14102008 jitmm a15 ramasoota tuberculosis
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Rapid detection of MDR-TB fromindoor air using
modified impinger andin-house nested PCR
Pannamthip Pitaksajjakula, Pholsak Piyatatb, Pongrama Ramasootaa
a Faculty of Tropical Medicine, Mahidol University, 420/6 Rajwithii road, Rachadhewee, Bangkok10310, ThailandbAlpine Filter Company (Thailand),
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Topics
Background & Rationale
Materials & MethodsResults
Discussions
Outputs & Application
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Background & RationaleTuberculosis (TB) is the cause of deaths worldwide,especially the emergence of Multi drug resistant (MDR)-TB
Airborne tubercle bacilli produced from pulmonary TBpatients, as droplet nuclei remain suspended in the air for
long time
Transmission of aerosal MTB have been long time reported
in many studies in different setting, such as hospitals(Schaal et al., 1991; Wan et al., 2004), Dental clinic (Bennettet al., 2000), and Air craft (Driver et al., 1994)
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In Thailand, the estimated number of TB cases was 204 cases per100,000 people with 12,000 deaths per year (WHO, 2004)
Report on indoor air transmission of MTB, among emergency
department personnel (ED) in Bangkok between 1988 - 2002
The TB incidence rate among ED personnel was 1,610 per
100,000 person-year (PY) (17.2 times higher than non-exposedworkers)
Background & Rationale
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MTB detection and diagnosis can be both conventional andadvance molecular methods
Culture-based method, which is laborious, and time-consuming (2-4 weeks), especially drug-susceptibilitytesting of MDR-TB took longer time
Rapid detection of MDR-TB from indoor air is importantfor controlling diseases transmission and surveillance
Background & Rationale
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RIFr-MTB can be use as marker for predicting
MDR- TB (Kim et al, 1997).
95 % of RIFr-MTB have distinct mutation in the
rpoB gene (Telenti et al, 1997).
Using high sensitivity PCR, and DNA sequencingfor rapid detection of RIFr-MTB, can reducedetection time to be only 1-2 days.
Background & Rationale
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To detect MTB from indoor air
Air collection
PCR detection
Detection of MTB in indoor air
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For air collection, several methods have been widelyused, but some limitation were reported
For filter air collection, dehydration is the majorproblem
For air impactor collection, the problem of funguscontamination, and drying of agar plate is also the major
problem
So, the air impinger collection was selected, since there
are no dehydration problem, and convenient for furtherPCR experiment
Background & Rationale
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Materials & Methods
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Air collection development
The modified air impinger collection wasdeveloped using in-house equipment
All equipments used in this invention was made inThailand (10 time less cost than import product)
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The high flow air pump was used to pump the air
at 40 L/min
The air was collected pass through the glassinlet, and the organism were trapped in liquidsolution inside glass impinger
This liquid solution will be further used for DNAisolation
Air collection development
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PCR development (nested PCR)PCR primer design
rpoB gene of MTB and other airborne bacteriawere obtained from GenBank database
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PCR primer designThe rpoB gene of all bacteria were aligned andcompared using clustalX software
The Rifampicin-Resistance Determining Region
(RRDRs) for RIFr
-MTB detection were selectedfor primer design
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PCR primer designBy using Primer 3 software, 3 paired of primer
were selectedAll primer were analyzed by Oligo-analyzersoftware
Self-priming
Loop forming
The best primer were selected for further PCR
optimization
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PCR optimizationAll PCR reagents and condition were optimized
Annealing temperature
Mg2+concentration
Primer concentration
dNTP concentration
Standard positive control were includes to determinedthe PCR detection limit (sensitivity)
All other 8 airborne bacteria were tested to determinedthe PCR specificity (Enterococcus spp., Psuedomonas aeruginosa,Streptococcus aureus, Enterobacter spp., Klebsiella pneumoniae,Streptococcus pyogenes, Acinetobacter spp., Legionella pneumophilla)
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Validation of Methods30 samples of air collections were performed inlaboratory ;spraying MTB (H37Ra ) in laminar flow
MTB containing solution was filtered through 0.45m polycarbonate filter
The filter were then rinse with distilled waterThis suspension will be further used for DNAisolation
MTB detection by nested PCR
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Results
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PCR primer design & optimizationTwo set of primers were designed to use as outer
and inner primer in nested PCR
Primer Sequence
Outer primer
rpoB-f 5-TGG TCC GCT TGC ACG AGG GTC AGA-3
rpoB-r 5- CTC AGG GGT TTC GAT CGG GCA CAT-3
Inner primer
rpoB-7 5 - GAT CAC ACC GCA GAC GTT GA-3
TB8 5 - TGC ACG TCG CGG ACC TCC A-3
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PCR conditionThe following PCR cycle were performed
First PCR95 C, 5 mins
95 C, 1 min, 72 C, min, 72 C min, 2 cycles
95 C, 1 min, 71 C, min, 72 C min, 2 cycles
95 C, 1 min, 70 C, min, 72 C min, 2 cycles
95 C, 1 min, 69 C, min, 72 C min, 25 cycles
72 C, 7 mins
Second PCR
95 C, 5 mins95 C, 1 min, 69 C, min, 72 C min, 2 cycles
95 C, 1 min, 68C, min, 72 C min, 2 cycles
95 C, 1 min, 67 C, min, 72 C min, 2 cycles
95 C, 1 min, 66 C, min, 72 C min, 30 cycles
72 C, 7 mins
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PCR optimizationBy nested PCR, as low as 2-20 bacilli can bedetected
All other 8 airborne bacteria were not amplified
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RIFr
-MTB detectionAfter PCR, the purified PCR product can be sentfor DNA-sequencing to identify mutation in hot
spot region
RIF
r
-MTB can be detected or suspected within 2days
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Discussion & Conclusion
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Discussion & ConclusionBy combination of both air sampling techniqueusing modified air impinger and in-house nestedPCR, as low as 2-20 tubercle bacilli can be detected
RIFr
-MTB also can be detected by direct DNA-sequencing of the purified PCR products
From this development, the suspected of MTB andRIFr-MTB contaminated in indoor air could bedetected
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Output
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Output & Application
Air collection & airborne bacteria and MTBdetection
Head office, Kasikorn Bank
Pranungklao Hospital, Nonthaburi Thonburi Hospital
Tesco Lotus Super Center, Ramindhra
Detection of MTB from Patient samples
Tropical Medicine Hospital, Faculty of Tropical
Medicine, Mahidol University
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National Inventor Award 2008 in MedicalSciences, National Research Council of Thailand
Output & Application
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Acknowledgement Thailand Tropical Disease Research Programme
(T2), National Research Council of Thailand
Mahidol University
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Thank You
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