Importance of real time pcr in diagnosis of infectious diseases
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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.
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Importance of real time pcr in diagnosis of infectious diseases
- 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
- 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
Editor's Notes
- Slide 23. The role of testing in diagnosis and treatment The HCV antibody test (Enzyme Immune Assay; EIA), which is used to detect the presence of anti-HCV, is the usual initial test for patients with clinical liver disease and is also acceptable for screening at-risk patients. To confirm the diagnosis, testing for serum HCV ribonucleic acid (RNA) by sensitive polymerase chain reaction (PCR) amplification is recommended. Prior to initiating treatment in patients infected with chronic hepatitis C, it is important to determine the patient’s baseline viral load with a quantitative assay as well as their HCV genotype. HCV genotype determines the length of therapy. While on treatment, patients’ progress can be monitored by determining the presence of HCV RNA, with a quantitative PCR assay. At the end of treatment and at follow-up a qualitative test will confirm the absence of HCV replication.
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