2. Background
• Leuser Virus epidemic in causing
‘yellow-eye’.
• 2 emerging strains, wild-type (mild
strain) and mutant (lethal strain).
• Initially prevent with mild-flu, rapid
decline, jaundice and death.
• Diagnostic ELISA-based blood test.
• Any positive result being quarantined
and treated.
• ELISA test is costly and detects both
strains, wild-type being unnecessarily
quarantined.
• Currently huge strain on resources.
• Urgent new diagnostic test needed.
• Pathogenicity not yet full understood.
• Further investigation into pathogenicity.
• Preliminary evidence suggests lethal
strain causes a rise in serum levels of
enzyme X.
• Mild strain doesn’t produce this rise.
• Proof of principle assay investigated.
• Other possible diagnostic markers
investigated.
3. Optimization
Substrate
(10mM) into
buffer
Aliquot (1ml)
incubated
Sample added
(0.1ml) &
incubated
Stop solution
(1ml) added
Absorbance read
Wavelength
determined
2nd Incubation
temperature
determined
2nd Incubation
time determined
Buffer pH
Human Serum
Blank
Parameters Using optimized
assay
Standards of
enzyme X
Sensitivity
(lowest enzyme
conc.)
Range (highest
& lowest conc.)
QC Samples
(accuracy)
Specificity
(enzyme Y)
Cut-off
Values
Using optimized
assay
Patient set A
Comparison
levels against
known strains
True/False
positives &
negatives
Determining cut
off values
Assay
differentiation
between strains
Effectiveness Using optimized
assay
Using
determined cut-
off values
Patient set B Blindly tested
ELISA
Optimized assay
compared to
ELISA
Standard ELISA
run
Patient set C
against ELISA
reagents
HPLC Standards HPLC
Serum levels of
metabolite P
Possible
diagnostic
marker
Further
pathogenesis
Dipstick
Urinanaylsis
Standard
Dipstick
Patient #90212
urine and
normal
Further
pathogenesis
H&E Stain Standard H&E
stain
Patient #90212
liver section and
normal
Further
pathogenesis
Hemolysis
Assay
Standard
Hemolysis
Assay
Patient #90212
sample, normal
and lysed
sample
Further
pathogenesis
Blood Smear Standard blood
smear
Patient #90212
and normal
blood sample
Further
pathogensis
Methods
4. Results – Assay Optimization & Parameters
Enzyme X Average
Absorbance (401nm)
Enzyme Y Average
Absorbance (401nm)
0.067 0.005
0
1
2
3
200 220 240 260 280 300 320 340 360 370 380 390 400 401 402 403 404 405 410 420 440 460 480 500
Absorbance(Arbitary
Units)
Wavelenth (nm)
Wavelength (nm)
Optimum Wavelength = 401nm
0
1
2
0 10 20 30 40 50 60
Absorabcneat
401nm
Tempetaure (◦C)
Optimum Temperature
Optimum Temperature = 37°C
0
0.5
1
1.5
2
2.5
3
3.5
pH 8 pH 9 pH 10 pH 11
Absorbanceat401nm
Optimum pH
Optimum pH & Incubation time
0 5 10 15 20
Optimum pH = 10
Optimum incubation time = 5mins
Specific for enzyme X
y = 0.1988x + 0.0003
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10 12
Absorbanceat401nm(ArbitaryUnits)
Enzyme Conc. (mg/l)
Enxyme X Standard Curve
Assay Range = 0.35mg/l – 8mg/l
Sensitive = 0.35mg/l
Accuracy = Accurate at a lower conc., less
so at a higher conc.
5. Results – Assay Cut-offs, Effectiveness & ELISA
Strain
1 (Wild-
Type)
Correct ID 17
Incorrect ID 3
2 (Mutant) Correct ID 17
Incorrect ID 3
Samples Average Abs
C1 0.044
C2 0.045
Positive 0.041
Negative 0.042
Reference 0.041
Effectiveness
ELISA Comparison
85% effectiveness, could be
improved accuracy using a
better cut-off value.
.
Assay Cut-off Values
Cut-Off Value was determined as 0.450ml/g
• Similar C1 & C2 values,
showing strains where
detected.
• Positive reference doesn’t
match samples.
• Positive and Negative
values the same.
• Reference sample the
same a positive.
Costly test not accurate or reliable.
6. HPLC
Results – Pathogenesis & Diagnostic markers
• Clear distinction levels between strain 1 and
strain 2.
• Overlapping values, no real difference between
the data.
• No distinct cut-off.
• No use as a diagnostic marker.
• Can be used as an indication of strain 2.
• Indicates pathogenesis.
• Further investigation for pathogenesis needed.
7. Results – Pathogenesis
Normal Patient #90212
Clearly shows mucin & cartilage
present, no blood cells. Blobs of
staining.
Not well stained, white and red
blood cells present. Excess
staining.
Normal Patient #90212
Red blood cells present, no white
blood cells. All standard shape.
Red blood cells shapes have
different structure, as well as
increased levels of white blood
cells present.
Blood Smear
H&E Stain
• Patient sample has more white blood cells, indicates infection.
• Irregular red blood cell/erythrocyte shape
• Haemolysis, virus attacking blood vessels, mainly erythrocytes.
• Directly attacks the liver, alter blood homeostasis
• Maybe the reason for such a rapid decline in the patients.
• Re-do normal sample with stain, better comparison.
• Patient #90212 contain blood cells.
• Damage to the liver.
• Causes low levels of white blood cells to enter the liver.
• Both aren’t clear staining, either excess stain or blobs present.
• Re-do for accuracy.
• Indicates ‘yellow-eye’ causes damage to the liver.
• Jaundice cause by a build up of bilirubin in the liver.
• Suggest this is due to the damage the virus causes to the liver.
8. Sample Average
absorbance
Result
Normal 0.053 Neg
Lysed 0.108 Pos
Patient
#90212
0.091 Neg
Patient
#90212
Normal
Glu Neg Neg
Bil + -
Ket Neg Neg
SG 1.005 1.005
Blood ++80 0
pH 8.5 8
Pro Neg Neg
Uro 2 1
Nit + -
Leu +70 Neg
Dipstick
Hemolysis Assay
Results – Pathogenesis
• kidney damBilirubin…
– Excess Bilirubin causes
Jaundice.
– Liver damage.
• Hematuria…
– Kidney infection
– Kidney damage
– UTI
• Urine pH…
– Diet (veggie/vegan).
– Glandular problem, links with
Hematuria.
– Kidneys producing ammonia to
neutralize the toxic acids.
• Urobilinogen..
- Liver disease or liver damage.
- Blocked bile flow from
gallbladder.
• Nitrate…
- UTI
• Leuocytes…
– Infection
– UTI
• Patient sample is not lysed.
• However value is close.
• Suggesting limited hemolysis.
- Low solute blood conc. (hypertonic).
- Dropped/Damaged sample
- Due to the virus.
- Hemoglobinemia due to haemoglobin into blood
plasma.
• Re-run close values, definitive answer.
• No definite hemolysis.
• Contradicted by blood smear
9. • Leuser Virus is an emerging virus which urgently needs a new diagnostic test.
• The proof of principle assay developed is shown to be…
- Accurate
- Specific
- Sensitive
- Reliable
• Metabolite P not useful as a diagnostic marker, diagnostic indicator
• Further pathogenesis investigation indicates…
- Damage to the liver, causing excess bilirubin build up causing presenting jaundice
- Erythrocyte damage
- Due to haemolysis
- Kidney Damage/Infection.
- UTI
- Conflicting results over haemolysis, evidence supports it. Assay repeat is needed.
Summary
Recent studies have shown the viral epidemic of the Leuser virus, causing ‘yellow-eye’, in South East Asia has both a wild-type (strain 1, mild strain) and mutant strain (strain 2, lethal strain) of the virus. Patients initially present with mild-flu like symptoms, followed by a ‘sudden rapid decline’1 then death.
The full pathogenicity of the virus is not yet understood, particularly the characteristic jaundice that precedes death, but those who present with the initial flu like symptoms are undergoing an ELISA-based blood test, with any positive results leading to the patient being quarantined and treated, with the treatment being costly and painful, but effective leading to lives being saved.
Despite this the ELSIA is 'costly and detects both strains'1, meaning people who only have the mild strain are being unnecessarily quarantined. The current containment strategy is putting a huge strain on local resources, becoming unstainable in the next 6 months.
There is an urgent need for a new diagnostic test that is cheaper and more specific, differentiating being the strains, as well as further research into the pathology of the disease, due to global 'data-sharing' initiatives; we are part of a research team who are co-operating to improve the diagnostic strategy, and further investigate the pathogenesis of the disease.
The research is based on the preliminary evidence that the lethal strain (strain 2) causes a rise in serum levels of enzyme X, but isn't yet seen in patients with the mild strain (strain 1), a proof of principle assay will be produced for further investigation by an independent research group, conducting the full-scale clinical evaluation.
We will also use other methods for investigate the further pathogenesis of the disease, as well as any other possible diagnostic markers.
