PAMM FOUNDATION LABORATORY FOR PATHOLOGY AND MEDICAL MICROBIOLOGY Development and evaluation of user-developed protocols for bacterial respiratory pathogens leveraging the snap-load-go workflow of BD MAX TM Ingrid Op den Buijs, Senior technologist PAMM, Laboratory for Medical Microbiology, The Netherlands
PAMM Laboratories, Veldhoven, The Netherlands Google Maps, 2013
Laboratory for Clinical Microbiology, Veldhoven, The Netherlands
Public Health Service regions The PAMM public laboratory of medical microbiology has an adherence area of 800,000 people in South-East Brabant. It serves four hospitals, nursing homes and 400 general practitioners. 137,000 molecular diagnostic (real-time PCR) tests in 2014 at the laboratory of medical microbiology. 5000 of those were on the BD MAX
Molecular Assays @ PAMM Respiratory infections Chlamydophila pneumoniae BD MAX Mycoplasma pneumoniae BD MAX Legionella pneumophilla BD MAX Chlamydophila psittaci BD MAX Mycobacterium tuberculosis complex Bordetella pertussis Bordetella parapertussis Pneumocystis carnii pneumonia Influenza A/B Respiratory syncytial virus (RSV) Rhinovirus Other Coxiella burnetti Streptococcus pneumoniae Dermatophytes Mumps Infection prevention ESBL Carbapenemases VRE MRSA STD Neisseria gonorrhoeae Chlamydia trachomatis Trichomonas vaginalis Mycoplasma genitalium Chlamydia trachomatis LGV CSF Herpes simplex virus 1/2 VZV Enterovirus Parechovirus Gastro-enteritis Salmonella spp Shigella spp Yersinia spp Campylobacter jejuni/lari/coli STEC Giardia lamblia Cryptosporidium parvum Dientamoeba fragilis Entamoeba histolytica Norovirus GI/GII BD MAX Rotavirus - BD MAX C.difficile toxins BD MAX Sequencing 16S sequencing LSU sequencing Next generation sequencing
Pre-BD MAX era at PAMM In-house developed assays easymag extraction followed by qpcr on ABI7500fast system Twice a week about 20 requests for qpcr bacterial respiratory pathogens: Mycobacterium tuberculosis complex Legionella pneumophila Mycoplasma pneumoniae Chlamydophila pneumoniae Chlamydophila psittaci
Migration to BD MAX Time-line (1-4 samples) BD MAX EasyMAG-ABI Sample prep Hands-on Extraction Hands-on2 PCR 00:00 00:14 00:28 00:43 00:57 01:12 01:26 01:40 01:55 02:09 02:24
BD MAX Tree different protocols: Closed BD IVD assays (CDIFF, Staph SR) Open BD mastermix, in-house primers and probes Research in-house primers, probes and mastermix
Leveraging the snap load go workflow Utilisation of dried primer-probe snap-ins to go with DNA kits
Migration b-cap to BD MAX Bacteria Target gene Sensitivity 1 QCMD12 L. pneumophila Outer membrane protein A 75 cp/ml 100% M. pneumoniae Macrophage infectivity potentiator protein 230 cp/ml 100% core 2 C. pneumoniae Outer membrane protein A 110 cp/ml 100% C. psittaci P1 adhesion protein gene 40 cp/ml 100% 3 1 Based on 500µl input. 2 One educational sample was not detected by the bcap assay (detected positive by 22.5% of participants). 3 For C.psittaci the 2013 panel was tested.
Experience b-cap Introduced to the routine-lab since october 1st 280 samples were tested 12 M.pneumoniae positives 4 L.pneumophila positives No C.psittaci or C.pneumoniae positives Due to easy of use: 7 days/week 100 90 80 70 60 50 40 30 20 10 0 oct-2014 nov-14 dec-14 jan-15 feb-15 Lpn Cps Cpn Mpn negatives
N M.tuberculosis complex diagnostics Total of 1891 TB culture requests in 2013. 1491 samples were also tested in PCR. Mean of 28 requests for PCR a week. Prevalence of 1.5% TB complex positive samples (23 positive patients) 50 45 40 35 30 25 20 PCR positive PCR negative 15 10 5 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 Week number
Multicenter study Collaboration of PAMM (Veldhoven), USC (Santiago de Compostella) & NHS Lothian (Edinburgh) External quality assessment: QCMD panel 2012 Analytical sensitivity with positive plasmid control Different pre-treatments Extended clinical evaluation
QCMD 2012 All sites ordered the QCMD 2012 panel at Qnostics Samples were proteinase K treated PAMM NHS Lothian USC QCMD Core? Sample Sample type MTB IC MTB IC MTB IC cells/vial MTB12-01 PRB 32.36 29.27 32.33 29.41 32.04 29.68 500 Y MTB12-02 PRB 32.67 29.63 33.03 29.38 33.85 29.38 100 N MTB12-03 PRB 33.49 30.18 31.44 28.98 31.42 29.52 1000 Y MTB12-04 PRB - 29.29-28.81-29.57 0 Y MTB12-05 PRB 29.68 28.78 27.75 28.98 28.18 29.15 10000 Y MTB12-06 Sputum 31.15 28.19 30.22 27.71 30.75 27.75 500 Y MTB12-07 Sputum 28.18 28.18 28.69 28.57 29.36 28.55 10000 Y MTB12-08 Sputum - 28.86-28.93-28.99 0 Y MTB12-09 Sputum 31.78 28.94 32.17 29.06 33.36 28.71 500 Y MTB12-10 Sputum 32.83 29.02 33.78 28.89 33.36 29.17 100 N All sites score 100%
Limit of detection Triplicates of IS6110 target-containing plasmids. Ten-fold dilutions tested in triplicates at all sites. The limit of detection was determined at 54-540 cp/ml at all laboratory sites. Further experiments showed a LoD of 270 cp/ml (13-54 genome copies). Copies IS6110 per genome Concept Dutch NvMM guidelines MTB
Pre-treatment of clinical specimens Tested both sputum as BAL samples. Pre-treatment methods that were used: Sputasol followed by 2% NaOH-NALC solution and phosphate buffer; BD Mycoprep decontamination treatment; Proteinase K incubation. After pre-treatment samples boiled to inactivate the MTB. Experiences with pre-treatments varies per laboratory. Most important is to liquefy the sample for pipetting purposes. One pre-treatment that worked for all participating laboratories: BD Mycoprep
Clinical evaluation Retrospective evaluation Pre-treatment methods customary for the specific laboratory sites: Sputasol followed by 2% NaOH-NALC solution and phosphate buffer; BD Mycoprep decontamination treatment; Proteinase K incubation. Prospective evaluation All the same treatmentmethod: BD Mycoprep Gold standard BBL MGIT source: BD.com Results were compared to smear results and culture A sample from a patient with proven active MTB was defined as true positive
Retrospective vs prospective study Culture Retrospective pos neg pos 78 3 81 BDmax neg 3 191 194 81 194 275 Sensitivity 96.3% Specificity 98.5% PPV 96.3% NPV 98.5% Culture Prospective pos neg pos 49 12 61 BDmax neg 11 227 238 60 239 299 Sensitivity 81.7% Specificity 95.0% PPV 80.3% NPV 95.4%
Retrospective vs prospective study True Retrospective pos neg pos 81 0 81 BDmax neg 3 191 194 84 193 275 Sensitivity 96.4% Specificity 100.0% PPV 100.0% NPV 98.5% True Prospective pos neg pos 61 0 61 BDmax neg 11 227 238 60 239 299 Sensitivity 84.7% Specificity 100.0% PPV 100.0% NPV 95.4%
Results clinical evaluation Retrospectively 25% of true positives were smear-microscopy negative All smear-microscopy positives were detected positive in qpcr Three (1%) of true positive samples not detected in qpcr From one patient 3 other samples were detected positive in qpcr All were smear-microscopy negative Prospectively 42% of true positives were smear-microscopy negative All smear-microscopy positives were detected positive in qpcr Eleven (3.7%) of true positive samples not detected in qpcr Eight different patients From four patients 1 other sample was detected positive in qpcr From the other four patients no other samples were tested All were smear-microscopy negative
Conclusion MTB BD MAX MTB assay is a sensitive and specific screening method of the detection of M.tuberculosis complex. The assay could provide a rapid alternative to smear-microscopy
Soon commercially available at Biolegio Detection of M.tuberculosis complex Quality control testing is ongoing Expected release-date: mid April 2015 Information & protocol ReadyMax TM snap-ins secretariaatmm@pamm.nl info@biolegio.com
Thanks to.. Jeroen van de Bovenkamp René Roymans Ingrid Op den Buijs Dominique Clarysse Ruud van der Steen Frank Spijkers Suzanne Paule