Clinical applications of Molecular

diagnostics
Diagnosis and monitoring of infectious diseases
Diagnosis and monitoring of HIV infection

I. Gobe
MLS210 -UB

I. Gobe; Univ Botswana

 INDICATIONS FOR MOLECULAR
TESTING
• Diagnosis
• Monitoring of response of viral infection to
therapy eg HIV viral load
• Providing a prognosis: mutation/genotype
profile predict the response pattern of a
particular strain to available drugs
• Tracing an outbreak of infection to its source

I. Gobe; Univ Botswana

 When are Molecular methods
necessary?
• 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

I. Gobe; Univ Botswana

 When are Molecular methods
necessary
• In situ detection of infectious agent
– Helicobacter pylori
– Toxoplasma gondii
• Agents present in low numbers
– HIV in antibody negative patients
– CMV in transplanted organs
• Organisms present in small volume specimens
– Intra-ocular fluid
– Forensic samples

I. Gobe; Univ Botswana

Fluorescence in situ hybridization
(FISH) : FISH

I. Gobe; Univ Botswana

 When are Molecular methods
necessary
• Differentiation of antigenically similar agents
– May be important for detecting specific virus genotypes
associated with human cancers (Papilloma viruses)
• Antiviral drug susceptibility testing
– May be important in helping to decide anti-viral therapy to
use in HIV infections
• Non-viable organisms
– Organisms tied up in immune complexes

I. Gobe; Univ Botswana

 What are the advantages of using a molecular
test?
• 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/rapidity
– Quicker than traditional culturing for certain organisms

I. Gobe; Univ Botswana

 What are the advantages of using a molecular
test?
• Simplicity and high throughput-Some assays
are now automated
• Applicability to archival material; do not
require viability of the pathogen for detection
and identification
• Drug targeting of specific mutations/proteins
• Better understanding of resistance
mechanisms

I. Gobe; Univ Botswana

What are the disadvantages of using a
molecular test?
• Expensive
• Will miss new organisms
• May be a problem with mixed cultures – would have
to assay for all organisms causing the infection.
• Too sensitive? Are the results clinically relevant?
• Inability to distinguish live from dead pathogens
• Requirement of technical skills

I. Gobe; Univ Botswana

 What are the different types of nucleic acid
molecular techniques that are used?
• 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

I. Gobe; Univ Botswana

Pathogens for which molecular testing
is part of standard care
• Human immunodeficiency virus Type 1
• Chlamydia trachomatis
• Neisseria gonorrhoeae
• Human papillomavirus
• Herpes simplex virus
• Mycobacteria tuberculosis
• Enteroviruses
• Cytomegalovirus
• Methicillin-resistant Staphylococcus aureus
• Hepatitis C virus

I. Gobe; Univ Botswana

 Chlamydia trachomatis and Neisseria
gonorrhoeae
• most common sexually transmitted bacterial
diseases
• worldwide estimates of approximately 50
million new cases annually

I. Gobe; Univ Botswana

 Chlamydia trachomatis
• A gram-negative, obligate intracellular bacteria.
• They form characteristic intracellular inclusions
which can be observed in cell culture by light
microscopy after special staining is applied
• Chlamydia trachomatis causes cervicitis,
salpingitis, proctitis and endometritis in women
and urethritis, epididymitis and proctitis in men.

I. Gobe; Univ Botswana

 Chlamydia trachomatis
• 70 - 80% of women and up to 50% of men
who are infected experience no symptoms.
• Many chlamydial infections in women remain
untreated which may result in low-grade
inflammation in the Fallopian tubes, a leading
contributor to infertility.
• transmitted in the birth canal, potentially
resulting in infant conjunctivitis and/or
chlamydial pneumonia in newborns

I. Gobe; Univ Botswana

 Neisseria gonorrhoeae
• gram-negative, oxidase positive diplococci
• can be observed in Gram-stained smears of
urethral discharges, usually within
neutrophils.
• Culture of N. gonorrhoeae can be difficult
because the organism does not survive long
outside its host and is highly susceptible to
adverse environmental conditions such as
drying and extreme temperatures

I. Gobe; Univ Botswana

 Neisseria gonorrhoeae
• causes acute urethritis in males, which if
untreated can develop into epididymitis,
prostatitis, and urethral stricture.
• In females, the primary site of infection is the
endocervix.
• An important complication in females is
development of pelvic inflammatory disease
which contributes to infertility
• Asymptomatic infections occur often in females
but infrequently in males.
I. Gobe; Univ Botswana
 Traditionl Detection Techniques For (CT) & (GC)

1. Cell Culture
2. Enzyme immunoassay(EI)
3. Direct antibody testing
• Replaced with amplified nucleic acid tests which offers
greater sensitivity for detecting Ct from genital
specimen
• Nucleic acid testing does not offer sig improvement in
sensitivity when culture is performed under ideal
conditions. However GC is a fastidious organism which
can lead to a decreased sensitivity of culture
particularly when specimen transport is required
before culturing

I. Gobe; Univ Botswana

 Advantages of nucleic acid
amplification testing (NAAT) for GC/CT
• Single specimen and single reaction can be
used
• Dna and Rna of GC and CT are more stable;
avoids the necessity of immediate transport to
the lab
• Urine samples can be used

I. Gobe; Univ Botswana

NAAT Technique Disadvantages:

• It is highly expensive.
• Specimen can contain amplification inhibitors that
result in false-negative (decreasing specificity).
• Cross-reactivity of primers used for N. gonorrhea
detection with non-gonococal Neisseria species.
• More susceptible than non-NAAT methods to
false positive results due to contamination, if
control procedure is not applied accurately.

I. Gobe; Univ Botswana

 Examples
• Amplicor CT/NG
• The AMPLICOR CT/NG Test for Neisseria gonorrhoeae is a
multiplex assay that permits the simultaneous
amplification of N. gonorrhoeae target DNA, C. trachomatis
target DNA, and CT/NG Internal Control (CT/NG IC) DNA.
• The Master Mix reagent contains biotinylated primer pairs
specific for N. gonorrhoeae and C. trachomatis.
• The CT/NG Internal Control contains identical primer binding
sequences as the C.trachomatis target DNA and uses the CT
primers for amplification.
• The detection reactions are performed independently for N.
gonorrhoeae, C. trachomatis and the CT/NG Internal Control.

I. Gobe; Univ Botswana

 Target selection:Amplicor CT/NG
Neisseria gonorrhoeae
• contain a highly-conserved DNA sequence (M·Ngo PII)
that,encodes a cytosine DNA methyltranferase, which
inhibits the digestion of chromosomal DNA by HaeIII
restriction endonuclease.
• The M·Ngo PII gene sequence (approximately 1044
base pairs) is present in the different strains of N.
gonorrhoeae, and not found in most other, non-
gonococcal Neisseria species.
• biotinylated primers SS01 and SS02 define a sequence
of approximately201 nucleotides within the M·Ngo PII
gene of N. gonorrhoeae.

I. Gobe; Univ Botswana

 Target selection:Amplicor CT/NG
Chlamydia trachomatis
• The AMPLICOR CT/NG Test for Chlamydia
trachomatis uses the biotinylated primers CP24 and
CP27 to define a sequence of approximately 207
nucleotides within the cryptic plasmid DNA of C.
trachomatis

I. Gobe; Univ Botswana

Human Immunodeficiency Virus Type 1
• Is an RNA virus (family Retrovirus, genus lentivirus)
• genomic RNA is copied to dsDNA by reverse transcriptase
• DNA molecule is integrated into host genome
• Reverse transcriptase does not have proof-reading
mechanisms, hence prone to error causing genetic
diversity of HIV
a. Major (M) group (predominant worldwide)
b. Outlier (O) group
c. Non-M, non-O (N) group)
• M group viruses further subdivided into subtypes (clades)
due to sequence diversity in HIV-1 gag and env genes
a. A to D, F to H, J, K
b. C is predominant subtype southern Africa
c. B is predominant subtype in Europe and North America
I. Gobe; Univ Botswana
Distribution of HIV-1 Subtypes in sub-Saharan
Africa
North
Poorly
Documented

Horn
A/G C
Western D
A/G
Eastern
Central A
Poorly
Documented
Southern C
Amoroso A; Inst of Human Virology; University Maryland, MD
I. Gobe; Univ Botswana
Molecular Diagnostics in HIV Infection
1. Viral Load Testing
• Quantification of HIV-1 RNA in plasma
• Used to predict time to progression to AIDS
• Monitor response to therapy
➢ HAART treatment expected to lower viral load by at least
2 log10 copies/ml (1/100th) of starting value
➢ results are reported as log10 copies/mL.
• HIV viral load testing is not used for HIV diagnosis in
adults.
• Sample type
➢ EDTA blood- anti-coagulant of choice
➢ Acid Citrate Dextrose
I. Gobe; Univ Botswana
 THE USE OF HIV VIRAL LOAD MEASUREMENTS
• HIV-1 RNA levels are a predictor of the time to progression to acquired
immunodeficiency syndrome (AIDS) and death that is independent of CD4 cell
counts

• Viral load measurements are primarily used for monitoring the response to
treatment
• changes in HIV-1 RNA levels must exceed 0.5log10 (at least threefold) in
magnitude to represent biologically relevant changes in viral replication. It is
important not to over interpret small increases or decreases in viral RNA.

Exogenous factors that may affect HIV RNA levels

• HIV Viral RNA levels can transiently rise during acute illness eg an outbreak of
herpes simplex infection, vaccination against a variety of pathogens including
influenza, pneumococcus, and tetanus.
• The increases may be quite dramatic, 1 log10 (tenfold) or greater; however,
values usually return to baseline within one month. Thus, plasma HIV-1 RNA
levels should not be measured within one month of any of these events.

I. Gobe; Univ Botswana

 HIV viral load technologies
Cobas Cobas Abott RT EasyQ EasyQ
Amplicor taqman Nuclisens V1.1 V2.0
NASBA
target gag gag Pol/integr gag gag gag
ase

Method qPCR qPCR qPCR Nasba-RT Nasba-RT Nasba-RT

(TA) (TA) (TA) (TA) (TA) (TA)

Linear 400- 40-10mil 40-10mil 176- 50-3mil 10-10mil

range 750000 3470000
Copies/m
l
I. Gobe; Univ Botswana
 Molecular Diagnostics in HIV Infection
2. Diagnosis of HIV infection in Neonates
• serological tests misleading because of presence of
maternal IgG antibodies
• testing done at birth and at 6 weeks.
• HIV proviral DNA detected; Molecular assay
3. Drug Resistance Testing
– Indications
➢patients who are failing on initial HAART regimen
➢changing from initial to second line of HAART
– Assays
➢Genotypic – detect mutations in RT and protease genes
➢Phenotypic – measures viral replication in presence drugs
I. Gobe; Univ Botswana
I. Gobe; Univ Botswana
I. Gobe; Univ Botswana
 CD4 count: flow cytometry

• Works on the principle of light scatter (due to different

size or granularity of the cell) combined with
fluorescence of cells after staining with monoclonal
antibodies to cellsurface markers tagged to fluorescent
dyes
• Population of interest can be identified and gated
• Percentage of CD4 T cells can be calculated (% of
lymphocytes, or % of leucocytes)
• Absolute CD4 count can then be determined using
either dual‐ or single‐platform methodology
I. Gobe; Univ Botswana
Cell subpopulations based on Fsc vs Ssc

Source:www.bdbiosciences.co
m/immunocytometry_systems
I. Gobe; Univ Botswana /support/training/online/ITF/s
tart.html
 IDENTIFICATION OF LYMPHOCYTES:
immunophenotyping
• In the laboratory, different types of lymphocytes can be
identified by the receptors on their surface

• The term ‘CD’ means cluster of differentiation. This is a

uniform nomenclature system which has been adopted to
identify these receptors e.g.
CD45 All White Blood Cells
CD3 T-cells
CD4 T-helper cells
CD8 T-cytotoxic cells
CD19 B-cells
CD56 Natural killer (NK) cells

I. Gobe; Univ Botswana

Dot Plot of CD3 FITC/CD4 PE

I. Gobe; Univ Botswana

Source:www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/start.html
 CD4 count: flow cytometry
Dual platform
• Uses a haematology analyser‐generated absolute
lymphocyte count
• Multiplying this with the CD4% ( from cytometer) gives
absolute CD4 count
Single platform:
• Derive absolute counts directly from flow cytometer
without need for haematology analyser
• by using a known number of fluorescent beads mixed with
a known volume of blood
• addition of beads (eg TruCount, FlowCount or Perfect Count
beads)
I. Gobe; Univ Botswana
I. Gobe; Univ Botswana
tuberculosis

I. Gobe; Univ Botswana

MTb- pathogenesis: THE GRANULOMA
• granuloma formation is a classical hallmark of
anti-tuberculosis host defense
• IFN-γ controls the infection by stimulating
infected macrophages to produce bactericidal
substances notably reactive nitrogen species
which kill intracellular M .tuberculosis.
• Activated macrophages also produce tumor
necrosis factor which leads to killing of kills
infected cells
• calcified lessions or fibrotically encapsulated
lesion
I. Gobe; Univ Botswana
 FAILURE OF THE GRANULOMA
• death of macrophages in the center of a
chronic granuloma, a process called caseous
necrosis
• tissue damage
• caseation destroys the alveolar
• liquefied caseum empties into the bronchial
tree, formings a lung cavity which results in
explosive growth of M. tuberculosis due to the
availability of oxygen.
I. Gobe; Univ Botswana
I. Gobe; Univ Botswana
I. Gobe; Univ Botswana
 TB incidence
Tuberculosis (TB)
remains one of the
world’s deadliest
communicable diseases.

Atleasr 1 million people

die every yr

I. Gobe; Univ Botswana

 TB notification rate by district/100 000 pop; 2010
(Botswana)

The incidence of TB was

estimated to be 503 per 100
000 population in 2010. This
is more than 3 times higher
than the global equivalent

I. Gobe; Univ Botswana

 TUBERCULOSIS DRUGNS

 ISONIAZID: binds tightly to the enoyl-acyl carrier

protein reductase known as InhA, thereby blocking
the natural enoyl-AcpM substrate and the action of
fatty acid synthase. This process inhibits the
synthesis of mycolic acid, required for the
mycobacterial cell wall.
 RIFAMPIN; inhibits bacterial DNA-dependent RNA
synthesis by inhibiting bacterial DNA-dependent
RNA polymerase

I. Gobe; Univ Botswana

 Tuberculosis; Xpert assay
• uses real-time PCR to amplify an M.
tuberculosis-specific sequence of the rpoB
gene.
• Ninety-five percent of mutations associated
with rifampin resistance occur in an 81-bp
core region of the bacterial RNA polymerase
gene, rpoB. All mutations that occur within
this region result in rifampin resistance.

I. Gobe; Univ Botswana

 Tuberculosis; Xpert assay
• Five different probes are used in the same reaction, each perfectly
complementary to a different target sequence within the rpoB gene.
• Together, their target sequences encompass the entire core region. The
generation of all five fluorescent colors during PCR amplification indicates
that rifampin-susceptible M. tuberculosis is present.
• The presence of any mutation in the core region prevents the binding of
one of the molecular beacons, resulting in the absence of one of the five
fluorescent colors
• Since molecular beacons fluoresce only when they are bound to their
targets, the absence of any one of the five colors in the assay indicates
that the bacilli in the sample are rifampin resistant.
• M. tuberculosis is identified when at least two of the five probes give
positive signals with a cycle threshold (CT) of ≤38 cycles

I. Gobe; Univ Botswana

Testing for RpoB mutation: The Xpert ® MTB/RIF

I. Gobe; Univ Botswana

 Testing for RpoB mutation: The Xpert ® MTB/RIF

- The 5 Probes used are molecular beacons.

I. Gobe; Univ Botswana
 Testing for RpoB mutation: The Xpert ® MTB/RIF

Proper test performance is ensured by 2 internal controls:

– Sample processing control (SPC) ensures adequate processing and monitors
presence of inhibition.
– Probe check control (PCC) verifies that the steps of the tests (rehydration, filling of
the cartridge, etc.) take place correctly.

When the test is completed the display can show:

– “MTB detected” expressed by levels (the higher the level, the higher the amount of
MTB detected in the sample) or “MTB not detected”;
– RIF results expressed as “detected”, “not detected” or “indeterminate” are available
only if MTB is detected.

Other possible results:

– Invalid: MTB invalid and SPC failed due to one of several reasons, such as inhibition;
– Error: MTB no result, SPC no result, PCC failed; fail of system components;
– No result: e.g. tests stopped during processing.

I. Gobe; Univ Botswana

 Testing for RpoB mutation: The Xpert ® MTB/RIF

• Sample processing control (SPC) ensures adequate processing and monitors presence
of inhibition.
• Assays that are negative for M. tuberculosis(atleast 2 probes NOT detected) and for
I. Gobe; Univ Botswana
the SPC internal control are reported as invalid assays.
 Tuberculosis;
Xpert assay
• reliability when
compared to sputum
microscopy
• speed

I. Gobe; Univ Botswana