Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

4.1 Medical Microbiology > Tuberculosis - Laboratory Investigation > Flashcards

Tuberculosis - Laboratory Investigation Flashcards

You may prefer our related Brainscape-certified flashcards:
USMLE® Step 1 flashcards Biology 101 flashcards
1
Q

What are the four levels to laboratory diagnosis of MTB?

A

Smear/Direct microscopy

Rapid molecular diagnostic tests

Culture media both solid and liquid

Drug susceptibility testing (molecular assays, liquid or solid medium methods)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are some of the benefits/downfalls of each diagnostic method for MTB?

A

Microscopy -> same day detection but poor sensitivity

Line probe assays -> first 24/48 hours

Direct culture and susceptiblity takes weeks to months

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is suggested as the best method of ast for tb and why?

A

Reference labs suggest direct susceptibility as the gold standard

Molecular methods can only detect the presence of genes known to confer resistance but this doesnt always represent the clinical picture

Molecular methods are also unable to identify resistance due to new mechanisms/genes -> if the mutation is new or unkown then it will come up falsely negative

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Give a quick breakdown of the lab investigation for tb

A

specimen receipt into ab within 24 hrs of specimen collection

process and concentrate sample (decontaminate)

Acid fast microscopy -> report <24hrs

Liquid culture e.g. automated MGIT

Recovery of organism (10-14 days)

Identification of species (21 days of recepit)

Susceptibility testing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Talk about decontamination step for TB

A

NACL NaOH used to decontaminate specimen of any respiratory tract flora/oral flora

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Talk about decontamination step for TB

A

NACL NaOH used to decontaminate specimen of any respiratory tract flora/oral flora

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

From a lab point of view how do we confirm a TB

A

Isolation of MTC (excluding M. bovis BCG) from a clinical specimen e.g. liquid culture positive

OR

Detection of MTC nucleic acid in a clinical specimen

AND

positive microscopy for AFB

i.e. positive liquid culture or NAAT as well as AFB on smear

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What kind of specimens are suitable for TB investigation?

A

Non Sterile Sites such as:
- pulmonary: sputa, BAL, BRW, BA
- renal: urine

Sterile sites:
- pleural fluids, joint fluids, CSF etc
- tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What kind of specimens are suitable for TB investigation?

A

Non Sterile Sites such as:
- pulmonary: sputa, BAL, BRW, BA
- renal: urine

Sterile sites:
- pleural fluids, joint fluids, CSF etc
- tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What kind of sample is required for query TB meningitis?

A

2 CSF samples needed to enhance senstivity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Give some exampls of inappropriate specimens for TB investigation

A

Faeces -> commensal mycobacteria, difficult to interpret, difficult to decontaminate

Urine for the investigation of pulmonary TB

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How should sputa or urine be collected?

A

clean, sterile plastic container
Before commencement of therapy
Early morning samples
Procured on 3 consecutive days
5-10mls ideally but min 2mls
Non salivary sputa
Not pooled
Refrigerated if delay in transport

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Why is an early morning sample ideal?

A

Lying down all night -> aids pooling of bacteria -> first expectorate/urine ideal -> collection of bacteria

Highest bacterial count

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What culture media is used for TB?

A

MGIT -> liquid culture

Lowenstein-Jensen (LJ) slopes - solid

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

If AFB negative smear what should you do?

A

Usually wait for consultant to request a molecular test as AFB neg doesnt always mean TB neg

High clinical suspicion important

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How do we carry out molecular detection of TB?

A

Direct detection of nucleic acid using the GeneXpert

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are some of the main safety considerations surrounding TB specimen processing safety?
(7)

A

Environmental persistence of TB - think of resistant cell wall

Aerosol formation + inhalation

Hazard Group 3 pathogen

Cat3 lab + biosafety cabinet

Centrifugation

Disinfection and autoclaving of equipment

Vaccination of staff and mantoux testing if necessary

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What kind of lab is TB processing done in?
(3)

A

Must be done in a category 3 containment lab

Must be done in a biosafety cabinet with an installed air filtration system with HEPA filters insalled

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How persistent is TB in the environment?

A

TB can resist disinfection and somatic stress

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What risk is there surrounding centrifugation of TB specimens?

A

Poses the risk of aerosols and therefore inhalation

Risk of breakages

Sealed buckets must be used to prevent aerosols

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How is equipment disinfected post TB processing?

A

Disinfected in hypochlorite solution and then autoclaved

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

How should staff be prepared for TB processing?

A

Staff should be appropriatly trained
BCG vaccinated

If exposed or signs of symptoms etc then Mantoux skin test should be carried out

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Why do we need to decontaminate sputa for TB testing?

A

There are lots of commensals present in sputa samples -> these will overgrow/outgrow any mycobacteria if allowed to do so

We want to kill as many of these commensals while preserving as much mycobacteria as we can

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is the most common method of decontamination for TB samples?

A

2% NaOH

This oftens involves a mucolytic agent such as N-acetyl-L-cysteine (NALC) or sputasol

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What is the ideal contamination rate of a sputa sample?

A

Between 2-5% required

<2% is too harsh and you will kill too much mycobacteria
>5% is too weak and the mycobacteria will become overgrown

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

How do we decon for TB?

A

NaCL NaOH made up daily

Equal volumes used

Vortex for 20 seconds

Let stand at room temperature for 15 mins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Does decon interfere with downstream culturing or detection methods?

A

NO decon using NaCl NaOH is validated on both the MGIT and the LPAs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What does MGIT stand for?

A

Mycobacterial Growth Indicaor Tube (MGIT)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Talk about the use of microscopy for TB
(3)

A

Microscopy should be performed after homogenisation but before decontamination, or it should be done directly from samples

Smear positives indicates presumptive diagnosis of TB and infectivity i.e. if smear positive then def positive and infectious

Smear negative doesnt necessariy mean negative but indicates less infectious

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

Talk about the use of microscopy for TB
(3)

A

Microscopy should be performed after homogenisation but before decontamination, or it should be done directly from samples

Smear positives indicates presumptive diagnosis of TB and infectivity i.e. if smear positive then def positive and infectious

Smear negative doesnt necessariy mean negative but indicates less infectious

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

What are the five main benefits of carrying out TB microscopy?

A

Used to determine whether isolation is required -> are they infectious or not

Influences the extent of contact tracing - more tracing needed if highly infecitous etc

Used to monitor treatment - negative smear good indicator that treatment is working

Results should be available within one working day

Both viable and non-viable organisms will stain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

What are the two smear stains available for TB?

A

Auramine-O stain
Ziehl Neelson

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Compare the use of Auramine O versus ZN?

A

Limit of detection about 10^4 bacilli/ml (10,000) for AFB but 5x10^3 (5000) for ZN i.e. need more bacteria for AO

However you dont need a fluorescent microscope for the ZN stain which makes it a better choice for high burden countries such as Africa

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Compare the use of Auramine O versus ZN?

A

Limit of detection about 10^4 bacilli/ml (10,000) for AFB but 5x10^3 (5000) for ZN i.e. need more bacteria for AO

However you dont need a fluorescent microscope for the ZN stain which makes it a better choice for high burden countries such as Africa

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

What are five factors that affect the sensitivity of smears?

A

Quality of specimen
If prepared prior to or post decontamination
Centrifugal force
Type of stain (auramine/direct Zn)
Experience of Reader

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

What is the requirement for a positive ZN?

A

Magnification at 1000 or >oil for 15 minutes or 300 fields

-> must see 1 bacillus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

What is required for a positive Auramine O?

A

Magnification of approximatel 150 dry (not oil) for 2-3 minutes 30 fields (80)

Must see 3 bacilli

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

In routine labs why is auramine O the preferred stain for TB?
(2)

A

10% more sensitive than ZN with similar specificity

Lower magnification and less time required

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

what % of culture positive specimens are smear positive?

A

About 50% of culture positives are smear positive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

What are five limitations of microscopy for TB?

A

Cannot distinguish between dead and living bacilli

High bacterial load >3000-5000AFB/ml is required for detection

Cannot distinguish between species

No indication of drug susceptibility

Some patient cohorts will have a much lower amount of TB in sputa -> HIV and children will always have a lower bacilli count

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

What is the go to solid media for TB?

A

Lowenstein-Jensen medium slopes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

What does the LJ slopes contain?

A

Eggs
Malachite green
Glycerol (for MTB) or pyruvate (M. bovis)
PANTA cocktail

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

What are the components of the PANTA cocktail?

A

Polymyxin
Amphotericin
Nalidixic acid
Trimethoprim
Azlocillin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

What does the malachite green component of LJ medium do?

A

Malachite green -> mycobacterium quite resistant to this but everything else is susceptible

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

What must be added to Lowenstein-Jensen media to favour growth of MTB vs M. bovis

A

For MTB add glycerol

For M. bovid add pyruvate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

What are the pros and cons of LJ slopes?
- 4 of each

A

Can detect as few as 10 viable cells
Easy and economical to prepare
Lower contamination rates then with liquid media
Isolated colonies with characterisitic tough, rough and buff colonial morphology can be observed

MTB takes between 14-28 days to grow but can take 8 weeks to grow if patient is on treatment
Need to be checked at regular intervals
If contamination does occur it covers the total surface of the medium
DST difficult to perform using egg-based media

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

Why is drug susceptibility testing more difficult to perform using egg-based media?

A

This is because some drugs must be adjusted to account for their loss by heating or by interaction with certain components of the egg

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

What is the go-to liquid media for TB?
(2)

A

Middlebrook 7H9 Broth

Its supplemented with glycerol to make it more suitable for M TB growth

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

What are the pros and cons of liquid media?

A

Growth is more rapid in liquid media vs solid

Contamination and mixed cultures are more common and difficult to interpret

50
Q

What is the go to method of automated mycobacterial culture in most labs?

A

Mycobacterial Growth Index Tube (MGIT) system

51
Q

Write a note on the MGIT.
- How does it work?
- How many and what samples?
(6)

A

Its a rapid liquid culture method

Utilises fluorescence technology triggered by O2 reduction

O2 continuously monitored over a 6 week protocol

Automatically reads cultures

Capable of holding 960 patient samples

Can be used for the majority of specimens in the TB lab

52
Q

How long does it usually take for a positive on the MGIT?
(2)

A

Usualy takes 10-12 days for a MGIT positive

The MGIT protocol is 6 weeks though so samples will stay onboard and be continuously monitored until then

53
Q

What kind of medium is used onboard the MGIT?

A

Middlebrook 7H9 broth containing OADC and PANTA Cocktail

54
Q

What technology is used onboard the MGIT?

A

Fluorometric technology

55
Q

What is the principle behind fluorometric measurement on the MGIT?

A

A fluorescent oxygen sensor is embedded in the base of each tube

This sensor detects any decrease in O2 dissolved in the broth

The oxygen sensor will emit light when exposed to UV

Actively respiring organisms consume O2, decreasing concentration

This reduction is detected by the MGIT

MGIT then flags tube as positive for scientist

56
Q

After a positive liqud culture what is the recommended next step?

A

To confirm with a ZN stain

-> centrifuge tube for 15 mins and stain using ZN
-> note appearance of mycobacteria (cording or non-cording/clumping)

57
Q

What does cording TB in a flagged MGIT tube indicate?

A

Typical of Mycobacterium TB

58
Q

What does non-cording/clumping bacteria in a ZN of a positive MGIT tube indicate?

A

Non-TB-Mycobacterium species

59
Q

What are the three limitations of culturing TB?

A

Turn around time (up to 8 weeks for solid and 6 for liquid)

Training -> staff need a lot of training

Cat 3 facilities are required

60
Q

When should susceptibility testing be done

A

When the patient is first found to have positive culture for TB

Ideally before treatment

61
Q

Write a note on Susceptibility testing for TB, where and why do we do it?

A

Only done in the referene lab

Really important to determine what is the best regime for the patient -> think long antibiotic course so want to get it right the first time

Phenotypic methods arent done as these would takes weeks ontop of the weeks/months for culture positives

62
Q

What methods are there of susceptibility testing for TB, what is the preferred method?

A

Solid media on LJ medium but this takes 3-4 weeks

Liquid media on the BACTEC or MGIT but this takes 7-10 days

Molecular methods - takes only a few hours - 1 day

Phenotypic DST remains Gold standard as molecular can miss mutations

63
Q

How is phenotypic ST carried out on the MGIT?
(3)

A

Multiple tubes used -> one growth control and one tube for each drug tested

A known concentration of each drug is added to a MGIT tube along with the specimen -> the growth of specimen is compared to the drug-free growth control -> same amount of specimen added to each tube aswell

If the drug is active growth will be inhibited and fluorescence will be suppressed while the drug free control will show increasing fluorescence

If the isolate is resistance growth and fluorescence will be commparable to that of the control

64
Q

Talk about broth microdilutions as a form of ST for TB?
(4)

A

A new method of ST

Trek Sensitre plates

Increasingly being used

Results were often interprete against EUCAST breakpoint established for other methods e.g. agar or MGIT but in 2020 EUCAST provided new guidelines for these as an MIC process

65
Q

What is the GeneXpert MTB/RIF Assay, what does it do?
(3)

A

Automated NAAT, Real time PCR for detection of TB and Rifampicin resistance detection

Endorced by WHO in December 2010

11 chamber self-contained PCR

66
Q

What are the main benefits of the GeneXpert MTB/RIF assay?
(7)

A

A closed system with minimal hands-on technical time

Provides rapid results in less than 2 hours

Does not require a Cat 3 lab -> good for developing countries

Sample preparation, amplification and detection all integrated on board

Dry interfaces - minimal contamination risk

Precise assay - computer controlled process and pipetting - less room for error

Quality assured - extraction controls, probe-check controls and each cartridge has its own PCR control

67
Q

What are the steps to using the MTB/RIF test on the GeneXpert
(8)

A

Sputum liquefaction and inactivation with 2:1 sample reagent -> incubated for 15 mins

Transfer of 2ml material into test cartridge

Insert onto test platform

Sample automatically filtered and washed

Ultrasonic lysis of filter-captured organisms to release DNA -> this chemically inactivates the bacteria

DNA molecules mixed with dry PCR reagents

Seminested rtpcr amplification and detection in integrated reaction tube

Printable test result

68
Q

What are the targets for MTB and RIF on the GeneXpert

A

rpoB gene of M. tuberculosis complex

69
Q

Talk about the use of the rpoB gene to detect TB and Rif on the GeneXpert
(6)

A

GeneXpert amplifies u a sequence of the rpoB gene where 95% of genes responsible for Rifampicin resistance are found

The GeneXpert utilises 3 specific primers and 5 unique molecular probes

Together the 5 probes cover the rifampicin resistant determining region of the rpoB gene sequence

The 5 probes bind to the wild type and not the resistance mutations => 5 probes binding = no resistance detected

At least 2 of the 5 probes within 2 cycles and a positive amplification control is needed for MTB detection

1 or more of the probes must fail for rifampicin resistance to be detected

70
Q

Other than MTB or Rif what does the geneXpert also determine?

A

It also determines the bacterial load through the Ct score

71
Q

How do you interpret Ct score?
(3)

A

A Ct <16 (low) means there is a high amount of Mycobacterium

A Ct >28 (high) means there is a low amount of Mycobacterium

i.e. Ct is inversibly proportional to Mycobacterium load

72
Q

What are the advantages of the gene xpert?
(5)

A

Real time PCR assay

Detects resistance to Rifampicin

Rapid

No need for Cat 3 facility

Only needs 131 CFU/ml compaired o 10^4 CFU/ml for smear

Can run pulmonary and extra-pulmonary samples

73
Q

What is the overall sensitivty of the GeneXpert MTB/RIF when compared to smears

A

Smear + were over 98% positive on GeneXpert

Smear - were about 75% sensitive on GeneXpert

But overall specificity was nearly 100%

74
Q

What is the overall sensitivty of the GeneXpert MTB/RIF for extra-pulmonary TB compared to culture
(3)

A

Overall sensitivty at 79 % and specificity at 97.3% when compared to culture

Sens in smear + was 99% but for smear - was 70%

Sens in children higher at 86% vs adults at 73%

75
Q

What extra-pulmonary samples work better on the GeneXpert?

A

Sensitivity remaines over 85% for CSF, biopsies, urines, pus samples and FNAs

76
Q

What extra-pulmonary samples have low sensitivity?

A

Pleural fluids @44%
Gastric aspirates @78%
Cavitary fluids @50.9%

77
Q

What are some limitations of the Gene Xoert MTB/RIF assay?
(4)

A

Less sensitive for smear-neg sputum (75%)
Limited sensitivity in some extrapulmonary samples
Decreased capacity for some Rif-R mutaions
Occasional false positive Rif-R

78
Q

What Rif-R mutations does the GeneXpert have a decreasede capacity to detect?

A

rpoB C533G mutation

-> just remember it cant detect all rpoB mutations - only 95%

79
Q

What might cause false positive Rif-R calls on the Gene-Xpert?
(2)

A

Paucibacillary (leprosy skin samples) occasionally false positive due to delays in the real-time signal generated by probes D and E

Fase recognition as a nonfunctional rpoB F51 4F silent mutation as conferring RIF-R

80
Q

What is the GeneXpert Ultra MTB/RIF assay
(3)

A

A new/improved assay for detection of TB and RIF-R

Uses two different multi-copy genes to detect MTBC DNA

Detects mutations within the rpoB gene for Rif-R

81
Q

What genes does the GeneXpert Ultra use to detect MTBC DNA?

A

IS6110 and IS1081

82
Q

What are the main improvements of the GeneXpert Ultra assay?
(6)

A

Increased cartridge capacity to hold double the amount of specimen and therefore DNA to improve sensitivity

Improvements in detecting trace amount of TB and RIF-R

Improved mutation detection chemistry - new probes

Improved sensitivity in children, HIV+ and extrapulmonary samples

Limit of detection decreased again to only 15.6 CFU/ml

Increased sensitivity for smear - specimens as well

83
Q

How did the GeneXpert compare to the GeneXpert Ultra in detecting MTB meningitis in a 2020 study?

A

The Ultra was not superior for HIV- patients in detecting MTB in CSF with tuberculous meningitis i.e. not more sensitive in HIV- infected

Ultra was more sensitive in detecting very low or trace levels of bacteria i.e. in those on antimicrobia treatment

Both Xpert and Ultra were more sensitive in HIV infected than in HIV-

84
Q

In general how does the GeneXpert compare to the Gene Ultra
(4)

A

The Ultra is 5% more sensitive but 3.2% less specific at detecting MTB

This may predispose to fals-positive results due to sample cross contamination

False-positives were seen when testing patients with a recent history of TB

Both sensitivity and specificity were the same for Rif-R detection

85
Q

Write a note on the Gene Xpert XDR, how does it work?

A

A further improved GeneXpert from the ultra

It analyses melting temperatures (Tms) using sloppy molecular beacon probes (SMB) to identify mutations associated with resistance against first and second line antimicrobials

86
Q

What are the main benefits of the Xoert XDR?

A

Doesnt just look for rifampicin resistance - looks at INH, FLQ, ETH and SLID resistance

Can differentiate between low versus high level resistance to INH and FLQ

Proved to be 94-100% sensitive and 100% specific for all drugs except for ETH when compared to sequencing

87
Q

What genes does the Xpert XDR detect and what resistance do they confer?

A

katG -> isoniazid
inhA -> isoniazid
gyrA -> fluoroquinolones
gyrB -> fluoroquinolones
rrs -> amikacin, kanamycin
eis -> kanamycin

88
Q

What genes are mutated to confer resistance against isoniazid?

A

katG
inhA

89
Q

What genes are mutated to confer resistance against isoniazid?

A

katG
inhA

90
Q

What genes are mutated to confer resistance against fluoroquinolones?

A

gyrA
gyrB

91
Q

What genes are mutated to confer resistance against amikacin?

A

rrs

92
Q

What genes are mutated to confer resistance against kanamycin?

A

rrs
eis

93
Q

What is the new point of care device for TB?

A

GeneXpert Omni

94
Q

What is the GeneXpert Omni?

A

A prototype point of care device

A single-module battery powdered platform

Equivalent sens and spec

Increased cost

95
Q

What are the three steps in using a line probe assay?

A

DNA extraction

amplification by PCR

Reverse hybridisation of amplified nucleic acids to specific DNA probes bound on strips

Evaluation

96
Q

What are the five line probe assays available?
(5)

A

GenoQuick MTB -> ID only
FluoroType MTB -> ID only

Genotype MTBDR/MTBDRplus - first line resistance
Genotype MTBDRsl - first and second line resistance

GenoScholar PZA-TB - first line resistance

97
Q

What are some benefits and cons of line probe assays

A

Some can detect resistance to a broader range of 1st line and 2nd line antimicrobials

Can provide mutation specific data for common variants

The are more prone to contamination as more handling is required

98
Q

What is the GenoQuick MTB line probe assay, what does it detect, how does it work?

A

LPA for the rapid direct detection of the MTC from pulmonary and extrapulmonary patient specimens

Detects 23S rRNA genes

Hybridisation of labelled amplicons to oligonucleotide probes arranged on a membrane strip

99
Q

What does the GenoQuick MTB detect?

A

Detects 23S rRNA genes

100
Q

How does the GenoQuick MTB work

A

Hybridisation of labelled amplicons to oligonucleotide probes arranged on a membrane strip

101
Q

How does the GenoQuick MTB work

A

Hybridisation of labelled amplicons to oligonucleotide probes arranged on a membrane strip

102
Q

Who makes the majority of line probe assays?

A

Hain Life Sciences

103
Q

What does the FluoroType MTB LPA do?
What are the pros of this LPA?

A

Rapid detection of the MTC from pulmonary and extrapulmonary patient specimens

Its an automated version of the GenoQuick LPA

It can have a result in about 3 hours

87-100% specific

104
Q

What is the GenoType MTBDRplus LPA, what does it do?

A

Identification of MTC
Identification of resistance to rifampicin (RMP) and/or isoniazid (INH)

105
Q

Talk about the GenoType MTBDRplus LPAs ability to detect resistance

A

Rifampicin: Detects mutations of the rpoB gene -> can detect 96% of mutations

Isoniazid: katG gene (high level resistance) + promoter region of the inhA gene (low level resistance) -> can detect 75% of mutations

106
Q

What is the KatG gene and what is it involved in?

A

It encodes a catalase peroxidase

This confers high levels of Isoniazid resistance

107
Q

What is the inhA gene and what is it involved in?

A

It encodes an NADH enoyl ACP reductase

It confers low levels of isoniazid resistance

108
Q

Explain how the GenoType MTBDR plus works to detect resistance

A

LPA has probes for both the wild type of the gene and mutated genes known to cause resistance

Susceptible strains will bind at all of the wild type probes

If resistance some of the wtprobes will be missing a signal and an additional signal will be present at the mutated probe indicating which resistance gene is present

Colorimetric assay

109
Q

Talk about the GenoType MTBDRsl

A

These LPAs are able to detect additional resistance in fluoroquinolones, aminoglycosides, cyclic peptides and ethambutol

Prior to the Xpert XDR these were considered the best but now the XDR is definitely less complicated and preferred as its a closed system

110
Q

What resistance does gyrB encode?

A

Fluoroquinolone resistance

111
Q

What does the eis gene confer resistance against?

A

Aminoglycoside resistance -> kanamycin resistance

112
Q

What does the emb8 gene confer resistance against?

A

Ethambutol resistance

113
Q

What are the pros and cons of the GenoType MTBDRsl?
(5)

A

Sensitivity, specificity and accuracy for detection of XDR-TB was 100% -> used to rule in XDR-TB rather than rule out

Ethambutol sensitivty only 65% => not recommended for the detection of ethambutol resistance

Might have to do more than one line probe assay to encompass all resistance

Not recommended for use directly on clinical specimens

Not a closed system

114
Q

What is the GenoScholar PZA-TB, how does it work?

A

A LPA for the rapid detection of resistance to pyrazinamide (PZA)

It taregts a 700bp fragment designed to cover the entire pncA gene

115
Q

Talk about PZA resistance

A

High rates of PZA resistance (39%-60%) in patients wih MDR-TB and XDR-TB

116
Q

What are the benefits of the GenoScholar PZA-TB?

A

DST for PZA is rarely performed as it requires stringent control of pH and inoculum size = LPA is a big strep forward => allows us to detect more of it

Good sensitivity of 93.2% and specificity of 91%

117
Q

What PCR method is used for TB?

A

Targeted NGS Deeplex-MycTB kit

118
Q

Talk about Targeted NGS Deeplex-MycTB kit

A

ultra-deep sequencing of a single 24-plex amplification of the main resistance targets

Identify the mycobacterial species + resistance

Genotypes the MBC -> not done in routine lab only in reference lab

Detects mutations across 18 genes associated with first and second line resistance

119
Q

Talk about Targeted NGS Deeplex-MycTB kit

A

ultra-deep sequencing of a single 24-plex amplification of the main resistance targets

Identify the mycobacterial species + resistance

Genotypes the MBC -> not done in routine lab only in reference lab

Detects mutations across 18 genes associated with first and second line resistance

120
Q

What are the pros and cons of the targeted NGS Deeplex-MycTB kit

A

Predicted 92% of resistance to first line adn 95% to second line

Issues with PYZ, ethambutol and low level rifampicin resistance

Offers species ID and resistance ID

Offers limited genotyping

4.1 Medical Microbiology (30 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions