Session 7 - TB diagnostics for DOH 2022 (2).pptx

T: +27(0)51 401 9111 | info@ufs.ac.za | www.ufs.ac.za
TB DIAGNOSTICS AND TREATMENT
AUTHOR: A VAN DER SPOEL VAN DIJK
DEPARTMENT: MEDICAL MICROBIOLOGY,
NHLS UNIVERSITAS, BLOEMFONTEIN.

Outcomes:
• To know the diagnostic processes used for TB testing in South
Africa
• To be able to draw a simple algorithm of the diagnostics of different
specimens
• To be able to describe the principals of the different methods
• To be able to analyse simple diagnostic results
• Know basics of treatment schedules and antibiotics used for
treatment of TB, MDR-TB, XDR-TB
• Be able to describe integrated questions about tuberculosis, the
different types and their treatment

DIAGNOSIS OF
TUBERCULOSIS

SPECIMEN COLLECTION AND TRANSPORT
• Types of specimens
– Pulmonary
• Sputum (One)
• Induced sputum
• Gastric lavage
• Urine : Early morning urine samples
• Others -CSF ; Lymph node aspirates; Tissue; Bone samples
• Blood cultures not for Xpert Ultra
All specimens to be transported on ice in a cooler box and kept in fridge for up to 5 days if not
processed immediately
All processing of ? TB specimens in lab - Class II Biological safety cabinet

DIAGNOSIS
Currently diagnosis in SA relies on Xpert Ultra (GXPU) using the
GeneXpert system
The current algorithm used by the National Department of Health (NDOH) of
South Africa (SA) are shown on the next slide

BACKGROUND CONT’D...
Do a LAM test and
as below
Do not treat
Collect one
specimen for
reflex testing
REFLEX testing
For reflex testing
algorithm see next
slide
TREAT according to
results of reflex
Follow –up MDR-TB
and XDR-TB with
monthly TB cultures
Regimen 1
is as in slide 64: 4 months
of INH, RIF, Etham, Z and 2
of RIF and INH

DIAGNOSIS CONT’D...
Smear microscopy
(Fluorescent auramine staining)
Culture method (liquid media) MGIT ,
Kinyoun stain, MPT64 antigen
Ultra (GXPU)
(RR)
DST for Levofloxacin (if susceptible to FQ),
for FQ resistant DST for Linezolid, BDQ
and CFZ (liquid media)
Genotype® MDRTBplus
(resistance to RIF and isoniazid (INH))
Genotype® MDRTBsl
(resistance to fluoroquinolones (FQ) and
injectable drugs)

ULTRA (GXPU) TEST
USING GENEXPERT
SYSTEM

First line test
Xpert MTB/RIF Ultra Assay:
 To to be requested for specimens from patients
with a clinical suspicion of tuberculosis (TB)
BUT
 only if the patient received NO anti-tuberculosis
therapy before
OR
 Less than 3 days of therapy in the last 6 months
BUT?
 This does not include isoniazid (H) preventative
therapy
Reason: Dead bacilli can be
detected resulting in false positives
results

First line test
Xpert MTB/RIF Ultra Assay:
 Extrapulmonary TB specimens include: fluids,
(pleural or pericardial) although pleural
biopsies are preferred, needle aspirates, tissues
and cerebrospinal fluid (CSF) and pus
 Generally does not apply to stool, pus swabs,
urine or blood.
 Often specimens with low
bacillary load do not pic
up RIF resistance

LABORATORY
DIAGNOSIS
• Important:
• Work with possible
tuberculosis specimens
only in a safety cabinet
(BSL2) with N95 masks,
double gloves and single
use gowns.
• Correlate clinical picture,
X-rays with special tests,
e.g. laboratory
investigations.

SPUTUM
• Take specimens early in morning before eating and
drinking
• Release of organisms varies and very low in HIV
patients
• Specimen must contain secretions from the lung and
not consist of saliva
• Use physiotherapist if difficult to obtain a
representative specimen

PAGE | 13
PAGE |
GX-I GX-IV GX-XVI GeneXpert Infinity Series
GeneXpert Module
Different systems are available and can do either one, 4, 16 or infinite numbers of specimens
by loading the specific number of cartridges at a time.

PAGE | 14
PAGE |
GENEXPERT®: SUMMARY OF 8 STEP
METHOD

XPERT® MTB/RIF ULTRA ASSAY
Detect MBTC and RIF resistance
From five bacilli/piece of bacilli to a billion

PAGE | 16
PAGE |
BASELINE
LOG-LINEAR
PLATEAU
EXPONENCIAL
CURVE GENERATED AS PCR PROGRESS IN REAL
TIME

-IS6110
-IS6110
-IS6110
-IS6110
• The genome of MBTC consist of
thousands of genes and the
Xpert utilise three of them for
the detection of TB and RIF
resistance
• The IS6110 element (except for
very few strains) are present in
1 to 25 copies in each TB bacilli
and using this element as
target, the GXPU are able to
detect TB even when only one
bacilli is present
• Of the IS1081 element eight
copies are found per TB bacilli
and even when no IS6110 is
absent the GXPU will still detect
TB and amplify it

-IS6110
-IS6110
-IS6110
-IS6110
• GXPU thus uses 6 probes
for detection of TB. One
each for the IS6110 and
IS1081 target sites and 4
probes for the core region
of the rpoB gene that are
targeted by rifampicin.
• In this way TB can be
detected as well as
resistance to RIF, when one
or more of the rpoB probes
are unable to bind to rpoB
gene. This indicate
resistance to RIF and can be
confirmed looking at the
melting curve

• 95% of R resistant isolates harbor mutations in the RRDR-1 (81bp
region in the rpoB gene) detected with Ultra
• Mutations at codon 516, 526 and 531 account for 86% R resistant
isolates
• 14% of mutations are rare and could result in low level R resistance not
detected by phenotypic culture methods
RIFAMPICIN

Xpert
LPA
fl
LPA
fl
424

IS6110
IS1081
Detected IS6110 = Yes
MTBC detected (High, Medium, Low, Very low)
All probes binded = RIF susceptible
XPERT RESULTS
Cycle threshold

IS6110
IS1081
All probes binded = RIF susceptible
XPERT RESULTS

IS6110
IS1081
Some probes did not attached = RIF resistant
XPERT RESULTS

Microscopy
Cepheid brochure, 2016
Why is smear microscopy done when XPERT®
MTB/RIF is positive?
To categorise patients for reporting as either smear
positive or smear negative using auramine staining
of follow-up samples
Patients with smear positive microscopy is highly
infective and can transmit TB to 20 people around
them by sneezing, laughing or even talking
Thus they are monitored for treatment success by
staining
Xpert pos/RIF (S)
XPERT® MTB/RIF SENSITIVE

XPERT® MTB/RIF ULTRA RESISTANT
When an GXPU test detects TB and rifampicin resistance:
ONLY then, REQUEST REFLEXS testing

DIAGNOSIS CONT’D...
Smear microscopy
(Fluorescent auramine staining)
Culture method (liquid media)
Ziehl-Neelsen, MPT64 antigen
Gene Xpert Ultra (GXPU)
(RR)
DST for Moxifloxacin high and Low,
Levofloxacin, Linezolid, BDQ and
CFZ (liquid media)
Genotype® MDRTBplus
(resistance R and isoniazid (H))
Genotype® MDRTBsl
(resistance to fluoroquinolones and
injectable drugs)

MICROSCOPY
FLORESCENT AURAMINE O STAIN
KINYOUN STAIN / ZIEHL NEELSEN (ZN)

KINYOUN (KN)
STAIN / ZIEHL
NEELSEN (ZN)
Advantage: - cheap – rapid
- Easy to perform
Disadvantages:
- sputum (need to contain 5000-10000
AFB/ ml.)
- Young children, elderly & HIV infected
persons may not produce cavities &
sputum containing AFB.

Kn staining
• Used to confirm growth in MGIT cultures
• Slides are stained with carbol fuchsin
• Decolourise with 3% acid alcohol
• Counter stained with methylene blue
• Slides are rinsed with water between stains and viewed
• under a microscope using a 1000x enlargement.
• The presence of serpentine corded bacilli with no other
bacteria present will indicate a pure culture.

FLORESCENT
AURAMINE O
STAIN
After Auramine or
Rhodamine stain bacilli
resist decolourisation by
and absolute alcohol for 10
min.
Therefore Acid and Alcohol
fast.

CULTURE METHODS
MYCOBACTERIA GROWTH INDICATOR TUBES (MGIT)

CULTURE
2. Liquid Media
– MGIT *
– Bac T/ Alert *
*Automated Continuous Growth Monitoring Systems
• *Detects 10 -100 organisms/ml
• * Growth detected in 10-21 days

MGIT Use mycobacteria growth indicator tubes (MGIT)
A liquid culture medium containing a cord factor
Enable rapid detection of the tubercle bacilli (7-14
days)
Easier contaminated than solid culture.

BACTEC MGIT 960 INSTRUMENT
Appear as long serpentine
cords in liquid medium due to
cord factor
Virulent strains grow in a
more dispersed manner
MPT64 antigen
to confirm and
KN/ZN stain

Microscopy
(N) await culture
LPAfl give MTB diagnosis and R/H resistance
LPA increases the amount of DNA in a
sample so that it can be detected
LPA is influenced by inhibitors / drugs
LPAfl DR or MDR-TB
Laboratory procedure for REFLEX
Sample
LPAsl
MGIT
poitive
LPAfl sensitive
then reported
Smear (P)

LPA MTBsl
Laboratory diagnosis
New TB case
Microscopy only
MTB/Sensitive
Microscopy and culture
Report result
Retreatment case – TB before
Auramine stain
Auramine/ZN stain
1+to 3+
MGIT culture Microscopy
TB PCR
Hain genotype®
LPA MTBDRplus
RIF Resistance or Dual INH
INH + RIF
Sensitive
TB PCR
Neg
MOTT PCR
Genotype ®
Mycobacterium CM
Loose bacilli
+
Serpentine cords
Report result
INH + RIF Resistance
FQ
SLIDS DST
S do DST for
levofloxacin
R to FQ
Xpert Ultra
Xpert Ultra
MTB/RR
REFLEX
R=XDR
DST for BDQ, CFZ, LZD

GenoType MTBDR plus (LPAfl)
Detect 8 WT probes and 4 mutation probes
rpoB gene:
 81 bp region 99% mutations

GenoType MTBDR plus (LPAfl)
Detect 8 WT probes and 4 mutation probesprobes
Difference between the region of the rpoB gene
detected by LPA (gray) and Xpert Ultra (coloured)
424-428
445-448
441-444
437-441
435-438
429-436
429-432
449-452
Q432ins
Phe433_Met43insPhe Ultra S
Asp441Val; Ser456Gln;His454Pro
572

• GenoType MTBDRsl (LPAsl)

LPAsl
• Detect MBTC
• Detect resistance to quinolones by detecting the most prominent
mutations in the gyrA and gyrB genes
• Detect resistance to aminoglycosides by detection the most
prominent mutations in the rrs and eis genes

TREATMENT AND RESISTANCE
• Need for prolonged therapy:
(1) the intracellular location of the organism makes treatment efficacy less
(2) caseous material, blocks penetration by the drug
(3) slow growth of the organism
(4) metabolically inactive "persisters" within the lesion
• treatment may not eradicate the infection
and reactivation of the disease may occur
in the future.

REASON FOR COMBINATION THERAPY
• Random (spontaneous) resistance NOT dependent on exposure to antibiotics
– E.g. random resistant mutants:
– 1/105 for INH; 1/106 for streptomycin
• If 1 of the organisms are resistant in vitro treatment will not be clinically
successful
• Two drugs: chance for a resistant mutant is much lower e.g. 1/105 x 1/106 =
1/1011
• Use of multiple-drug therapy prevents the emergence of drug-resistant mutants during the
long duration of treatment
• Organisms that become resistant to one drug will be inhibited by another drug.

• Sensitive TB – no resistance to rifampicin
• Treat with regimen 1: 1st line drugs namely 2
months - RHZE followed by 4 months - RH
• R/RIF = Rifampicin; H/INH = Isoniazid; Z =
Pyrazinamide; E = ethambutol

• MDR
• Multi-drug resistance (MDR)
• The most common pattern is resistance to both isoniazid
and rifampicin
• Some isolates are resistant to 3 or more drugs
• pre-XDR-TB: TB strains MDR/RR-TB) and which are also
resistant to any fluoroquinolone.
• XDR
– extensively multiresistant TB – resistant against rifampicin,
isoniazid, a quinolone (levofloxacin, moxifloxacin) and one of
bedaquiline, linezolid
• Predisposing factors for MDR:
– Previous treatment for tuberculosis
– Non-compliance – non-completion of course

• MDR
• Treatment for uncomplicated MDRTB are with new drugs
combined with old as on treatment slide 65
• A lot of excluding criteria exist, however and most patients
receive the long treatment regimen
• XDR
• Treatment of XDR with only resistance to FQ are on slide 65
• Treatment mostly rely on results of molecular and phenotypic
drug susceptibility (pDST) testing combined with adverse
effect experienced by the patients.
• Treatment drugs are added according to the categories of
drugs on the next slide.
• Follow-up
• Treatment monitoring are by sputum microscopy, and culture
every month and molecular and pDST every third month

TREATMENT OF MDR- AND XDR-TB
WHO classification of drugs used for treatment of MDR- and XDR-TB

Table: Summary of the different treatment regimens still used for MDR- and
XDR-TB patients in SA
Current 2020 TB treatment regimens
1 2 3 4 5 6 7 8 9 10 11
Short MDR Regimen
hd Isoniazid (INH)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16- 20
FluoroquinoloneResistant
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16- 20
Long MDR Regimen
Ciprofloxacin (Cfx)
Intensive Phase Cont. Phase 5months
Linezolid (LZD)
Bedaquiline (BDQ)
Levofloxacin (Lfx)
Continuation 6months of 4-6 drug
intensive Phase 12 months
Bedaquiline
Linezolid
Pyrazinamide (PZA)
Ethambutol
levofloxacin
Ciprofloxacin
para-aminosalicylic acid (PAS)
Teridazone/PAS
Continuation Phase 12months of 3-4 drugs
BDQ
LZD
Dlm (± ano Group C)
Cfx
Intensive Phase 6-8months

Session 7 - TB diagnostics for DOH 2022 (2).pptx

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