Asim Farooq Shizza Fatima

Molecular Diagnosis Of Infectious Diseases

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

Diagnosis

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

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

Why Molecular Diagnosis Need an accurate and timely diagnosisImportant for initiating the proper treatment

Important for preventing the spread of a contagious disease

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 Francisella tularensis

Brucella species Coccidioidis immitis

Continued… In situ detection of infectious agents

Helicobacter pylori Toxoplasma gondii

Agents present in low numbers HIV at early stages

CMV in transplanted organsOrganisms present in small volume specimens

Intra-ocular fluid Forensic samples

Continued…

Molecular epidemiologyTo identify point sources for hospital and

community-based outbreaksTo predict virulence

Culture confirmation

Molecular TechniquesDirect 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 amplificationProbe amplificationSignal amplification

Combinations of the above

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.

AdvantagesHigh sensitivity

Can theoretically detect the presence of a single organism

High specificityCan detect specific genotypesCan determine drug resistance

Can predict virulenceSpeed

Quicker than traditional culturing for certain organisms

Continued…

SimplicitySome assays are now automated

DisadvantagesExpensiveSo 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.

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

Molecular Diagnosis Viral selectionViral loads maximum in lower tract specimensAlso found in gastrointestinal tract and fecesSARS-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

RNA ExtractionTesting multiple specimens Nucleocapsid transcripts nuc and pol genes

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

Pertussis

An acute, highly contagious respiratory disease characterized by paroxysmal coughing that ends in a

loud whooping inspiration.

Definition

Bordetella pertussis, is a small, nonmotile gram-negative coccobacillus

nonmotile

gram-negative coccobacillus

Causative agent

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.

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

Utilizing PCR technique

Each PCR mixture consisted of A 300 nM concentration of each primer10 μl of DNA extract High-fidelity PCR master mix (containing 1.5

mM MgCl2 and a 0.2 mM concentration of each deoxynucleoside triphosphate)

And an enzyme mixture of Taq and Tgo DNA polymerases in a 50-μl volume.

DENATURATION

• THE DNA WAS DENATURED FOR 3 MIN AT 94°C

ANNEALING

• THEN SUBJECTED TO 40 CYCLES OF AMPLIFICATION (94°C FOR 15 S, 60°C FOR 20 S, AND 72°C FOR 30 S)

EXTENSION

• FOLLOWED BY A FINAL EXTENSION OF 10 MIN AT 72°C

Steps of PCR

When DNAs extracted from clinical samples of lung tissue infected with

Bacillus anthracis Group A Streptococcus Group B Streptococcus Haemophilus influenzae  Legionella pneumophila  Staphylococcus aureus Streptococcus pneumoniae, or Yersinia pestis  And from liver tissue infected with spotted-fever-

group Rickettsia

The IS481 PCR assay did not generate an amplicon.

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.

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

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.

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.

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

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. 

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. pertussis by PCR tests 

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

Viral Influenza

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

Definition

Influenza virus is a negative-sense single-

stranded RNA virus

Belonging to the family Orthomyxoviridae

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.

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

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

Molecular diagnosis

Molecular tests for influenza virus detection include

Reverse transcriptase PCR (RT-PCR) NASBA LAMP

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

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.

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.

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.

Uses

Particularly useful method for infectious disease diagnosis in low and middle income countries

In LAMP, the target sequence is amplified at a constant temperature of 60 - 65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity

Typically, 4 different primers are used to identify 6 distinct regions on the target gene, which adds highly to the specificity