MODULE 2: UNIT 2.

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LABORATORY DIAGNOSIS OF TB

IN-SERVICE COURSE IN INTEGRATED AND COMPREHENSIVE TB AN

D LEPROSY MANAGEMENT 1
 Brief introduction

• Laboratory diagnosis depends on identifying the infective organism in

secretions or tissues of diseased individuals.
• This unit covers specimen Collection procedures, instructions for
collection and storage of specimens, methods used in laboratory
diagnosis of TB, Ziehl-Neelsen and Fluorescent staining procedures,
reporting microscopy and molecular results.

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 Unit Objectives

By the end of this unit participants should be able to:

• Explain to presumptive clients specimen collection procedures
• Identify the components of a typical laboratory requisition form
• Explain the various methods used in laboratory diagnosis of TB
• Explain the steps for diagnosis and monitoring TB patients
• Explain how reporting and recording of microscopy and molecular results
is done
• Appreciate the importance of recording TB data in the laboratory register
• Record patient data in the laboratory register

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 Specimen collection procedures

• Collect specimens in clean, sterile, clear, appropriate

container.
• Label the container with the patient’s name, number,
date and time of collection.
• Complete all available lab request form and collect
initial specimens before antimicrobial therapy is
started. 4
 Laboratory requisition form
Information needed on the form

• The patient’s name, identification number (presumptive TB

numbers & OPD numbers) and location
• Date and time of collection of specimen
• Source of specimen
• Type of specimen
• Requested investigations
• Other relevant information.

Participants should completely fill the request form with patient information
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 Specimen collection procedures

• Show the patient how to open and close the falcon tube/sputum
cup, instruct and demonstrate fully and slowly the steps below on
how to collect sputum:
• Rinse mouth with clean water
• Collect the sputum sample in the designated booth or in the court
yard away from other people and with sufficient privacy,
• Take 3-4 deep breaths, holding a breath for 3-5 seconds after each
inhalation, 7
 Specimen collection procedures

• Cough after the last inhalation and empty at least 3-5 mL of sputum into the falcon
tube/sputum cup while taking care not to contaminate the outside of the tube/cup.
• Close and return the falcon tube/sputum cup to the laboratory and wash your hands
thereafter.
• The lab staff should wipe the outside of the tube with cotton wool soaked in
tuberculocidal disinfectant (Phenol, Lysol, or Bleach/Jik)
• And wash hands thereafter using water and soap, or an alcohol-based hand rub, if
available.

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 Specimen collection procedures

• One sample should be produced on spot (spot

sample) and the second sample on early
morning of day two (early morning sample)
when a patient wakes up after rinsing the
mouth with water (before brushing).
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Basic Triple Packaging System

Given the risks

associated with TB
specimens, NTLP
recommends Triple
packaging system
for safe
transportation of
infectious
materials.
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Modified Triple Packaging

Modified Triple Packaging ① ②

• ① A leak-proof sputum
container - primary
receptacle.
• ② Wrap container in
absorbent cotton material. ③ ④
• ③ A leak-proof zip lock bag -
secondary packaging.
• ④Safety box - tertiary outer
packaging.
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 Samples for GeneXpert MTB/RIF

• Fresh Sputum (Induced/Expectorated)

• Gastric Aspirates
• CSF
• Lymph node Aspirate
• Pleural Fluid

Each type of sample should be processed using the recommended

protocol to avoid errors.

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 Criteria for rejection of specimens
No Reason for Rejection Defaulter No. Rejected %age
• Missing or inadequate 1 No ward/clinic of origin 4A, 4B, 4C 120 31
identification 2 Clotted specimens (in EDTA) ACU, SOPD 96 24
3 Wrong container Ward 7 48 12
• Contamination 4 No doctor’s name /initials MAC 52 13
• Specimen collected in the 5 Unlabeled specimen A/E, 3BE 25 6

wrong collection tube 6 Incomplete Request A/E 28 7

7 Haemolysed sample 5C 12 3
• Inappropriate transport or 8 Insufficient sample ACU 08 2
storage 9 Contaminated request/container 5AA, ACU 05 1
10 Un-matching request & specimen 4B 05 1
• Insufficient volume Total 397 100%

• Unknown time delay

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 Methods used in lab diagnosis of TB

Available Tests When Used

• Microscopy- light and • None availability or Non
fluorescent functional Xpert MTB/RIF and
• X-pert MTB/Rif assay treatment monitoring
• Initial test for ALL presumptive
• Culture ( MGIT and LJ TB cases if available. NOT FOR
media) FOLLOW UP.
• Treatment monitoring of DR-
TB
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 Methods used in lab diagnosis of TB .....

Available Tests When Used

• Line Probe Assay (LPA) • Rapid ID of MTB for FLR and
• Lipoarabinomannan assay SLR
(TB LAM) • Diagnosis and screening of
• Histopathology active TB in PLHIV, CD4≤200
• TB cytology

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 AFB Microscopy Systems
Method Description

• Bright field/ordinary microscopy and

Fluorescence microscopy.
• Different stains used - based on the same
principle
• Acid Fast Bacilli appear RED in ZN and
ORANGE/YELLOW in FM Microscopy.
• Smear microscopy is mainstay for diagnosis
although it is now being replaced by Xpert
MTB/RIF.

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 Ziehl-Neelsen (ZN) Method

• Highly specific, but not sensitive

• Detects roughly 50% of all the active cases.
• Sensitivity can be as low as 20% in children
and HIV patients.
• Cannot detect resistance to drugs.
• Ziehl-Neelsen staining detects bacilli > 10,000
per ml of sputum

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 Fluorescent (FM) Microscopy

• Higher sensitivity than ZN (10%)

• FM takes less time
• High cost - need for regular maintenance
• Not as specific for acid-fast organisms (i.e. TB
or Nocardia).
• Used at labs with large specimen numbers as
a screening tool.

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 Xpert MTB/RIF

Method Description
• Classic NAAT based on real time PCR technology
• Allows automated sample processing, DNA amplification
and detection of TB and screening for rifampicin resistance
• For detection of MTB/RIF among other diseases
• Rapid and easy to use (approx. 90 min test)
• Proven safety
• Precise, highly sensitive and specific
• Designed to suit client needs (minimum training required)
• Few infrastructural requirements

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 Line-Probe Assays for MDR TB
Method Description
• Fast, but expensive and exhibits variable degrees of
sensitivity
• Best for smear positive sputum specimens or culture
sediments
• Detects MTB and genetic mutations conferring
rifampin and isoniazid resistance
• High sensitivity (>90%) and specificity (>95%) for MDR
TB
• Requires PCR Equipment

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 When to use molecular tests

• MDR TB • All previously treated cases (Treatment

SURVEILLANCE failure, Return after LTF, Relapse)

• Smear positive refugees

• DR TB contacts • Smear positive after 2, 5 and 6 months of
treatment

• HIV positive patients

• DIAGNOSIS • Children< 14 years

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 Mycobacteria Growth Indicator Tube
(MGIT 960)
Method Description
• Detects TB within 4 – 14 days. Bottom of bottles contain a
fluorescent material in Silicone, quenched by O2 in media
until used up gradually.
• Performs antimicrobial susceptibility testing
Compared to solid culture/DST:
• More rapid time to detection
• More sensitive
• Higher contamination rate
• Rapid method for species
• ID needed

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 Culture (LJ)
Method Description
• Culture on liquid or solid media is the GOLD STANDARD.
It is sensitive to 10-100 bacilli/ml.
• The bigger the sample, the better the chances of
isolating TB.
• Sensitive and specific
• Permits testing for drug resistance.
• Requires biosafety facilities and trained personnel.
• Requires 6 to 8 weeks incubation.
• Performs antimicrobial susceptibility testing

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 TB LAM
Method Description
For the diagnosis and screening of active tuberculosis in
people living with HIV:
• Signs and symptoms of TB with CD4 cell count ≤ 200 cells/ul
• PLHIV - ‘’seriously ill’’ regardless of CD4 count/unknown CD4
count**
LAM is Lipoarabinomannan, a protein produced by cell
walls of TB bacilli and excreted in urine
• “seriously ill” is defined based on four danger signs** :
• respiratory rate > 30/min,
• temperature >39 °C,
• heart rate >120/min and
• unable to walk unaided.

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 Histopathology
Method Description
• Tissue biopsy: Morphologically the
inflammation is suggestive of TB.
• Fine Needle Aspirate Cytology (FNAC): ZN
or fluorescent microscopy

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 Steps for lab diagnosis of TB patients

• All presumptive TB cases should undergo Xpert MTB/RIF as initial test if

available or smear microscopy if Xpert MTB/RIF is unavailable or non-
functional.
• A smear negative sample from high-risk category should be referred for
Xpert MTB/RIF test.
• A smear positive sample should be referred for Xpert MTB/RIF test to
rule out Rifampicin Resistance.
• If Xpert MTB/RIF or microscopy result are negative do further clinical
evaluation and X-ray.
• HIV positive patients who have CD4 ≤ 200 or very sick should be tested
for TB using Urine TB LAM test.
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 Recording into the Laboratory TB register

• Know the importance of recording TB

data in the laboratory register
• Record patient data in the laboratory
register

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 Description and Instructions

1. LABORATORY SERIAL NUMBER:

• This is the unique serial number issued to Presumptive TB patient. The
number begins with 1 at the beginning of the calendar year (January)
and ends at the end of the calendar year (December)
2. DATE:
• Write the date for each sample tested
3. PRESUMPTIVE TB/UNIT NUMBER:
• Write the presumptive TB number
4. NIN:
• Write the National Identification Number (NIN) of the patient.
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 Description and Instructions …..

5. NAME IN FULL/TELEPHONE NUMBER:

• Write the patients names in full both surname and other names and record the
patient’s telephone number as well.
6. SEX:
• Write in the patient’s sex, record M if patient is Male and F if patient is Female
7. AGE:
• Write the patient’s age in complete years if over one year of age. Use months if the
patient is under one year of age, clearly writing “MTH” after the age, and “Days” if less
than 1 month.
8. ADDRESS:
• Write the patient’s address. The patients address or location is given by the district (1),
sub-county (2), parish (3) and village (4) for better follow up
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 Description and Instructions …..

9. NAME OF FACILITY/ UNIT

• Write the name of the Treatment Unit (May not necessarily
be the facility where laboratory test is done)

10. EXAMINATION TYPE:

• Put a tick under diagnosis if specimen is for Pre-treatment
examination or Follow-up (FU) sputum examinations collected
at 2, 5 and 6 months (record FU2, FU5, FU6)

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 Description and Instructions …..

11. EXAMINATION RESULTS:

• SPECIMEN RESULT 1: write the results of the Ziehl Nielsen test
on the first sputum collected. Spot specimen microscopic
findings

• SPECIMEN RESULT 2: write in the results of the second Ziehl

Neelsen test. Early morning specimen microscopic findings.

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 Reporting ZN Microscopy Results

IUATLD Quantification for AFB Microscopy

AFB Counts under X100 oil immersion Recording / Reporting
No AFB/100 fields 0/No acid-fast bacilli observed
1 to 9 AFB/100 fields Actual AFB count
10 to 99 AFB/100 fields +
1 to 10 AFB/ field 2+
> 10 AFB/ field 3+
If using fluorochrome staining, use appropriate conversion factors. See
http://www.sahealthinfo.org/tb/tbmicroscopy.htm 33
 Description and Instructions …..

12. GENEXPERT EXAMINATION RESULTS: record as follows

GENEXPERT RESULTS/REPORT RECORDING CODE

MTB NOT DETECTED N

MTB DETECTED, RIFAMPICIN RESISTANCE NOT DETECTED T

MTB DETECTED, RIFAMPICIN RESISTANCE TI

INDETERMINATE
MTB DETECTED, RIFAMPICIN RESISTANCE DETECTED RR
INVALID/ERRORS/ NO RESULTS I
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 Interpretation of Xpert
MTB/RIF results

• Lower Cycle threshold (Ct) values represent a higher starting MTB Ct Range
RESULT
concentration of DNA template
• Higher Ct values represent a lower concentration of DNA High <16
template.
• MTB Not Detected- Sample has no target TB DNA Medium 16-22
• MTB Detected- MTB target DNA is detected.
• MTB Detected—displayed as: Low 22-28
• High
• Medium
• Low or Very Low >28
• Very Low depending on the Ct value of the MTB target present in the
sample.
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 Description and Instructions ….

13. HIV STATUS

• HIV test results should be recorded as follows:
HIV TEST RESULTS RECORDING CODE
POSITIVE RESULT Pos
NEGATIVE RESULT Neg
14. Linked to TB care?
• Write name of health facility and Unit TB No. of where patient started treatment from.
15. REMARKS:
• Write in any remarks/comments arising from the results of the test, e.g. Report presence of
yeast cells

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 Refer to Case study 1

Results of Rukundo Patra:

• The GeneXpert test is MTB not detected and Rifampicin Resistance
Not detected.
• HIV test – Negative

• Complete the laboratory register

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 Exercise

1. Discuss the procedure for patient preparation and

specimen collection.
2. How is specimen for microscopy and Xpert MTB/RIF
stored?
3. List essential methods used in laboratory diagnosis of TB
4. How is microscopy and molecular result reported?

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 Conclusion

Laboratory methods used in the diagnosis of TB include:

• Microscopy- light and fluorescent.
• X-pert MTB/Rif assay
• Culture ( MGIT and LJ media)
• Line probe assay
• Lipoarabinomannan assay (LF-LAM)
• Histopathology

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 References

• MOH NTLP, October 2017. Tuberculosis and Leprosy Case

Management Desk Guide, 3rd Edition.
• MOH NTLP, June 2017. Facilitor’s Manual for Health Workers in the
Management of Recording and Reporting of Tuberculosis Data.
• Ministry of Health, 2015. Central Public Health Laboratories (CPHL)
Biosafety and Biosecurity Manual.

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