Diagnosis of Tuberculosis in Children: A Cooperation Study ... · Spine confirmed Spine unconfirmed...

University of Berne

Kantha BophaFoundationCambodia

21. Tuberculosis Symposium Münchenwiler22.03.2012

Diagnosis of Tuberculosis in Children:

A Cooperation Study between Switzerland and Cambodia

Kurt Schopfer

Former Director of the Institute of Infectious Diseases (ifik), University of Bern, Switzerland

University of Berne



PEDIATRIC TUBERCULOSIS SURVEY KANTHA BOPHA HOSPITALS(SIEM REAP AND PHNOM PENH), CAMBODIA: TIME SCHEDULE

2003 Project planning and decision making(B. Richner, P. Studer, Kantha Bopha Foundation; K. Schopfer, University of Berne, Switzerland)

2004 Project initiation in the Kantha Bopha Children’s Hospitals in Phnom Penh and Siem Reap, Cambodia

2008 Project conclusion2011 End of evaluation and publication work (K. Schopfer, H. Rieder)

Overview Cambodia and Kantha Bopha Hospitals

Investigation planning and logistics

Result presentation and discussion

OUTLINE PRESENTATION

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Area 1‘818‘040 km2

Arable land 20.44%

Predominantly rural

Mekong „Mother of all Waters“, „The Nine tailed Dragon“ Longest river in SE AsiaSpecies richest river on earth

Tonle Sap 2‘500 (dry season) to 25‘000 km2 (rainy season)Downward (dry season) – upward (rainy season)

Largest fresh water lake in SE AsiaMost important ecosystemMost productive inland fishery of the worldFish: ~75% of protein consumed in Cambodia

Cambodia: Geography

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Ancient History Khmer EmpireLargest sacral complexes on earthMost densly populated urban areas on earth

Recent History Vietnam War, „Khmer Rouge“, UNCTAD (HIV)

Population 13‘995‘904 (census 2008)

Ethnic groups Khmer 90% Vietnamese 5%Chinese 1%Others 4%

Religion Theravadda Buddhism 95%

Net migration 0 migrants/1000 population

Cambodia: History and Demography

Kantha Bopha Hospitals Phnom Penh and Siem Reap Largest pediatric hospital complexes in Cambodia>80% of all children hospitalized in Cambodia>100’000 hospitalised in 2008

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The build up of a TB laboratory: logistics

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The build up of a “Tuberculosis Laboratory Unit”: logistics

2003 Decision making

2004 Start building up «TB unit»Training staff membersDiagnostic flow schedule

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K. Schopfer et al, Int J Tuberc Lung Dis; 16(4):503-506,2012

Phase 1:Objective: Operational infrastructure and logistics:building laboratory, implementing technologies, diagnostic flow schedule

88 children

Phase 2:Objective: Diagnosis of tuberculosis:

Clinical diagnosisLaboratory confirmation by Ziehl-Neelsen, rRNAamplification within 48h (on site)

Aliquot refrigeration and periodic shipments to Switzerlandfor cultivation (DST, Molecular Biology, isolate collection)

405 children

Phase 3:Objective: M. tuberculosis isolate collectionSampling reduced to defined workdays in order to reduceworkload

96 children

Overview Project Organization from 2004 to July 2008

Total of 589 children investigated

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Phase 2, Siem Reap Hospital:

9 month periode: July 1st, 2005 to March 31st, 2006

Top row in box numerators, bottom row denominatorsCircles with vertical lines: monthly point estimates with 95% CIStraight horizontal line: average proportion of confirmed cases

Dotted horizontal line: 95% CI (18.7 -26.8)

Diagnostic Performance during study phase 2

405 children: clinical diagnosis of tuberculosis

91 children: microbiologically «confirmed» diagnosis of M. tuberculosis infection(ZN, rRNA, cultivation)

Rate per thousand admissions:Clinical cases: 7.7 / 1000“Confirmed” cases: 1.7 / 1000

rRNA identified 91.2% of all culture “confirmed” cases

Culture alone contributed 7.1% to all laboratory confirmed cases

Maximum turn-around time for on-site laboratory diagnosis was 48h for 97.4% of all children

54’000 children hospitalized in the Siem Reap Hospital during this phase 2 periode


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dashes horizontal lines:95% CI interval around mean proportion over the entire phase

Demographic data (Study Phase 2)

Among all cases: Boys slightly predominated: female/male ratio: 0.80

“Confirmed”: Females slightly predominated: 1.17Clinical : Opposite being true: 0.71

Male predominance among all cases:

Partially explained by sex imbalance at admission:0.78 (as compared to census with close to 1)

Mean age with clinical diagnosis (both sexes): 6.3 years

Mean age with “confirmed” diagnosis: girls: 8.6 yearsboys: 8.7 years

Children with confirmed diagnosis significantly older than those with a clinical diagnosis


Age distribution:

Rate of infection:

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1.75 Mio children in population (served by the Siem Reap KB Hospital:4’5 Mio; 38.7% aged 16 years or less according census 2008)

54’000 children hospitalized in Phase 2

Phase 2

«Confirmed» in Phase 2

Age specific proportion of«confirmed» in Phase 2

< 1 year 1 to < years 3 to <5 years 5 to <16 years

Age groups in years

0

20

80

60

40

Perc

ent o

f pop

ulat

ion

Age distribution of childhood population

Demographic data (Study Phase 2)

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Control chart:

Interval of sampling and arrival in thecollaborating institute in Switzerland

Of 1516 specimens collected 404 had valid cultureresults and positive ZN and/or rRNA results

Of those 383 had valid storage and shipment data

Of those 282 (75.2%) confirmed by culture

No evidence of negative impact of storage length, refrigeration and resulting delay between samplingand cultivation results in Switzerland

Straight horizontal line: Positivity over entire study periodeDashed lines: Upper and lower control limits (3 SD from mean in each interval)

Hollow circles: point proportion of culture positivity at each interval

Shipment delay and cultivation yield


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Clinical Analysis (Study Phase 2)

Refinded data analysis in preparation!

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Dominant extra-respiratory manifestations, Phase 2

Per c

ent o

f cas

es

0

20

40

60

80

100404 239

Extra-respiratory

only

Allcases

Extra-respiratorycases

Musculo-skeletal, not spine

Other extra-respiratory

Gastro-intestinal

Spine with psoasSpine without psoasPsoas, no spine

Lymphatic

(Sub-)cutaneous

Nervous system

Respiratoryonly

Both



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Dominant extra-respiratory manifestations, Phase 2

Per c

ent o

f cas

es

0

20

40

60

80

100404 239

Extra-respiratory

only

Allcases

Extra-respiratorycases

Musculo-skeletal, not spine

Other extra-respiratory

Gastro-intestinal

Spine with psoasSpine without psoasPsoas, no spine

Lymphatic

(Sub-)cutaneous

Nervous system

Respiratoryonly

BothNot-specified


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CRP in tuberculosis of the spine and psoas abscess

CRP value

0

100

200

300

n=11 n=10 n=3 n=8

Spineconfirmed

Spineunconfirmed

Psoas onlyconfirmed

Psoas onlyunconfirmed

90th75th

25th10th

Median

Percentile



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IGRA positivity by age and case status

Age group (years)

0 1 3 5

Per c

ent p

ositive

0

20

40

60

80

100

5 years and older

Total

Confirmed

Not confirmed

Interferon Release Assay (IGRA)

IGRA in children with a clinical diagnosis of tuberculosis stratified into age groups

IGRA in all children with a clinical diagnosis of tuberculosis

IGRA (Quantiferon Gold, in tube) in 338 (75.9%) of405 patients in study phase 2

Clinical cases (n 257): IGRA positivity: 10.9%

Confirmed cases (n 81): IGRA positivity : 53.1%


No significant age dependence

IGRA in children with «confirmed» tuberculosis stratified into age groups

IGRA in all children with «confirmed» tuberculosis

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Drug susceptibility test results among M tuberculosisstrains isolated from 159 children

Per c

ent r

esis

tant

0

5

10

15

20

anyresistance

any SMYresistance any INH

resistanceany RMPresistance

any MDR

1.9%

Drug susceptibility testing

Resistant strains (any drug) more prevalent in samples from the Phnom Penh Hospitals (southern urban region of Cambodia, mostdensely populated) than in the Siem Reap region (more rural area).

Drug resistance situation: of no significant impact for treatment


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M. tuberculosis strain prevalence:

Cambodia

Beijing: 12.9%EAI: 60.8%

Siem Reap Hospitals:


Phnom Penh Hospitals:


Spoligotype families among 159 M tuberculosis strainsisolated from children, Siem Reap compared to Phnom Penh

Per

cen

t with

spo

ligot

ype

0

10

20

30

40

50

60

70

Beijing EAI Other

Siem ReapPhnom Penh

Genotyping by spoligotype testing


Collaborative Investigation withDick van Soolingen and Kristin Kremer; Tuberculosis Reference Laboratory, National Institute of Public Health andEnvironment, Bilthoven, The Netherlands

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China > 50%

Vietnam ~ 50%

Thailand ~ 50%

Cambodia ~ 13%

Beijing strain prevalence:

Genotyping: Beijing strain prevalence pattern


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?

Genotyping: strain prevalence pattern by spoligotype analysis

?

Dangrek mountains

North to South «gradient» of strain prevalence(Beijing, EAI) within Cambodia (Phnom Penh region: 25.7% to Siem Reap region: 8.9%)

«Gradient» Cambodia to neigbouring countries (12.9% to ~50% or more)

Explanations ? (Cohorts? Geography? Demography? …?)

(Comparable finding regarding resistance pattern distribution)


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?

Genotyping: strain prevalence pattern by spoligotype analysis

?

Dangrek mountains

„Children play a special role in the understanding of theepidemiology of tuberculosis. … has very limited impact on thedynamics of the tuberculosis epidemic in a community …, tuberculosis in children is more informative about the epidemic‘sdynamic than any other manifestation of the disease. Tuberculosisin children always points to recent transmission …“H.L. Rieder, Epidemiology of tuberculosis in children, Annales Nestlé, 55:1-9, 1997

«unselected children population» such as it is the case in a general pediatrichospital may more closely mirror the true pattern of M. tuberculosis straincirculation as any adult cohort …???

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Stratification into 3 study phases (589 patients overall):

Phase 1: Preparation (88 patients) Phase 2: Clinical trial (52’400 patients admitted; among them 405 with a clinical diagnosis of tuberculosis

(excluding 6 from Phnom Penh), 91 «confirmed» as M. tuberculosis infection by microbiology (22.5%)Phase 3: M. tuberculosis isolate collection (96 patients)

Samples: 1516 samples analyzed (2.5 per child): 1473 ZN (21.8% pos), 1421 rRNA (30.8% pos), 1082 cultures (28.2% pos). – Total of 159 different M. tuberculosis isolates available at present

On site availability : rRNA within 48 hours in 97.4% of the patients in clinical trial (phase 2)(rRNA identified 91.2%; rRNA 90.5 % positive in culture confirmed cases)

Cultivation: Added 7.1% to all laboratory confirmed cases

Shipment: No impact of storage length (up to six months)

Clinical: Mean age: clinical diagnosis 6.3 yearsconfirmed 8.6 (girls) resp. 8.7 years (boys)

Sex ratio: female/male 0.8; confirmed 1.17, clinical 0.71 (admission 0.8)

Rates per 1000: clinical 7.7, confirmed 1.7Clinical presentation: respiratory only: ~45%

extrarespiratory only: ~45%mixed ~10%

Summary I


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IGRA: Positive: in 10.9% of clinical casesin 53.1% of confirmed cases,

No age dependency

DST: Any resistance: 15% of all strains isolatedSelected drugs: SMY 8%, INH 7%, RMP 1.9%Of no impact for treatment

Slightly more resistant strains in the Phnom Penh Hospitals

Genotyping: Spoligotyping: Beijing Cambodia: 12.9%Phnom Penh Hospital: 25.7%Siem Reap Hospital: 8.9%

EAI Cambodia: 60.8%Phnom Penh Hospital: 42.9%Siem Reap Hospital: 63.7%

Compare to neighbouring countries

Summary II


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Acknowledgement

Data analysis H.L. Rieder, International Union against Tuberculosis and Lung Diseases, Paris, France

Spoligo- andMIRU-VNTR-Typing Dick van Soolingen and Kristin Kremer; Tuberculosis Reference Laboratory, National Institute of

Public Health and Environment, Bilthoven, The Netherlands

Cambodian Team B. RichnerP. StuderD. LaurentY. ChantanaT. Somathea

Many collaborators of the medical and the nursing staff, the laboratory crew and administrative team of the Kantha Bopha Hospitals in Phnom Penh and Siem Reap, Cambodia

Swiss Team J. PortmannA. HiltyS. LüthiT. BodmerJ. Steinlin-SchopferS. DrozD. Schopfer

Many collaborators, especially of the Mycobacteriology Unit, of the Institute of InfectiousDiseases, University of Bern, Switzerland

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Culture positivity among microscopyand / or RNA positive results

Per c

ent c

ulture positive

0

20

40

60

80

100

Microscopy posrRNA neg

Microscopy negrRNA pos

Microscopy posrRNA pos

Culture positive Institute of Infectious Diseases, Bern

Phase 2 in the Siem Reap laboratories

Entire study periode in the Siem Reap laboratories

Analysis by specimen rather than children


Diagnosis of Tuberculosis in Children: A Cooperation Study ... · Spine confirmed Spine unconfirmed...

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