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Technologies for Rapid Diagnosis of UTIsg

Mohammad Reza Bakhtiari, DCLS, PhD

Drawbacks ofConventional Urine Culture (CUC)

• Cost• Labor‐Intensive: extensive amount of laboratory work

• Slow: time lapse between specimen collection and the detection of bacterial growthbacterial growth

• Delayed antibiotic susceptibility reports• Special Training Required• Many samples from suspected UTI patients ultimately test negative for bacteria

Automated Plate Streaking Automated Gram Staining

PREVI™ Isola system automates and standardizes the tedious task of inoculating 

plated media.

Automated Identification and Antibiotic Susceptibility Testing(ID/AST)

MicroScan WalkAway plus System

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Urine Technologies

• Urine Macroscopy• Urine Sediment Image Analysis

• Urine Flow Cytometry

The role of Urine Macroscopy?

• In women with classic UTI symptoms, particularly in the absence of vaginal symptoms, the probability of a UTI is so high that empiric treatment may be considered without dipstick testing. 

• In a patient with nonspecific complaints such as dysuria and vaginal 

discharge, a dipstick might be useful.

• In low‐risk patients with a low pretest probability of UTI, the dipstick d t l l t i f ti h b th l k t t d it itadequately rules out infection when both leukocyte esterase and nitrites 

are negative.

• Reduction of costs.• Reduction of Lab work load.

Urine Dipstick for Diagnosing Urinary Tract InfectionAm Fam Physician. 2006 Jan 1;73(1):129-132.

Validation of a method for the rapid diagnosis of urinary tract infection suitable for use in general practice.Br J Gen Pract. 1990 October; 40(339): 403–405.

The urine dipstick test useful to rule out infections. A meta-analysis of the accuracy.BMC Urol. 2004 Jun 2;4:4.

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Causes of false positive and false negative results in urine dipstick testing

Pediatr Clin N Am 53 (2006) 325– 337

Fluorescence Urine Analyzers

iQ®200 Series UrineUF‐1000i

Main result screen of the UF‐100

40 WBCs/μL

125 bacteria/μL

/μ

Diagnostic Microbiology and Infectious Disease 65 (2009) 103–107

Cutoff values for bacteria and leukocytes for urine flow cytometer SysmexUF‐1000i in urinary tract infections

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Automated Urine Screening Systems

Manual Urine Screening Systems

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www.cmft.nhs.uk/media/438403/urine_microbiology_com%20_2_.pdf

New Technologies for Identificationof Uropathogens

• Pyro Sequencing• Capillary Electrophoresis• Micro Calorimetry• GC‐MS• MALDI‐TOF• Probe Hybridization

– FISH– Biosensors/Biochips

Proteomics Methodology

Sample

Homogenization

LC for Protein Concentration

2‐D Gel Electrophoresis

Protein Separation

p

Spot Visualization

In Gel Tryptic Digestion

Mass Spectrometry

Protein Database Search

Protein‐Protein Interaction Analysis

Protein Identification

Bioinformatics

Functional Proteomics

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2‐D Gel Electrophoresis

Isoelectric Focusing + SDS Page (ISO-DALT)

Mass Spectrometry

Mass Ionizers

MALDI: Matrix Assisted Laser Desorption Ionization

ESI: Electrospray Ionization

Mass Analyzers

TOF: Time OF Flight

Quadropole MS

Ion trap/Fourier Transform Ion Cyclotron MS

MALDI/TOF

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Probe Hybridization

1. ssDNA probe is used to find DNA or RNA with a homologous sequence.

2. This will tell us if a specific sequence is present.

3. The substrate with the genetic material in question is fixed on a membrane

Dot Blot / Reverse Dot BlotDot Blot / Reverse Dot Blot

in question is fixed on a membrane.

4. The membrane is blocked with non-specific proteins.

5. Small drops of a known sequence of DNA with a radioactive or fluorescent marker is added.

6. The plate is rinsed and read in the proper instrument.

DNA Arrays

Technological advances in biotechnology, electronics, and robotics have made it possible to miniaturize the probe hybridization and detection methods. Instead of t ddi t tistudding one gene at a time.

DNA chips allow thousands or tens of thousands of specific DNA or RNA sequences to be detected simultaneously on a small glass or silica slide only 1-2 cm square.

Types of DNA Arrays

MacroarraysMacroarrays

Membrane blots Membrane blots

11--10 10 K GenesK Genes

MicroarraysMicroarrays

Glass or polypropyleneGlass or polypropylene

1010KK--100100K genesK genes

HighHigh--density density OligonucleotideOligonucleotide Arrays Arrays

Silicon Silicon

potentially up to potentially up to 4 4 million genesmillion genes

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Trends in Pharmacological Sciences June 2011, Vol. 32, No. 6

Thank you for your Attention

Mohammad Reza Bakhtiari, CLD, PhDbakhtiari@irost.ir