who, why, what and when of the wcm ruddy.pdf · wcm: who we are national mycobacterium reference...
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Who, Why, What and
When of the WCM
Dr Michael Ruddy
Wales Centre for Mycobacteria
Public Health Wales Microbiology
WCM
Who we are
Why we exist
What we do
When and how to access and use us
WCM: who we are
National mycobacterium reference unit
for Wales
Regional mycobacterium reference unit
for SW Region England
Primary mycobacterial analysis majority
of samples in Wales
Reference services to Channel Islands/
Isle of Man/ private Vet labs/ parts of
Eire
Llandough Hospital ~1933 (courtesy Cardiff University)
Microbiologylaboratories
NHSNPHS
StaffWCM
Rhian Williams
Gwyneth Samuels
Kay Parry
Dave Tucker
WMC
Lewis White
Michael Perry
Sally Corden
PHW Micro ABM
Jenny Bayliss
Mike Isaac
WCM Reporting/Collaboration
HPS SMT via PHW Microbiology Cardiff
PHW TB Programme Group/CDSC
DH via HPA CDSC TB section/TB
Programme Board/HPA NMRL
UK Mycobacterial reference laboratory
network
UK TB DAME Group
UK MDRTB Group
HPA LARS
ECDC
PHW TB Programme
Promote best practice for the prevention
and control of tuberculosis
Co-ordinate the National TB
Programme for Wales
Provide specialist TB microbiological
diagnostic services through the Wales
Centre for Mycobacteria
Why
TB
MDRTB
MOTT/NTM
TB
Clinical disease caused by Mycobacterium tuberculosis (M tb)
“consumption”
“Captain of the men of death”
“Great white plague”
Killed 1/4 Europeans 19th Century
killed 1 billion worldwide in last 200 years
TB
One third world infected (~2 billion)
~10% immune competent develop disease
~8 million new cases pa
2-3 million deaths pa
major cause death 15-49 year olds (inc HIV)
>95% burden in poorest countries
Estimated TB Incidence Rates, 2001
25 - 49
50 - 99
100 - 299
< 10
10 - 24
No estimate
per 100 000 pop
300 or more
The designations employed and the presentation of material on this map do not imply the expression of any opinion whatsoever on the part of the World Health
Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.
White lines on maps represent approximate border lines for which there may not yet be full agreement.
© WHO 2003
Predicting Risk of Infection
Nature & duration contact infectious case
smear status estimate of bacilli excretion
survival of bacilli in droplets:
– UV light (kills bacilli)
– air movement (disperses droplets)
overcrowding; ventilation; sunlight
prompt detection and management of cases
screening
Predicting Risk of Disease
Compromised immune competence to M
tb:
– HIV; diabetes; chemotherapy
– malnutrition
– extremes of age
Enhanced immune competence to M tb:
– exposure to atypical mycobacteria
– vaccination (BCG)
– health and nutrition
TB Drug Resistance
Spontaneous mutations (1x10-7/8)
selection of bacilli resistant mutations (“Acquired Resistance”)– poor drug prescribing; poor adherence;
poor supply; poor quality
transmission of resistant strain to new case (“Primary Resistance”)
Multi Drug Resistance (MDR): H+R
Extensive Drug Resistance (XDR): H + R + injectable + fluoroquinolone
MDR TB: WHO IUATLD 2008
Globally 4.8%
Range MDR TB (new cases) 0 – 22.3%
– Baku, Azerbaijan 22.3%
– Rep Moldova 19.4%
– Donetsk, Ukraine ~ 18%
– Estonia ~ 14%
– Latvia ~ 12%
– Lithuania ~ 10%
– Inner Mongolia ~ 7%
LuxembourgMalta*
NetherlandsSlovenia
United KingdomFinland
BelgiumDenmarkFrance
Greece*Czech Republic*Ireland*Portugal*Austria
SlovakiaGermany
Italy*Bulgaria*
Spain*Hungary*
RomaniaCyprus
LatviaLithuania
Estonia
0 5 10 15 20 25
Multi drug resistance (MDR),
EU 2005
Percentage per tested cases
* Countries with incomplete data
Source: EuroTB
Source: WHO
Chemotherapy
BCG vaccination
Tuberculosis ( all forms )- Notifications, annual totals
England and Wales 1913 - 2000#
Year
Port Health Authorities not included - 1913-1938 classified as pulmonary TB
Figures for 1982 onwards exclude notifications for chemoprophylaxis
# Data for 2000 provisional
Source: Statutory Notifications to the Communicable Disease Surveillance Centre
TB Notifications England & Wales 1982 -
2005
Source: HPA ETS Sept 07
TB Notifications UK By Region
2005
Source: HPA ETS Sept 07
Source: NPHS CDSC
TB & Drug Resistance Wales
& England 2009
9,040 total TB notifications
–15 cases per 100,000 population
214 TB notifications Wales
–7.1cases per 100,000 population
Drug resistance 7.8%
MDR TB 1.2%
Summary of TB epidemiology
~2 billion people infected with Mtb
~8 million new cases annually
~9000 new cases UK annually
(increasing)
Increasing rates MDR worldwide
Increasing globalisation
Classification Mycobacteria
(TB or not TB)
1880s-1980s: phenotypic
Post 1990s: genotypic (16S rRNA sequencing) >100 species
MTBC: – M tuberculosis, M bovis, M africanum, M microti
(M leprae)
MOTT/NTM: – slow growing mycobacteria
– Rapid growing mycobacteria (RGM)
Epidemiology MOTT/NTM
Free living, ubiquitous in environment
Water, dust, soil, food, milk
Associated with animals (wild, domestic, birds)
No significant person-to-person spread
Oligotrophic (grow in metal cleaning fluids)
Waxy/lipid walls – hydrophobic
Epidemiology MOTT/NTM
Numbers isolates increasing:– 0.5-30% ref lab reports
– >35% reports CDC Atlanta
– >50% isolates NMRL
– ~40% isolates WCM
Increasing number of different species isolated from clinical samples
Majority of disease relates to few common species (eg MAC >90% disease in HIV/AIDS)
Clinical-Pathological Features
Pulmonary disease
Superficial lymph node infection
Skin & soft tissue lesions
Other extra pulmonary sites
Disseminated infection in the immune-compromised
Newer disease presentations
Treatment: overview
Manage under expert in mycobacterial disease
Consider surgery
Basis many MOTT/NTM regimens is prolonged rifampicin & ethambutol – (+\- macrolides/quinolones)
In vitro DST results not helpful (accept M kansasii & RGM)
? Role newer agents eg linezolid, moxifloaxcin, tigecycline
Laboratory role
What we do
Robert Koch 1843-1910
Nobel Prize (1905)
(Physiology or Medicine)
WCM: What we do
Role of Laboratory
Microscopy (auramine/ZN stain for AAFB)
Culture (solid & liquid)
Identification (conventional & molecular)
Drug sensitivity testing (conventional &
molecular)
“Serology” (gamma interferon testing)
Molecular strain typing (MIRU-VNTR/RFLP)
Fluorescent acid-fast bacilli seen in auramine stain of a sputum
specimen (M.Tb)
Pink / red acid-fast bacilli seen in Ziehl-Neelsen stain
of sputum. (M.Tb)
Geneprobe luminometer for rapid mycobacterial culture identification
Automated geneprobe for ID of
Mycobacterial species
“HAIN GenoType”
HAIN GenoType
Mycobacterium
M.Tb susceptibility test after 2 weeks incubation: drug free control slope on far left shows
growth of M.Tb, isoniazid containing slopes (blue labels) show no growth = SENSITIVE to
Isoniazid
M.Tb susceptibility test after 2 weeks incubation: drug free control slope on far left shows growth
of M.Tb, isoniazid containing slopes (blue labels) all show growth. RESISTANT to Isoniazid.
Direct molecular detection MTBC
GenoType MTBDRplus system (Hain
Lifescience) (FIND)
– Smear positive respiratory samples
– CSF
Can detect common rifampicin and isoniazid
resistance mutations in rpoB, KatG & InhA
Good correlation culture and DST
? ?Smear negative respiratory samples?
Interferon-gamma
testing
IFN-gamma for TB
Commercial blood tests to detect
infection with M tb
Expose patient blood sample to
specific antigens from M tb
Measure the release of IFN-gamma
from the peripheral blood monocytes
TB-specific antigens: ESAT-6 &
CFP-10
Encoded by RD-1 genes
Absent from BCG
Absent from most MOTT/NTM
Induce IFN- responses
Species specificity of ESAT-6 and
CFP-10Environmental
strains
Antigens
ESAT CFP
M abcessus - -M avium - -M branderi - -M celatum - -M chelonae - -M fortuitum - -M gordonii - -M intracellulare - -M kansasii + +M malmoense - -M marinum + +M oenavense - -M scrofulaceum - -M smegmatis - -M szulgai + +M terrae - -M vaccae - -M xenopi - -
Tuberculosis
complex
Antigens
ESAT CFP
M tuberculosis + +
M africanum + +
M bovis + +
BCG substrain
gothenburg - -
moreau - -
tice - -
tokyo - -
danish - -
glaxo - -
montreal - -
pasteur - -
Andersen P et al, Lancet 2000
IFNg release: rationale
Benefits IFN-gamma vs TST
Not influenced by prior BCG
Less influenced by exposure to
MOTT/NTM
No need for repeat visit for reading
Recommendations For
Usage 2005 CDC (Atlanta): QFT-G = TST
2006 NICE:– LTBI (if positive TST)
– 6 week repeat test in initial TST negative TB contacts
– “rule out infection” if no cultures
2007 DH “Toolkit”:– not cost effective for active TB
– potential value in difficult cases (children, immune compromised)
2007 DH new h/c workers OcH screening:– can use in place of TST
? Asylum seekers
? Pre immune suppression (eg anti-TNF alpha)
Molecular Typing M tb
Mtb Typing: Why perform?
Local outbreak investigation (confirm suspected epidemiological links)
Detect unknown links – targeted interventions
Exclude lab cross contamination
Detect emergence of new strains
Detect globally prevalent strains (?inform vaccine strategies/treatment protocols)
MIRU-VNTR Mycobacterial Interspersed Repetitive
Units Variable Number Tandem Repeats
Mini satellite regions of repeating sequences of nucleotides
?function, highly conserved
Number of times the sequence repeats at each region varies
>40 such regions (MIRU) useful to type strains
15 specific loci adopted across UK
24 loci new international standard
Combined results
ETR MIRU
Isolate Patient A B C D E 2 4 1
0
1
6
2
0
2
3
2
4
2
6
2
7
3
1
3
9
4
0
A 3 1 4 3 3 2 2 2 3 2 5 1 4 3 3 2 3
B 6 4 4 6 5 2 5 4 3 2 1
0
2 2 3 5 3 3
C 2 2 2 3 2 1 2 4 3 2 6 1 5 3 2 2 4
D 4 2 2 3 4 2 2 4 5 2 5 1 7 3 4 3 3
E 3 1 4 3 3 2 2 2 3 2 4 1 4 3 3 2 3
F 3 2 3 3 3 1 2 3 3 2 5 1 5 3 3 2 3
G 4 2 2 3 4 2 2 7 4 2 5 - 1 3 4 3 4
H 2 2 2 3 2 1 2 4 3 2 6 1 5 3 2 2 4
VNTR-
MIRU
Generate a
15(24) digit
strain
designation
number
Collated in
UK National
database
Geographical
incidence of TB
isolates with 15-
VNTR profile
‘422225435173533’,
which corresponds to
the Leicester outbreak
strain.
Summary
Key laboratory role:
–Prompt diagnosis
–Assist expert clinical case management
–Guide Public Health interventions
–Help break chain of infection
–?? Future “search and destroy”
WCM Info & Contact Details
PHW: http://howis.wales.nhs.uk/sitesplus/888/WCM user manual
WCM: 029 20716408 [email protected]
WMC: 029 20746581 [email protected]
Acknowledgements
Daniel Thomas, Eleri Davies, Gwen Lowe,
Gwyneth Samuel, Jenny Bayliss, Kay Parry,
Lewis White, Lika Nehaul, Michael Perry,
Mike Isaac, Paula Brookes, Peter James,
Rhian Williams, Robin Howe, Sally Corden
WCM/WMC
CDSC
PHW TB Programme Group
HPA MRU London