1) Real time PCR provides a rapid, sensitive and specific method for diagnosis of infectious diseases compared to conventional methods. It can detect pathogens within 1 hour compared to days or months for cultures. 2) The document discusses various infectious diseases and the recommended tests for their diagnosis. Real time PCR is highlighted as the most effective method for diagnosis of diseases like tuberculosis, hepatitis B, hepatitis C and human papillomavirus. 3) Point-of-care testing using technologies like TrueNat provide portable, easy-to-use and low-cost real time PCR diagnostics that can be used even in remote locations.

1. IMPORTANCE OF MOLECULAR
METHODS IN DIAGNOSIS OF
INFECTIOUS DISEASES – A Special
emphasis on Real Time PCR
Maj (Dr) Shilpi Gupta
Graded Specialist (Microbiology)

2. TOPIC FOR DISCUSSION
Introduction
Conventional Vs Molecular methods
Conventional PCR Vs Real Time PCR
Special Cases

3. INFECTIOUS DISEASES (LIST)
Bacterial
• Typhoid
• Cholera
• Tuberculosis
• Leprosy
• Tetanus
• Diphtheria
• Gonococcal
• Chlamydia
• Syphilis
• Leptospirosis
• Scrub typhus
• UTI
• Meningitis
• Bacterial dysentery
Viral
• Hepatitis A,B,C,D,E
• HIV
• Chicken pox
• Dengue
• Chikungunya
• Japanese
encephalitis (JE)
• H1N1
• SARS
• Ebola
• Nipah
• HPV
• Measles
• Encephalitis
Fungal
• Candidiasis
• Cryptococcosis
• Aspergillosis
• Penicilliosis
Parasitic
• Malaria
• Filaria
• Amoebiasis
• Leishmaniasis
• Kala azar

4. What are the available
options for Laboratory
Diagnosis?

5. LAB DIAGNOSIS OF INFECTIOUS DISEASES
MICROSCOPY
Gram Stain
ZN Stain
Fluorescence
stain
Electron
Microscopy
CULTURE
TECHNIQUES
Solid
Liquid
SEROLOGICAL
METHODS
Agglutination
Precipitation
ELISA
NUCLEIC ACID
AMPLIFICATION TEST
LAMP
PCR
Real Time PCR
OTHER
Biochemical
tests
TST/ Mantoux
test
Adenosine
Deaminase
(ADA)

6. Advantages of Molecular over conventional
methods
Molecular Conventional
Rapid (1h – 1 day) Days to month
More sensitive Less sensitive
More specific Less specific
Less laborious Laborious, requires manpower
Less chances of contamination Chances of contamination more
Quantification possible Qualitative

7. Conventional PCR Set up
REAL TIME PCR

8. Conventional PCR & Real Time PCR
Conventional PCR Real Time PCR
Semi-automated Totally automated
5-6 hrs to complete one test 1hrs or <1 hr for one test
Less sensitive, specific and reproducible
than REAL TIME
Most specific, sensitive , reliable and
reproducible
Requires sophisticated infrastructure Lesser space

10. POINT OF CARE TESTING (POCT)
Point-of-care testing (POCT): is defined as medical testing
at or near the site of patient care
• Simple, Automated & Easy to
read
• Independent power source
• Cost effective • Long shelf life reagents
• Microfluidic (less sample) • Short sample processing time
• Quantitative • High sensitivity and specificity
• Portable or near patient
diagnosis
• No need of skilled trained lab
technician

11. POCT (list)
Generation Methods Examples
First generation Rapid Detection tests (RDTs) Dengue, Chikungunya,
Hepatitis A, B, C, E
Second generation Cartridge based NAAT (CB NAAT) GeneXpert (MTB, H1N1, HIV,
HBV, HCV)
Third generation Hand held portable chip based
Real Time NAAT
TrueNat (HBV, HCV, Dengue,
Chikungunya, Malaria, HPV)
Pai, N.P
., et al. 2012, PLOS Medicine
Pai, N.P and Pai, M. Discovery medicine 2012

12. EVOLUTION OF NAAT: From reference lab
to Point-of-care
NAAT
• RT PCR
CB-NAAT
• Gene Xpert
• Xpert MTB/RIF
POCT
• Real time micro
system

13. 13 Real Time PCR - Xpert MTB/RIF (Cepheid)

14. The Truelab™ Real Time micro PCR system

15. INDIGENOUS PRODUCT – TrueNAT (Molbio
Diagnostics)

16. ADVANTAGES OF TrueNAT over Xpert MTB/RIF
Xpert MTB/RIF TrueNAT
Method No step for removal of PCR
inhibitors
PCR inhibitors removed thus
increase positivity rate
Conditions for machine Non portable (bulky) system Potable
No battery backup Battery backup available
Strict temp conditions required Any temp
Senstivity & Specificity Slightly lesser Comparable or better

17. CASE SCENARIO
 27 y/o male, HIV-negative, presented to a hospital with 2 months of
cough
 CXR: “Diffuse micronodular pattern”
 Treated for Community Acquired Pneumonia and discharged
 Team did considered MTB
 Sputum AFB smear-negative
 Cultures pending
 No Sputum molecular method done
 Culture grew M. tuberculosis 1 month later
 Already hospitalized at another hospital in ICU
 Being treated for bacterial sepsis
 He not only tested positive for TB, but had also developed MDR-TB
 ATT is started, but patient passes away a few days later

18. Concerns in Diagnostics
 Medical diagnostics take time, are costly and often give
inconclusive results.
 Many doctors prefer to administer drugs on poor/average patients
on the basis of symptoms rather than recommending expensive
tests.
 By the time the disease is diagnosed, patients turn resistant to the
drugs or their disease worsens.

19. Real Time PCR is most
specific, sensitive, rapid, reliable,
reproducible, POCT and extremely
powerful method among all types of diagnostic
modalities

20. RECOMMENDED TESTS FOR PUL & EPTB
CONDITION FREQUENCY SPECIMEN RECOMMENDED TESTS REMARKS
Pulmonary TB 75% Sputum • Liquid culture (Gold
standard)
• Real Time PCR
• In MDR suspected
cases molecular
method should be 1st
choice
Tubercular
lymphadenitis
35-40% Lymph node
aspirate
• Liquid culture (~70%
positivity)
• Real Time PCR
Pleural TB 20% Pleural fluid • Biochemical analysis
• ADA is useful screening
• IGRA may be helpful
• Liquid culture (~80%
positivity)
• Real Time PCR has
low sensitivity
• Smear microscopy
lower sensitivity

21. RECOMMENDED TESTS FOR PUL & EPTB Contd..
CONDITION FREQUENCY SPECIMEN RECOMMENDED TESTS REMARKS
Genitourinary
TB
10-15% Urine • Culture (80% positivity)
• Real Time PCR (90%
positivity)
• Culture negative pyuria with
acidic urine
• 75% patients have abnormal
CXR
• 3 morning urine samples
collected (~100 ml)
Males Prostatic
secretion
• Culture (~40% positivity)
• Real Time PCR (~36%
positivity)
• Leucocyturia and hematuria
are seen in ~50%
Females Endometrial
biopsy tissue
Menstrual blood
Pelvic fluid
• Real Time PCR (~30%
positivity)
• Culture (~40% positivity)
• Patient present with infertility,
pelvic pain, menstrual
disorders
Potts disease 10% Pus drained
from abscess
Synovial fluid
Bone biopsy
• Culture
• Histology
• Radiological (CT MRI) reveals
characteristic lesions

22. RECOMMENDED TESTS FOR PUL & EPTB Contd..
CONDITION FREQUENCY SPECIMEN RECOMMENDED TESTS REMARKS
Tubercular
meningitis
5% CSF • Biochemical analysis
• Real Time PCR (~ 70%
positivity)
• Culture (~ 80 %
• Negative result does not
exclude diagnosis of
TBM
Tubercular
peritonitis
3.5% Ascitic fluid • Biochemical analysis
• Culture (low sensitivity)
• Peritoneal biopsy (by
laparoscopy) is often
needed to establish the
diagnosis
Tubercular
pericarditis
Very low Pericardial
fluid
• Biochemical analysis
• Culture (~ 70% positivity)
• ADA
• IFN Y assay
• Disease of elderly with
low TB prevalence
• Seen in HIV +ve

23. HEPATITIS B INFECTION
• Acute: Initial infection with the hepatitis B virus
Mild or Severe
• Chronic: Hepatitis B virus remains in the blood for more
than 6 months or for years CARRIERS
Recovery or Chronic hepatitis
(9 of 10) (1 of 10)
Why Chronic Hepatitis B is dangerous?
Chronic
Hepatitis B
Liver
cirrhosis
Liver
failure
Liver
cancer

24. HEPATITIS B

25. Algorithm for diagnosis of Hep B in Jaundice
patients
HBV
HBsAg
Reactive Non-reactive
IgM Anti HBc
Reactive
If HbsAg is Reactive and lgM anti HBc is Nonreactive: HBV positive
If lgM Anti HBc is Reactive and HBsAg is Nonreactive: HBV positive
If both Reactive: HBV positive
If both Non-reactive: HBV negative
Non-reactive
National Laboratory Guidelines for Testing of Viral Hepatitis,2018 MOHFW

26. Algorithm for diagnosis of Hep B in patients without
Jaundice
HBV
HBsAg
Reactive
Report: HBV
Positive
Non-
Report: HBV
Negative
National Laboratory Guidelines for Testing of Viral Hepatitis,2018 MOHFW

27. LAB DIAGNOSIS

28. Role of HBV DNA
 Most sensitive index of viral replication
 Better marker of level of viraemia as compared to HBeAg
 HBV detection based on Real Time PCR approach can detect
as few as 10² – 10³ genome copies.
 Useful in following the course of HBV replication in patients
with chronic hep B receiving antiviral chemotherapy
 Patients with HBV pre-core mutant are HBeAg negative and
HBV DNA positive

29. OCCULT HEPATITIS B INFECTION
 Occult Hepatitis B: Infection with detectable HBV DNA and
undetectable surface antigen (HBsAg) in blood (very low levels
(invariably <200 IU/mL).
 Most are also anti-HBc positive.
 HBV DNA amplification by Real Time PCR is a gold standard assay for
detection & quantification of Occult HBV infection.
2011, World J Gastroenterology

31. HEPATITIS C INFECTION
Fibrosis1
Chronic HCV
infection can lead to
the development of
fibrous scar tissue
within the liver
Fibrosis Cirrhosis Hepatocellular Carcinoma
(with cirrhosis)
Cirrhosis1,2
Over time, fibrosis can progress,
causing severe scarring of the
liver, restricted blood flow,
impaired liver function, and
eventually liver failure
HCC3
Cancer of the liver can
develop after years of
chronic HCV infection

32. Total
No.
Infected
(millions)
Diagnosed
Undiagnosed
2.7 to 5 Million1
75% Unaware of Infection
1.1 Million1
21% Unaware of Infection
~800,000 to 1.4 Million1
65% Unaware of Infection
HIV HBV HCV
4
3
2
1
0
Prevalence of Chronic Viral Infections
HCV is Nearly 4 Times as Prevalent as HIV and HBV
• A 2011 study estimated that as many as 5.2 million persons are
living with HCV in the United States2
HBV=hepatitis B virus; HCV=hepatitis C virus; HIV=human immunodeficiency virus.
1. Institute of Medicine. Washington, DC: The National Academies Press; 2010.
2. Chak E, et al. Liver Int. 2011;31(8):1090-1101.
3. Gish Hepatology 2015

33. Algorithm for diagnosis of Hep C in patients with or
without Jaundice
HCV
Anti HCV
Reactive
Report: *HCV Ab Positive
Non-reactive
Report: HCV Ab Negative
*Should be followed by testing for HCV RNA using NAT (RT-PCR) for confirmation
If HCV RNA is detected: current/active HCV infection
If HCV RNA is not detected: Past, resolved infection or false HCV antibody positivity

34. Method Screen Confirm
Duration
of therapy
Assessing
treatment
response
HCV antibody test
(ELISA)
X
PCR: HCV genotype X
Real Time PCR:
HCV RNA
X X
Australian Government National Hepatitis C Testing Policy 2007. Available at: http://www.health.gov.au

35. Mosquito borne illness

36. What causes Malaria?
 Malaria is caused by Plasmodium of which 4 species infect humans.
-P. vivax
-P. ovale
-P. malariae
-P. falciparum :causes the most severe form of malaria
Cerebral malaria
Acute renal failure
Acute respiratory distress syndrome (ARDS)
Severe anaemia (Hb < 5g%)
Hemoglobinuria
Hypotension, Shock
Hyperpyrexia
Hemolysis

37. DIAGNOSIS
•Fever, Chills,
Headache,
Malaise, Vomiting,
Diarrhea, jaundice,
Body & Joint Pain
CLINICAL
• Technical and expert skill
required
• Early infection parasite
not detected
• Qualitative
MICROSCOPY
• False negative results
• overall sensitivity of <
81%.
• Cross reactivity with
rheumatoid factor
RDT RT PCR
• Most specific,
sensitive , reliable
and reproducible
• Exact quantification
of DNA in sample

39. DENGUE FEVER AND CHIKUNGUNYA

40. LAB DIAGNOSIS
Method Detection Test
Antigen
detection
Detect the NS1 antigen RDT, ELISA, Real Time PCR
Antibody
detection
Detect IgM and IgG
antibodies
RDT, ELISA
Viral isolation Grows virus Viral culture

43. LAB DIAGNOSIS
 Virus isolation
 Must only be carried in Level-3 laboratories
 Results can take between 1-2 weeks.
 Real Time PCR
 Rapid
 Reliable
 Specific

44. Human Papilloma Virus (HPV)
 More than 100 types of HPV, of which atleast 14 are cancer
causing.
 Two HPV types (16 and 18) cause 70% of cervical cancers
and pre-cancerous cervical lesions.
 Cervical cancer is the second most common cancer in
women in developing countries.

45. PREVENTION
PRIMARY SECONDARY TERTIARY
Girls 9-14 yrs Women 30 years old or older All women as needed
HPV vaccination Screen and treat Treatment of invasive cancer
at any age
Health and sex education 1. HPV testing for high risk
HPV types – POCT
2. Visual inspection with
Acetic acid (VIA)
3. PAP test and liquid based
cytology (LBC)
Surgery
Radiotherapy
Chemotherapy
Palliative
Condom promotion Followed by treatment
Male circumcision

46. abnormal cells in micropscope
CxCa Prevention (screening)
When positive triage, diagnosis, therapy
1) PAP smear
2) HPV Test
High-risk HPV16,18,31,33,…
Low-risk HPV6, 11, 42,…..

47. Take Home Message
 Conventional Microbiological diagnostic methods are time taking
and inconclusive at times
 Advance Molecular methods are rapid, specific and most reliable in
current scenario
 To date Real Time PCR is the only available method for diagnosis in
certain conditions

48. THANK YOU