How a diagnostic system operates in Ghana

Transcription

1 How a diagnostic system operates in Ghana

2 Case finding: Community based volunteers Suspect cases are referred to treatment centres Clinical diagnosis established by local surgeons Eligible for laboratory diagnosis: Early lesions (duration of the disease < 6 months) Complete sets of specimens with clinical information

3 Diagnostic network External Reference Laboratory DITM EQA MIC, PCR specimens Local Reference Laboratory KCCR MIC, Culture, PCR specimens External Reference Laboratory BNITM Histopathology Diagnostic specimens Agogo Hospital Dunkwa Hospital Goaso Hospital Agroyesum Hospital BUD Treatment Centres

4 Specimen collection Standardized protocol for specimen collection: Non-ulcerative lesions 3 x tissue specimens * 1 x tissue microscopy/culture culture 1 x tissue DRB-PCR** 1 x tissue histopathology (differential diagnosis) x swab smear microscopy (locally) 1 x swab culture Ulcerative lesions: 3 x swab specimens 3 x tissue specimens * 1 x swab DRB-PCR** 1 x tissue microscopy/culture culture 1 x tissue DRB-PCR ** 1 x tissue histopathology (differential diagnosis) *Tissue specimens are either post-surgically excised specimens or punch biopsies ** one additional swab and tissue specimen each for standard reference PCR

5 Specimen collection Specimen bags drug treatment sites = DT surgical treatment sites = ST Purpose: Specimen containers And other items for specimen collection transport of specimens in bags to laboratory

6 Specimen collection: specimen bags drug treatment sites = DT surgical treatment sites = ST Specimen containers with different storage/transport media Swabs taped to specimen containers Biopsy punches Sterile scalpel Sterile forceps Ruler Data entry form Specimen containers with different storage/transport media Swabs taped to specimen containers Sterile scalpel Sterile forceps Ruler Data entry form

7 Specimen containers Surgical treatment (ST*) non-ulcerative lesions tissue specimen containers ulcerative lesions tissue & swab specimen containers 3 x tissue 3 x swab 3 x tissue * Treatment centres that are not part of the BURULICO drug trial

8 Specimen containers Drug treatment (DT*) non-ulcerative lesions tissue specimen containers ulcerative lesions tissue & swab specimen containers 3 x tissue 2 x biopsy punch 3 x swab 3 x tissue * Drug trial centres receive only specimen bags with biopsy punches

9 Microscopy/ Culture Media PCR Histo- pathology Transport media (+ PANTA) (stable at RT) Becton Dickinson Dubos Broth Base + Glycerin + PANTA Cell Lysis Solution (stable at RT) Gentra Systems (part of the DNA extraction kit) 10 % Formalin (stable at RT) Fixation solution

10 Specimen collection non-ulcerative lesions (nodules) 1. Tissue specimen (microscopy/culture) 3 2. Tissue specimen (DRB-PCR) 3. Tissue specimen (histopathology) 1 2 histopathology Segments from centre of the lesion microscopy & culture PCR

11 Specimen collection ulcerative lesions 1) Diagnostic swab (ZN microscopy) 2) Diagnostic swab (culture) 3) Diagnostic swab (DRB-PCR) Circling the undermined edge of ulcer! 1,2,3 4) Tissue specimen (micoscopy/culture) 4,5,6 5) Tissue specimen (DRB-PCR) 6) Tissue specimen (histopathology) Edge of ulcer below the end of the undermined edge of the lesion

12 Specimen collection tissue specimens Standardized size and quality of tissue specimens: 3 mm punch biopsies surgical specimens: 10 x 10 mm all tissue specimens must contain subcutaneous adipose tissue!

13 Data collection: Data entry form

14 BU1/BU2 forms

15 Laboratory analysis Swab and tissue specimens Microscopy Culture DRB-PCR Histopathology

16 Processing of specimens Diagnostic specimens Local Reference Laboratory MIC, PCR, Culture MIC, PCR specimens External Reference Laboratories EQA MIC, PCR Histopathology Preliminary result Final result Consensus result not determined Hospital

17 External Quality Assurance Microscopy PCR Reading of slides at test laboratory Blinded re-reading of slides by controller Sample size: 100 % (no baseline data available) Discrepant results re-read by controller 2 nd controller result = final result Determination of false negatives and false positives Corrective action: Re-reading of discrepant slides at test laboratory under supervision Testing of PCR specimens at test laboratory Blinded parallel testing by controller Sample size: 100 % (no baseline data available) Discrepant results re-tested by controller 2nd controller result = final result Determination of false negatives and false positives Corrective action: Re-testing of discrepant specimens at test laboratory

18 External Quality Assurance Microscopy (n = 287 slides) PCR (n = 265 specimens) Concordance rate: 82.9 % Discordance rate: 17.1 % False negatives test laboratory: 11.1 % High false negatives test laboratory: 7.3 % False positives test laboratory: 5.9 % High false positives test laboratory: 5.9 % Corrective action: Re-reading of discordant slides under supervision Second reading test laboratory: False negatives test laboratory: 1.7 % High false negatives test laboratory: 1.4 % False positives test laboratory: 0.3 % High false positives test laboratory: 0 % Concordance rate: 97.9 % Discordance rate: 2.1 % Concordance rate: 87.9 % Discordance rate: 12.1 % False negatives test laboratory: 5.3 % False positives test laboratory: 6.8 % Corrective action: Re-testing of discordant specimens Second PCR test laboratory: False negatives test laboratory: 3.8 % False positives test laboratory: 0.8 % Concordance rate: 95.5 % Discordance rate: 4.5 %

19 Laboratory confirmation January 2003 August 2005: Diagnostic specimens from 185 clinically diagnosed BUD patients subjected to laboratory analysis (ulcers: n= 115, nodules: n=70) One positive laboratory test: 71 % laboratory confirmed cases Two positive laboratory tests: 53 % laboratory confirmed cases one positive laboratory test: 20 % more laboratory confirmed cases

20 Laboratory confirmation Pre-ulcerative cases: Combination of : Tissue microscopy (40 %) + Tissue PCR (additional 25 %) = 65 % positive results 65 % laboratory confirmed cases (post-surgery) Ulcerative cases: Combination of Swab smear microscopy (30 %) + Swab PCR (additional 40 %) = 70 % positive results 70 % laboratory diagnosis (pre-surgery) Tissue Microscopy + Tissue PCR (additional 5 % post-surgery)

21 20 % more laboratory confirmed cases by one positive laboratory test Combination of MIC and PCR detects 65 % of pre-ulcerative and 70 % of ulcerative cases Stepwise approach to the laboratory diagnosis of BUD Pre-ulcerative: Swab smear MIC + Swab PCR + Histopathology Ulcerative: Tissue MIC + Tissue PCR + Histopathology Benefits: Pre-treatment diagnosis for ulcers by nonivasive test Cost savings compared to simultaneous testing - Pre-ulcerative: 35 % - Ulcerative: 60 %