacettepe University Faculty of MedicineDepartment of Medical Microbiology

Tuberculosis in the World • WHO 2009:

• 14 million cases• 1.68 million

death

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Undetected probable cases in 2009 (WHO)

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WHO 2009:

• WHO; case detection rate in 2009: 63%

• Half of TB cases are detectable in Africa.

• Patients do not admit• Inadequate diagnostic tools

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Laboratory Diagnosis

• Smear microscopy: 20-80% sensitivity in culture-confirmed cases.

• At least two sputum specimens are necessary.

• Problem in HIV-infected and pediatric patients.

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Laboratory Diagnosis

• Culture: Results can be obtained in several weeks.

• Well-equipped laboratories are necessary.

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Rate limiting step in TB control: Case Detection

• 625.000 life/year could be saved by using tests with 100% accuracy.

• 392.000 life/year could be saved by using tests with 85% sensitivity and 97% specificity…22.4% of deaths due to TB.

For the laboratory diagnosis of TB;

Is there an IDEAL TEST?

• with high sensitivity,• with high specificity,• easy to perform,• rapid,• cheap…

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For the laboratory diagnosis of TB;

There is NO IDEAL TEST !

• Molecular methods– Diagnostic tests– Tests detecting resistance

• Point of care tests

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Possible candidates could arise from which test group?

Drug Susceptibility Tests

• Phenotypic Methods:– Solid Media

• Löwenstein-Jensen medium• Middlebrook agar

– Rapid culture systems (liquid media)• Bactec 460• MGIT• Mycolor-TK

• Genotypic Methods:– Mutation detection methods

• Reverse hybridization (Strip tests)• Real-time PCR• DNA sequencing

Molecular Methods

• 1-2 days• Well-standardized commercial methods

• Inno-Lipa Rif TB• Hain MDRTBplus• Cepheid Xpert MTB/RIF

Direct susceptibility testing

• Should be performed in smear-positive cases.• Rapid; reflects bacteria population better;

cheaper.• Calibration of inoculum is more difficult;

normal respiratory flora can grow despite decontamination process

• Overall rate of failure can be 10-15% or more.

Direct susceptibility testing

• MTBDR strip test• MTBDRplus strip test• Nitrate reduction assay• MODS: Microscopic observation drug

susceptibility

Direct Susceptibility TestsMeta-Analysis Study

• The studies in which INH and RIF resistance detected by direct method were selected.

• Eighteen studies out of 64, were included in meta-analysis.

Direct Susceptibility TestsMeta-Analysis Study

Method Sensitivity (%) Specificity (%)

MTBDR 99 98

MTBDRplus 99 99

Nitrate Red. Assay 99 100

MODS 96 96

Detection of rifampicin resistance:

Direct Susceptibility TestsMeta-Analysis Study

Method Sensitivity (%) Specificity (%)

MTBDR 71 100

MTBDRplus 96 100

Nitrate Red. Assay 94 100

MODS 92 96

Detection of isoniazide resistance:

Direct Susceptibility TestsMeta-Analysis Study

Method Turn around time

MTBDR 1-2 days

MTBDRplus 1-2 days

Nitrat Red. Assay 3 weeks

MODS 3 weeks

WHO Recommendations for Strip Tests-2008

• Adoption of line probe assays for rapid detection of MDR-TB should be decided by Ministries of Health.

• Direct testing of sputum smear-positive specimens and indirect test on isolates grown from smear-negative and smear-positive specimens are adequately validated.

• Direct use on smear-negative specimens is not recommended.

• Adoption of line probe assays does not eliminate the need for conventional culture and DST.

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Point of Care Tests

• Tests performed instantly, near the patient

• Should be cheap, accurate, practical• Should be performed in any treatment

site without any need for a specialized laboratory.

• Should be performed on different specimens like blood, urine and breath

• Duration:<3saat (even 15min…5min)

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Lipoarabinomannan

• Lipoarabinomannan (LAM): within Mtb cell wall,

• 17.5 kDa, heat-stable, glycolipid

• 15% of bacterial weight • Immunogenic, virulence

factor

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Detection of LAM in Urine

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LAM ..Clerview-TB-ELISA

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Dheda 2010: 440 TB, 31% infected with HIV

Determine TB-LAM (Alere)

• Will be sold within 2012

• LAM Ag in urine

• 30 minutes• 3-3.5 USD

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Detection of LAM in Urine

Peter 2011:Active TB, HIV (+) patients

Clearview TB-ELISA

• Overall sensitivity: 58%

• Sensitivity in CD4<100cells/ml: 72%

Determine TB-LAM

• Overall sensitivity: 74%

• Sensitivity in CD4<100cells/ml: 85%

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“TB-Breathalyzer” (Rapid Biosensor Systems Inc.)

• Mtb Ag85B in cough• Collection tube containing Ab;

fluorescent response• Sensitivity 74%, specificity 79%

(31 TB patients)• Sensitivity together with smear

microscopy: 93.5%

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Xpert MTB/RIF Assay

• Product of a collaborative study:– FIND (Foundation for Innovative New Diagnostics)

– Cepheid (USA)

– University of Medicine and Dentistry of New Jersey

• Detection of M. tuberculosis and rifampicin resistance in two hours.

• A “hemi-nested real-time PCR” that uses three primers and five probes.

Xpert MTB/RIF Assay

Satisfactory results were obtained in the studies that were performed in 2010:

• Sensitivity in smear-positive culture-positive specimens: 99%

• Sensitivity in smear-negative culture-positive specimens: 90%

Xpert MTB/RIF Assay Meta-Analysis Study

• Eighteen study had been chosen among 90 studies.

• A total of 10.224 specimen– 2983 bacteriological TB diagnosis– 6183 smear and culture-negative

• Average sensitivity for diagnosis of TB: 90.4%• Average specificity for diagnosis of TB : 98.4%

Xpert MTB/RIF Assay Meta-Analysis Study

• Average sensitivity for detection of rifampicin resistance: 94.1%

• Average specificity for detection of rifampicin resistance: 97.0%

• Average sensitivity in extra-pulmonary specimens: 80.4%

• Average specificity in extra-pulmonary specimens : 86.1%

Xpert MTB/RIF AssayMeta-Analysis Study

COMMENT:

• Xpert MTB/RIF assay fulfills the requirements of rapidly and effectively diagnosing TB and RIF resistance.

• Since the mutation points in 5% RIF-resistant Mtb isolates occur outside core gene region, they would not be identified by Xpert MTB/RIF assay.

TB Scent• Giant African pouched rats trained to

detect TB evaluated sputum samples from 10.523 patients whose sputum had previously been evaluated by smear microscopy.

• Microscopists found 13.3% of the patients to be positive.

• Screening of the samples by rats revealed 620 new TB-positive patients, increasing the case detection rate by 44%.

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TB Scent

• 10 well-trained giant African pouched rats (Cricetomys gambianus)

• 23.101 sputum samples• Smear microscopy: 2.487 positive

sputum• Rats identified 2.274 of these

samples; an additional 927 samples were also found to be positive by the rats, increasing the case detection rate by 44%.

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TB Scent

• Daily sensitivity of rats to detect Mtb: 72-100%

• Daily false positivity: 0.7-8.1%

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TB Scent

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• Metil fenilasetat• Metil p-anisat• Metil nikotinat• o-fenilanizol

Electronical Nose !

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