MDR-TB Diagnosis: How new and currenttools can be used to improve diagnosis

Claude Muvunyi MD, PhD

Meeting the Experts on MDR‐TB 8th December 2012, ITM, Antwerp

OUTLINE

• Part I: Sample Collection, Transport and Processing

• Part II: Overview of New lab techniques

Dr. Affolabi Dissou

Introducing New Technology• Infrastructure, biosafety measures and maintenance

• Equipment validation and maintenance

• Specimen transport and referral mechanisms

• Management of laboratory commodities and supplies

• Laboratory information data and management system

• Laboratory quality management

• Strategies for HR development and retention

Specimen• Appropriateness

• Collection

• Transport to laboratory

• Processing (Inoculation of media, Identification, DST, molecular test)

• Report

Appropriate Specimen• From relevant body site

• Lungs (Sputum)• Pleura

• Central nervous system• Lymphatic system• Genitourinary systems• Bones and joints• Disseminated (miliary

TB)• Pericardial disease

Type of sample

Sputum (resp. sample), CSF, gastric washings, lymph nodes (tissues), urine, faeces…

Source: CDC, 2001.

Collection (1)• Appropriate equipment (Proper containers for sputa)

Collection (2)• Good instructions to patient• Explain and demonstrate procedure

Wrong position

Collection (3)

- Sputum collection outside or in an empty room with very good ventilation

- The health worker should supervise,but should NOT stand in front of the patient

Collection (4)

How many? 2 or 3 for sputum? (spot/ morning)

Quality? salivary (white, watery, frothy)?

Quantity? at least 3 ml (3 – 5 ml)

Quality and amount of samples are crucial

Specimen Quality

Poor quality sputum

Better quality Source: CDC, 2007

Patient instruction for sputum sampling can improvetuberculosis diagnosis

A 16% increase in diagnostic yield Sputum samples from the intervention group were of greater volume

int j tuberc lung dis 9(7):814–817; 2005

TotalOdd Ratio P-value(95% Cl)

Gross appearance of sputum specimenssputum (versus saliva ) 1.69 (1.15-2.49) 0.007purulent, bloody (versus saliva, mucoid) 2.26 (1.58-3.23) <0.001

Volume of sputum specimens≥1ml (<1ml versus ≥1ml) 1.44 (1.02-2.03) 0.039≥2ml (<2ml versus ≥2ml) 1.30 (0.97-1.74) 0.080 ≥3ml (<3ml versus ≥3ml) 1.15 (0.84-1.58) 0.387 ≥4ml (<4ml versus ≥4ml) 1.44 (1.08-1.92) 0.014≥5ml (<5ml versus ≥5ml) 1.39 (0.76-2.53) 0.287

correlation of smear results with quality and quantity of sputum specimens

Yoon et al. BMC Infectious Diseases 2012, 12:172

Studies Method of collection

Number of specimens

% of positive smear

% of positive culture

Pande, et al. 1974 Spot 160 52 98Morning 160 85 92

Kim, et al. 1989 Spot 2,736 7Morning 2,968 12

Otto D. Schoch et al. 2007

Spot 83 54 39Morning 84 62 49

Case-yields of sputum specimens collected on the spot and morning

Case Yields of Multiple Sputum Specimenson Smear and Culture Examinations

(Review of 37 studies)

(Review of 4 studies)

Mase S, et al. I J TLD 2007;11(5):485-95.

Transport to Laboratory

• Safe packaging

• Temperature• Good labeling

Safe; Quick; Always in a cold box

Viability of M. tuberculosis in sputum stored at different temperatures

0

10

20

30

40

50

60

70

80

90

100

12

34

56

10090

81

63

29

10

100 100

100

9081

73

Room Refrigerator

Day 0Day 3 Day 7

Day 14Day 21

Day 28

Paramasivan CN et al. Tubercle. 1983 Jun;64(2):119-24.

No. of sputa tested: 41

0102030405060708090

100

12

34

5

99

87

5040

37

99 97

8067 67

Room Refrigerator

Day 0Day 3

Day 7Day 14 Day 28

Viability of M. tuberculosis in sputum stored at different temperatures

H. T. Banda et al. Int j tuberc lung dis 4(3):272–274 2000

No. of sputa tested: 30

Transport ≤ 4 days for ≥ 90% of specimens

Yes No

Ship to reference laboratory

Decontaminate with 4% NaOH

Centrifuge at 300g and decent

Inoculate on LJ medium

Inoculate on Ogawa Medium

Add cetylpyridinium and ship to reference laboratory

Centrifuge at 3000g with cooling turned off and decant repeat

centrifugation after suspending the pellet in sterile 2% NaCl solution

Inoculate on LJ medium

Processing: Decision for transport medium, decontamination, centrifugation, and choice of medium.

Reporting

• Recod result in TB laboratory register

• Verify lab serial number on specimen report form and register are the same before reporting

• Submit timely reports to the treatment facilities

• Maintain an accurate TB laboratory register and worksheet register

• Signature

20

Specimen Transportation in the Lab Network

DH Laboratories

HC Laboratories

NRL , CHUK, (CHUB in near future)

Samples sent for CD4 count, Hematology, clinical chemistry and QC

Daily basis with motorbike

Feedback

Samples sent  for  specialized analysis ( DBS, VL Tb culture, DST and epidemic  disease)

Samples sent  for  specialized analysis ( DBS, VL Tb culture, DST and epidemics  diseases)

Tests   performed and feedback sent twice per week to different levels by TRAC NET, phone call and  courier

21

Map Of Rwanda Showing the District Hospital Selected for Sample transportation (Pilot)

DistrictHospital

ByumbaRutongoKibogoraBushenge

Shyira

Ruhengeri

Kaduha

Gitwe

Gakoma

Nyanza

Kagbayi

Nyagatare

Ngarama

KiziguroGahini

Summary• Good quality specimens are the cornerstone for high quality

tuberculosis diagnosis and treatment monitoring.

• Proper collection procedures and containers, adequate volumes and appropriate specimen transport conditions can all affect TB lab results.

• AFB in specimens do not lose their acid-fastness over time, but they rapidly lose their viability

• Recovery of viable bacilli decreases because of the ability to recover from other contaminating organisms

MDR-TB Diagnosis: How new and currenttools can be used to improve diagnosis

Dissou Affolabi MD, PhD

Meeting the Experts on MDR‐TB 8th December 2012, ITM, Antwerp