51
Multiplex PCR: Multiplex PCR: A Game changer A Game changer in in infectious disease diagnostics infectious disease diagnostics

Multipex for viral and atypical pneumonia

Embed Size (px)

DESCRIPTION

Diagnosis of pneumonia can be challeging, especially if pathogens other than Streptococcus pneumoniae are involved Multiplex PCR with results available within the same day can investigate the presence or absence of 16 viruses and 5 bacteria, enablng the physician to make informed decisions about treatment, prognosis and public health and infection control measures.

Citation preview

Page 1: Multipex for viral and atypical pneumonia

Multiplex PCR:Multiplex PCR:A Game changer A Game changer

in in infectious disease diagnosticsinfectious disease diagnostics

Page 2: Multipex for viral and atypical pneumonia

Diagnosis Diagnosis of of

atypical pneumoniaatypical pneumonia

Page 3: Multipex for viral and atypical pneumonia

Pneumonia

• Commonest infection• Important cause of morbidity & mortality

• Bacterial pathogens: 85%– Streptococcus pneumoniae– Haemophilus influenzae– Morexella catarrahis

Page 4: Multipex for viral and atypical pneumonia

Streptococcus pneumoniae

Page 5: Multipex for viral and atypical pneumonia

Acute lobar pneumonia

• Purulent sputum• Rales are heard over involved lobe• Patient is sick

• After Influenza : Staphylococcus aureus• Ch. Alcoholism : Kleb pneumoniae• Bronchiectasis : Pseud aeruginosa• Cystic fibrosis : Pseud aeruginosa

Page 6: Multipex for viral and atypical pneumonia

Atypical pneumonia

• Also known as Pneuminitides

• Diagnosed when:

– Bacterial pathogen not identified on smear examination

– Pneumonia did no respond to β lactam drugs

Page 7: Multipex for viral and atypical pneumonia

Atypical Pneumonia

• 1940s Eaton Agent : Mycoplasma pneumoniae• 1970s Legionnaire’s : Legionella pneumophila• 1980s TWAR : Chlamydia pneumoniae• 1990s AIDS infection: Pneumocystis jerivoci• 2000s SARS• 2010s MERS

Page 8: Multipex for viral and atypical pneumonia

Clinical featuresWalking pneumonia

• Patient with a variety of pulmonary & extra pulmonary symptoms

• Mental confusion• Prominent headache• Myalgia• Ear pain• Diarrhoea• Rash• Cardiac involvement

Page 9: Multipex for viral and atypical pneumonia

Investigations• X Ray chest PA view• Sputum: Gram stain & culture• Blood culture• Serum transaminase• Serum phosphorus levels• Urinanalysis• Ferritin level• Creatinine phosphokinase (CPK)• CRP

Page 10: Multipex for viral and atypical pneumonia

Atypical pneumonia

Zoonotic Non Zoonotic

-Psittacosis - Mycoplasma-Q fever - Legionella-Tularemia - Chlamydia

Page 11: Multipex for viral and atypical pneumonia

Atypical pneumonia• Clinical manifestations are

– fever, dyspnea & cough– unilateral patchy segmental infiltrations

• No pathogenic organism isolated• But knowing etiological agent determines

– Potential prognosis– Optimal treatment– Public health precautions

Page 12: Multipex for viral and atypical pneumonia

Mycoplasma pneumoniae(Eaton agent)

• A common cause of CAP• A disease of gradual & incidious onset• Characterized by prolonged paroxysmal cough

and prolonged resolution of symptoms in an otherwise healthy patient, usually below 40 yrs, most common between 5 and 20 yrs

• Large outbreaks in late summer & fall• Incubation period : 3 weeks (smoldering)• Only 5 to 10% of infected develop pneumonia

Page 13: Multipex for viral and atypical pneumonia

Mycoplasma pneumoniae

• c/f include fever, malaise, persistent slowly worsening cough, headache, chills, sore throat, pleuratic chest pain

• Mycoplasma pneumoniae is also implicated in– Acute hepatitis– Immune thrombocytopenic purpura– Severe autoimmune hemolytic anaemia– Stevens Johnson syndrome– Arthritis– Transverse myelitis

Page 14: Multipex for viral and atypical pneumonia

Chlamydia pneumoniae• Mild pneumonia & bronchitis in young adults• In USA 300,000 cases are diagnosed annually• 10% of all CAP cases amongst adults• More common in males (cigarette smoking)• Primary infection: 7 to 40 yrs• Reinfection pneumonia: in elderly• Incubation period is 3 to 4 wks• Bronchitis, persistent cough, maliase over weeks, fever not a

prominent feature• WBC not elevated, Alkaline phosphatase maybe

Page 15: Multipex for viral and atypical pneumonia

Legionella pneumophila

• False but enduring status as an exotic infection• Failure to diagnose is due to lack of clinical

awareness and absence of lab facilities• In USA 8000 to 1800 pts are admitted annually• Named in 1979after an epidemic at a Legionnaires

annual meet in Philadelphia in 1976• Two types of infections:

– Severe multisystem diseases including pneumonia– A self limiting flu like illness: Pontaic fever

Page 16: Multipex for viral and atypical pneumonia

Legionella pneumophila

• c/f: nothing pathgnomic• Fever, non productive cough, headache, myalgia,

rigor, dyspnoea, diarrhoea & delirium• X-ray: alveolar infiltration• Fram negative bacilli, grows between 25 & 42 C

with optimum temp as 35 C• Inside free living protozoa in acquatic

environment, mostly in slime• Sensitive to macrolites

Page 17: Multipex for viral and atypical pneumonia

Pertusis in TunisiaAsma Zourani et al Diag Micro & ID 2012; 72 (4): 303 - 317

• Between 2007 & 2011• 626 samples from 599 infants < 1 yr• 126 (21%) positive by PCR, 1 by culture• B. pertusis 82%• B. parapertusis 6% ; both in 8%• Reported throughout the year• With a peak in summer• Mothers seemed to be likely source of infection

Page 18: Multipex for viral and atypical pneumonia

Viral pneumonia

• Largest proportion of childhood pneumonia• Decreases in frequency in healthy young &

middle aged adults• Increases in frequency amongst the elderly• Second commonest cause of pneumonia (after

Strep pneumoniae) 13 to 50%

Page 19: Multipex for viral and atypical pneumonia

Viral pneumoniaimmuno competent child

• Influenza A & B• Respiratory syncytial virus• Adenovirus (especially in military recruits)• Parainfluenza virus• Other viruses implicated as when molecular

diagnostic facilities improve & are used

Page 20: Multipex for viral and atypical pneumonia

Pathophysiology of viral pneumonia

Some virus are • cytopathic & directly affect pneumocytes or bronchial cellsOthers, either cause inflammation due to over excuberant

immune response– Type 1 cytokines (CMI)– Type 2 cytokines (allergic response)

Children infected with RSV who develop acute bronchiolitis rather than URTI have impaired type 1 immunity or agumented type 2 immunity

Page 21: Multipex for viral and atypical pneumonia

NexGen MoDxsEnd point PCR:• 1st Generation : PCR • 2nd Generation: Isothermal PCR

» NASBA» SDA

• 3rd Generation: Real Time PCR (21st Century):• Next Generation Multiplex Real Time PCR

• DPO technology• TOCE technology

Page 22: Multipex for viral and atypical pneumonia

DPO TM

Dual Primer Oligonucleotide

TOCE TM

Tagging Oligonucleotide Cleavage & Extension

Page 23: Multipex for viral and atypical pneumonia

• How to increase specificity with out changing the basic thermodynamics and kinetics of PCR?• Increased primer length increases specificity• BUT increased primer length increases the Tm

Principle of DPO™

Page 24: Multipex for viral and atypical pneumonia

NexGen MoDx Solutions

Page 25: Multipex for viral and atypical pneumonia

DPO TM

Dual Primer Oligonucleotide

TOCE TM

Tagging Oligonucleotide Cleavage & Extension

Page 26: Multipex for viral and atypical pneumonia

Principles of TOCE™

Page 27: Multipex for viral and atypical pneumonia

NexGen MoDx Solutions

Page 28: Multipex for viral and atypical pneumonia

Key Features and Benefits of DPO™/TOCE™

Page 29: Multipex for viral and atypical pneumonia

Key Features and Benefits of DPO™/TOCE™

Page 30: Multipex for viral and atypical pneumonia

What is the Paradigm Shift ?

Page 31: Multipex for viral and atypical pneumonia

Current Offering• Respiratory:

– RV 16– RB 5

• Genital– STI 7– HPV 28

Page 32: Multipex for viral and atypical pneumonia
Page 33: Multipex for viral and atypical pneumonia

• Each swab contains– Specimen collection

swab with a tip flocked with soft nylon fibre

– Polypropylene screw cap tube with 2 ml of eNAT transport medium

– Each swab has a molded breakpoint in the shaft

Page 34: Multipex for viral and atypical pneumonia

Sample collection & Transportation

• All sample must be collected using eNAT Swabs (COPAN)• Three different swabs are available

– eNAT Regular applicator (606CS01 R)– eNAT L Shaped Applicator (606CS01 L)– eNAT Pernasal applicator (606CS01 P)

• eNAT medium stabilizes & preserves RNA/DNA for prolonged time periods

• eNAT medium contains detergent & protein denaturant, so not suitable for culture based tests

• Transport at 5 to 25 C (in cold)

Page 35: Multipex for viral and atypical pneumonia
Page 36: Multipex for viral and atypical pneumonia
Page 37: Multipex for viral and atypical pneumonia
Page 38: Multipex for viral and atypical pneumonia
Page 39: Multipex for viral and atypical pneumonia
Page 40: Multipex for viral and atypical pneumonia
Page 41: Multipex for viral and atypical pneumonia
Page 42: Multipex for viral and atypical pneumonia
Page 43: Multipex for viral and atypical pneumonia

RV 16 ReportNasopharyngeal Swab is positive for

Human Rinovirus, Adenovirus and Parainfluenza 1

Page 44: Multipex for viral and atypical pneumonia
Page 45: Multipex for viral and atypical pneumonia

RB 5 Results

• Sample is positive for Bordetella parapertusis

Page 46: Multipex for viral and atypical pneumonia
Page 47: Multipex for viral and atypical pneumonia
Page 48: Multipex for viral and atypical pneumonia
Page 49: Multipex for viral and atypical pneumonia
Page 50: Multipex for viral and atypical pneumonia

Current Offering• Respiratory:

– RV 16– RB 5

• Genital– STI 7– HPV 28

Page 51: Multipex for viral and atypical pneumonia