Multipex for viral and atypical pneumonia
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Multipex for viral and atypical pneumonia

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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......

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.

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  • 1. Multiplex PCR:Multiplex PCR: A Game changerA Game changer inin infectious disease diagnosticsinfectious disease diagnostics
  • 2. DiagnosisDiagnosis ofof atypical pneumoniaatypical pneumonia
  • 3. Pneumonia • Commonest infection • Important cause of morbidity & mortality • Bacterial pathogens: 85% – Streptococcus pneumoniae – Haemophilus influenzae – Morexella catarrahis
  • 4. Streptococcus pneumoniae
  • 5. 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
  • 6. Atypical pneumonia • Also known as Pneuminitides • Diagnosed when: – Bacterial pathogen not identified on smear examination – Pneumonia did no respond to β lactam drugs
  • 7. 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
  • 8. Clinical features Walking pneumonia • Patient with a variety of pulmonary & extra pulmonary symptoms • Mental confusion • Prominent headache • Myalgia • Ear pain • Diarrhoea • Rash • Cardiac involvement
  • 9. Investigations • X Ray chest PA view • Sputum: Gram stain & culture • Blood culture • Serum transaminase • Serum phosphorus levels • Urinanalysis • Ferritin level • Creatinine phosphokinase (CPK) • CRP
  • 10. Atypical pneumonia Zoonotic Non Zoonotic -Psittacosis - Mycoplasma -Q fever - Legionella -Tularemia - Chlamydia
  • 11. 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
  • 12. 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
  • 13. 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
  • 14. 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
  • 15. 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
  • 16. 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
  • 17. Pertusis in Tunisia Asma 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
  • 18. 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%
  • 19. Viral pneumonia immuno 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
  • 20. Pathophysiology of viral pneumonia Some virus are • cytopathic & directly affect pneumocytes or bronchial cells Others, 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
  • 21. NexGen MoDxs End 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
  • 22. DPO TM Dual Primer Oligonucleotide TOCE TM Tagging Oligonucleotide Cleavage & Extension
  • 23. • 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™
  • 24. NexGen MoDx Solutions
  • 25. DPO TM Dual Primer Oligonucleotide TOCE TM Tagging Oligonucleotide Cleavage & Extension
  • 26. Principles of TOCE™
  • 27. NexGen MoDx Solutions
  • 28. Key Features and Benefits of DPO™/TOCE™
  • 29. Key Features and Benefits of DPO™/TOCE™
  • 30. What is the Paradigm Shift ?
  • 31. Current Offering • Respiratory: – RV 16 – RB 5 • Genital – STI 7 – HPV 28
  • 32. • 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
  • 33. 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)
  • 34. RV 16 Report Nasopharyngeal Swab is positive for Human Rinovirus, Adenovirus and Parainfluenza 1
  • 35. RB 5 Results • Sample is positive for Bordetella parapertusis
  • 36. Current Offering • Respiratory: – RV 16 – RB 5 • Genital – STI 7 – HPV 28