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.

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

33. • 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

34. 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)

43. RV 16 Report
Nasopharyngeal Swab is positive for
Human Rinovirus, Adenovirus and Parainfluenza 1

45. RB 5 Results
• Sample is positive for Bordetella parapertusis

50. Current Offering
• Respiratory:
– RV 16
– RB 5
• Genital
– STI 7
– HPV 28