Asim Farooq Shizza Fatima

1. Molecular Diagnosis Of Infectious Diseases AsimFarooq ShizzaFatima

2. Contents • Introduction • Need, Advantages and Disadvantages • SARS and its Diagnosis • Pertussis and Viral Pneumonia and their Diagnosis

3. Infectious Disease • Clinically evident illness • Infection and presence of pathogenic agent in host organism • Sometimes contagious • Viruses, Bacteria, Fungi, Protozoa, Multicellular Parasites and Prions • Primary and opportunistic pathogens

4. Molecular Diagnosis • A technique of identifying organisms on the basis of their genetic makeup • Molecules specific to a particular organism • Molecular sizes, structure, mass • DNA, RNA and Proteins • Cellular and molecular interactions

5. Why Molecular Diagnosis • Need an accurate and timely diagnosis • Important for initiating the proper treatment • Important for preventing the spread of a contagious disease

6. Continued… • Nonculturable agents • Human papilloma virus • Hepatitis B virus • Fastidious, slow-growing agents • Mycobacterium tuberculosis • Legionella pneumophilia • Highly infectious agents that are dangerous to culture • Francisellatularensis • Brucella species • Coccidioidisimmitis

7. Continued… • In situ detection of infectious agents • Helicobacter pylori • Toxoplasma gondii • Agents present in low numbers • HIV at early stages • CMV in transplanted organs • Organisms present in small volume specimens • Intra-ocular fluid • Forensic samples

8. Continued… • Molecular epidemiology • To identify point sources for hospital and community-based outbreaks • To predict virulence • Culture confirmation

9. Molecular Techniques • Direct probe testing – better for identification than for detection because it is not as sensitive as amplification methods • Amplification methods – used to improve the sensitivity of the nucleic acid testing technique Target amplification Probe amplification Signal amplification Combinations of the above

10. Target Amplification • Target amplification requires that the DNA to be tested for be amplified, i.e., the number of copies of the DNA is increased.

11. Advantages • High sensitivity Can theoretically detect the presence of a single organism • High specificity Can detect specific genotypes Can determine drug resistance Can predict virulence • Speed Quicker than traditional culturing for certain organisms

12. Continued… • Simplicity Some assays are now automated

13. Disadvantages • Expensive • So specific that must have good clinical data to support infection by that organism before testing is initiated. • Will miss new organisms unless sequencing is done as we will be doing in the lab for our molecular unknowns • May be a problem with mixed cultures – would have to assay for all organisms causing the infection.

14. Severe Acute Respiratory Syndrome • SARS coronavirus • Outbreak in China and Hong Kong in 2002 • Flu, fever, myalgia, lethargy, cough, sore throat, shortness of breath • Positive-strand, enveloped RNA viruses • 13 known genes and 14 known proteins • Large pleomorphic spherical particles with bulbous surface projections that form a corona

15. Molecular Diagnosis • Viral selection • Viral loads maximum in lower tract specimens • Also found in gastrointestinal tract and feces • SARS-CoV RNA has been detected in blood, cerebrospinal fluid, urine, and tears • Viruses obtained from different sources are then subjected to RNA extraction and then amplification through PCR

16. RNA Extraction • Testing multiple specimens • Nucleocapsid transcripts • nuc and pol genes

17. Interpretation of Results • For a positive result, repeat it again or repeat it with a different genomic locus • False-positive specimens can occur with poorly designed primers • A negative result from an infected patient could be due to the presence of PCR inhibitors that co-purify with RNA, a poor quality specimen, or a specimen lacking virus • Negative PCR results for specimens from the upper respiratory tract could trigger sampling from the lower respiratory tract where the titers of virus are higher

19. Pertussis

21. Causative agent

22. Molecular Diagnosis • A study was carried out on 5 patients for molecular diagnosis of pertussis • Age of the patients ranged from 35 days to 3 months, and one patient was 13 years old. • The clinical histories of the five patients varied, but all had a cough and other respiratory symptoms.

23. Hematoxylin and eosin staining of lung tissues from the patients showed bronchopneumonia • Silver staining (Steiner's method) demonstrated coccobacilli in all patients, while Gram's staining showed gram-negative bacilli in only patients 2 and 4. • From the tissue specimen β-globin gene was amplified

24. Utilizing PCR technique • Each PCR mixture consisted of • A300 nM concentration of each primer • 10 μl of DNA extract • High-fidelity PCR master mix (containing 1.5 mM MgCl2 and a 0.2 mM concentration of each deoxynucleosidetriphosphate) • And an enzyme mixture of Taq and Tgo DNA polymerases in a 50-μl volume.

25. Steps of PCR

26. When DNAs extracted from clinical samples of lung tissue infected with • Bacillus anthracis • Group A Streptococcus • Group B Streptococcus • Haemophilusinfluenzae • Legionellapneumophila • Staphylococcus aureus • Streptococcus pneumoniae, or Yersiniapestis • And from liver tissue infected with spotted-fever-group Rickettsia

27. The IS481 PCR assay did not generate an amplicon.

28. The IS481PCR assay amplified a 181-bp fragment from lung or trachea specimens from the five patients, suggesting that either B. pertussis or B. holmesii was present.

29. Sequencing the 181-bp amplicons of the IS481 gene from the clinical isolates and the five patients.

30. Results of sequencing • At nucleotide 100 in all five clinical isolates of B. holmesii, a mixture of nucleotide bases C and A occurred, with C slightly more predominant than A. • In contrast, an A was always present at the same nucleotide position in all five patient and clinical isolates of B. pertussis. • An analysis of the sequence from the reverse strand confirmed that a mixture of nucleotide bases G and T occurred at the homologous position in all five B. holmesii isolates, while only a T occurred in theB. pertussis isolates and the five patient isolates.

31. The patient history and clinical information are beneficial in differentiating between B. holmesii and B. pertussis. Although B. holmesii is known to cause septicemia and, in some instances, respiratory illnesses in adolescents and adults, the respiratory illness caused by B. holmesii is mild compared with that caused by B. pertussis, and no deaths have been attributed to B. holmesii.

32. To quantify the amounts of DNA extracted from human tissues, a real-time assay to detect an 80-bp region of the human RNase P gene was performed

33. Results of PCR • The specific real-time pertussis toxin assay, which targets a single-copy gene, demonstrated that all specimens from the five patients were positive for B. 

34. Results of diagnosis • Of the five patients diagnosed with B. pertussis infection in this study, the epidemiologic data support the PCR results for four who were in contact with ill family members • Patient 2 confirmed influenza case without an epidemiologic link but was determined to be infected with B. pertussisby PCR tests 

35. Patient 4 presented as a presumed SIDS-related fatality; however, conventional and real-time PCR tests, an immunohistochemicalassay, and an epidemiologic link to a known B. pertussis case established that the infant was infected with B. pertussis.

36. Viral Influenza

37. Definition Influenza is a febrile illness characterized by fever; cough; upper respiratory symptoms including sore throat, rhinorrhea, and nasal congestion; and systemic symptoms including headache, myalgia, and malaise that results in a significant number of hospitalizations in all age groups

39. Antiviral agents • Specific antiviral agents such as M2 channel inhibitors (amantidine and rimantidine) or • NA inhibitors (oseltamivir and zanamavir) can be prescribed; however, these drugs are effective only when given within the first 24 h following infection.

40. Diagnostic approaches • Serological tests such as the HAI test have been used to detect seroconversion of influenza virus • Nasopharyngeal swabs and NPA are the preferred specimens for influenza virus detection

41. Isolation of influenza virus was historically performed in embryonated hen eggs or tube cultures of primary monkey kidney, Madin-Darby canine kidney (MDCK), or A549 cells. CPE consistent with influenza virus can be visualized by light microscopy

42. Molecular diagnosis • Molecular tests for influenza virus detection include • Reverse transcriptase PCR (RT-PCR) • NASBA • LAMP

43. RT-PCR • In the case of RT-PCR, nucleic acid is reverse transcribed into cDNA using virus-specific oligonucleotide primers • Several different gene targets have been used for amplification including the matrix, HA, and NS protein genes

44. NASBA(Nucleic acid sequence based amplification) • A primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature.

45. Working • RNA template is given to the reaction mixture, the first primer attaches to its complementary site at the 3' end of the template • Reverse transcriptase synthesizes the opposite, complementary DNA strand • RNAse H destroys the RNA template (RNAse H only destroys RNA in RNA-DNA hybrids, but not single-stranded RNA) • the second primer attaches to the 5' end of the DNA strand • T7 RNA polymerase produces a complementary RNA strand which can be used again in step 1, so this reaction is cyclic.

47. LAMP(Loop mediated isothermal amplification) • LAMP is a novel approach to nucleic acid amplification which uses a single temperature incubation thereby obviating the need for expensive thermal cyclers.

48. Uses