In the name of GOD

Automated methods in the MicrobiologyLab-Issues and Troubleshooting!

Presented By: Dr B. Oboudi

Background/history Plate streakers and Gram stainers Blood cultures Automated Susceptibility Testing Automated ID testing Automated Urine Analysers Molecular assays MALDI-TOF

Overview

Background

Manual streaking of plates has not really changed sincesolid agar plates were first used

Microscopy is mostly unchanged Incubators while probably more reliable are essentially the

same Plate reading has not really changed over the years

Obstacles to automation in Microbiology

Microbiology is too complex to automate: blood,sterile body fluids, urine, catheter tips, tissues,prosthetic devices, lower respiratory tract specimens Variations in the processing of specimens: tissue

digestion, urine colony count, impression smearspreparation Machines are programmable but humans are flexible Costs of automation Microbiology labs are small for automation

Pre analytical

Specimen separators Media pouring Issues with each are unique

Issues and solutions

Sterility at every point or risk of contamination Tubings need to be autoclaved UV light needs to be regularly inspected and

maintained

Conventional blood culture

7 days

Blood Cultures

Standard in most diagnostic labs, varying sizes. Better detection times More advanced media Reduced total incubation before calling a bottle a

final negative Suppliers: BacT/Alert (BioMerieux), BACTEC FX

(BD), Versa TREK

Issues with blood culture systems

False positives due to high TLC count Continuous power supply needed Cost

Conventional ID and susceptibility testing

24-48hours

Automated ID and Sens

Automated ID testing available since 1977 Biochemical substrates miniaturised and read by

colorimetric or fluorometric means Available in many labs Multiple Antibiotics in different dilutions available on Cards

or panels to ascertain MIC Suppliers: Microscan Walkaway (Dade Behring) Vitek2

(BioMerieux) BD Phoenix (BD)

Issues with identification systems

Pure culture Simple tests are not covered like oxidase, catalase,

motility, pigment production Variability of identification in rarer organisms Only organisms covered in database Inoculum size can affect MIC interpretations MICs of some antibiotics are not actual MICs but

extrapolations from related antibiotics

Automated Urine Analysers

Automated Dip-strip inoculation and reading Cell counts performed automatically – either by flow

cytometry or (more recently) high resolution opticstaking pictures of cells High Negative predictive value for urine cultures

Issues with urine analysers

Back to basics Appropriate specimen collection Early transport

Molecular methods

GeneXpert- not just for TB, MRSA, C. difficile, etc.

Issues with molecular methods

Cannot differentiate live from dead Negative results do not rule out infection

MALDI-TOF(Matrix assisted LASER DesorptionIonisation-Time of Flight Mass spectrometry)

Protein based spectral identification of bacteria Identifications available in literally minutes – not hours Tiny amount of bacterial growth needed – not affected by

media or incubation conditions Minimal cost per test (in cents not even dollars), virtually no

consumables Suppliers : Bruker, BioMerieux

Issues with MALDI-TOF

Pure culture In mixed cultures, will pick up the dominant

organism Antimicrobial susceptibility not possible Viruses not identified Identifies only organisms in database

Plate Streakers

Select appropriate media Loads the samples Spreading the inoculum to obtain isolated single colonies

following incubation Suppliers: WASP (Copan) Previ-Isola (BioMerieux) Innova

(BD) and Inoqula (KIESTRA) Not all systems include Gram stain preparation

DL-96II Microbial ID/AST System

Provide bacteria ID and AST tests results at thesame time with automated reading and analysisEffective identification of wide range coveringvast routine organisms (over 600 in total)including major clinical pathogenic bacteria andfungiAdvanced expert system presents testing resultswith high accuracy and instructive interpretation

Identification range: 5 categories of test cards covering Micrococcaceae, Streptococcus,Enterobacteriaceae, Non-fermentative bacteria, Yeast-like fungi

Test Principles: Identification: colorimetry / Susceptibility testing: turbidimetryThroughput: 60--120 sample per hourOptional Configuration : Automated sampling device, sampling 60-80 plates per hourPrinter: External printerInterface: 17 inch display , support LIS/HISEnvironmental temperature: 5℃--40℃Relative humidity: 10%--90%;Atmospheric pressure: 76kPa--106kPa;

DL-Bt64 Auto Blood Culture System

Auto blood culture system for both blood and sterile body fluidspecimensExpendable modular design to meet different size laboratory’scapacity needsHigh sensitivity & specificity: able to detect anaerobic/aerobic andfungi for both adult and child, characterized for its excellentrecovery of fungiFAN bottle (Fastidious Antibiotic Neutralization ) with unique resindesign enables absorbing most of antibiotics, consequently risesup the sensitivity of detection rate.

Test Principles: Colorimetry & CO2 sensor.Sample Type: Blood and various sterile body fluid specimens.Capacity: 64 bottles / 112bottles / 224 bottlesDetection Method: Adopt continuous swinging vibration mode, non-stopmonitoring. The system automatically reads the color data of the sensor at thebottom of each bottle every 10 minutes by each independent detectorReal-time detection and warning, with audio, visual and software alarm ofpositive samples or negative samples.Interpretation Method: Analyze sustained acceleration, Speed, Initialthreshold.Sample Loading:

Able to accept samples in random position and at any time in any rack.Able to resume the procedure if a sample is picked up and reinserted within 2

hours.Incubation detection period: to be set by the operator, and at most 7 days.

System hardware: Main instrument with 17' internal touch-pad, barcodescanner.

DL-Bt 112 Auto Blood CultureSystem

Auto blood culture system for both blood and sterile body fluidspecimensExpendable modular design to meet different size laboratory’scapacity needsHigh sensitivity & specificity: able to detect anaerobic/aerobicand fungi for both adult and child, characterized for its excellentrecovery of fungiFAN bottle (Fastidious Antibiotic Neutralization ) with uniqueresin design enables absorbing most of antibiotics,consequently rises up the sensitivity of detection rate

Test Principles: Colorimetry & CO2 sensor.Sample Type: Blood and various sterile body fluid specimens.Capacity: 64 bottles / 112bottles / 224 bottlesDetection Method: Adopt continuous swinging vibration mode, non-stopmonitoring. The system automatically reads the color data of the sensor at thebottom of each bottle every 10 minutes by each independent detectorReal-time detection and warning, with audio, visual and software alarm ofpositive samples or negative samples.Interpretation Method: Analyze sustained acceleration, Speed, Initialthreshold.Sample Loading:

Able to accept samples in random position and at any time in any rack.Able to resume the procedure if a sample is picked up and reinserted within 2

hours.Incubation detection period: to be set by the operator, and at most 7 days.

System hardware: Main instrument with 17' internal touch-pad, barcodescanner

Way ahead………………..

Total lab automation???????

Obstacles to automation in Microbiology

Microbiology is too complex to automate: blood,sterile body fluids, urine, catheter tips, tissues,prosthetic devices, lower respiratory tract specimens Variations in the processing of specimens: tissue

digestion, urine colony count, impression smearspreparation…….……….BUT THE SIMPLERMETHODS CAN BE AUTOMATED Machines are programmable but humans are flexible Costs of automation Microbiology labs are small for automation..........Not

all!!!

CONCLUSION-TWO QUOTES

“The first rule of any technology used in a business is thatautomation applied to an efficient operation will magnifythe efficiency. The second is that automation applied to aninefficient operation will magnify the inefficiency.”

-Bill Gates

CONCLUSION-TWO QUOTES

THANK YOU