Immunological and Molecular Assessment of

Umbilical Cord Blood and Maternal Blood

Admixture in a Group of Cameroonian Neonates
Putatively Primed in utero to P.falciparum
Antigens.
Thesis presented in partial fulfillment for the award of the
Medicinae Doctorae Degree by:

BESONG MICHEAL EBANGHA

Director:

Co-Directors:

Pr. Rose G.F. LEKE

Pr. Judith TORIMIRO

Dr. Samuel TASSI YUNGA
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Plan
Introduction
Objectives
Methods
Results and Discussion
Conclusions
Recommendations
Summary in French

INTRODUCTION (1)

Malaria: parasitic disease, Plasmodium sp., female Anopheles sp.

NMCP: 41% OP, 43% deaths in 2008.
More vulnerable: Children < 5yrs and pregnant women.
Pregnancy: Parasitaemia, placental malaria and placental

inflammation, adverse pregnancy outcomes.

Better prevention: Method of detecting malaria parasites

and placental inflammation, especially in asymptomatic women.

Current methods: Do not inform on inflammation; require blood.
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INTRODUCTION (2)

sTNF-R1 and sTNF-R2

in plasma: relate to peripheral parasitaemia, placental malaria

/inflammation, pregnancy outcome.

extensively shed by placental tissue
regulate the bioactivity of TNF-

If detection in urine is accurate/discriminative: opportunity

for easy, rapid, non-invasive and routine screening for

malaria during pregnancy.
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OBJECTIVES

General objective: Assess the possibility of using sTNF-

R1 and -R2 in urine to screen for malaria in pregnancy.

Specific objectives
Determine the influence of malaria in pregnancy on the

levels of sTNF-R1 and sTNF-R2 in urine.

Study the association between peripheral parasitaemia and

urine levels of sTNF-R1 and sTNF-R2.

Determine the performances of urine sTNF-R1 and sTNF-

R2 assays in screening for malaria in pregnancy.

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METHODS (1)

Study type: Cross-sectional

Study duration: 6 months (from August 2009 to January 2010).
Setting:
Yaounde (Baptist HC, Pres. HC, Biyem-Assi DH, CHY);
Soa HD (Ngali HC, Ntouessong HC).

Sample size: 66 participants (Lehrs equation).

Sampling: Consecutive
Ethics: Approval by the IRB of the BTC UY1.
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METHODS (2)

Inclusion criteria
Pregnant
Non-pregnant of child-bearing age.
Signed informed consent

Exclusion criteria.
Parturient
Vaginal bleeding in pregnancy
Non-pregnant with on-going menstrual flow.
Women on anti-malaria curative treatment.

METHODS (3)

Procedure
SI
T
E

ASSESSMENT
FOR
ELIGIBILITY

RAPID MALARIA
SCREENING
Matching

RECRUITMENT PROPER
Enrolment forms filled.
Body temperature
SAMPLE COLLECTION
(Austgulen
Austgulen et
et al.
al. 1992
1992)

Same
day
LA
BO
RA
TO
RY

Urine: aliquots, storage at -25oC.

Blood: PCV, WBC count, Thick/Thin blood films made.

Next day
Staining and Reading of slides (Gold standard)
Sample size
attained
ELISA for sTNF-R1 and sTNF-R2 in urine...
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METHODS (4)

Procedure (Human sTNF-R1 and -R2 ELISA)

OD
450nm

STOP!
TMB.
S-P
conjugate.
Biotinylate
d tracer
antibodies.
Samples
and
standards
in
Pre-coated

R2 standard curve
Av. R2 Ods

Linear (Av. R2 Ods)

12
10
8

Av R2 OD

6
4
2
0

= 400 600 800 1000 1200

0 f(x)
200
R = 0

R2 standard conc (pg/ml)

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METHODS (5)

Data Management
Data entry: CRFs and MS Excel 2007; Analysis: SPSS 17.0
Central tendency: means (normal), median (skewed).
Frequencies and proportions used to characterize our study

population.
Non-parametric tests: comparisons (Kruskal-Wallis / Mann-

Whitney U) and associations (Spearmans rank / Partial).

p values <0.05 were considered statistically significant.
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Performance of screening tests: 22 tables and ROC

Ideal Test:
Perfect
Discrimination
AUC = 1
Intermediate
Discrimination
1 > AUC > 0.5

True positive Rate

(Sensitivity)

METHODS (6)

Sensitivity, Specificity

No
Discrimination.
Useless
AUC = 0.5
False Positive Rate (1 Specificity)

Predictive values: Prevalence of periph. parasitaemia of 22.6%

(Walker-Abbey et al., 2005)

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RESULTS/DISCUSSION (1)

Total 66 sample sets from:

46 pregnant women (23 positive, Pg+Mal+ and 23 negative Pg+Mal)
20 non-pregnant (10 positive, Pg Mal+ and 10 negative, PgMal )

Mean age: 25 5years.

Mean GA of Mal+ and Mal were similar (p=0.589)
40.9% primi- and 59.1% multi-gravid, Mal+ = Mal
12.1% had temp. 38.0oC : all Mal+ (Pg > Pg+ p=0.036).
Pg+Mal+ and PgMal+ had similar parasitaemia (p=0.259)
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RESULTS/DISCUSSION (2)

Comparison of urine sTNF-R1 and -R2 of groups

p<
0.001
p<
0.001

*p <
0.001

*Davison et al., 2006; McGuire et al., 1998; Arntzen et al., 1995; Molyneux et al., 1993;...
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RESULTS/DISCUSSION (3)

Influence of pregnancy
p=0.000
06

p=0.00
5
5.95
(4.00)

0.00
(0.00)

0.73
(4.87)

Median
(IQR)
ng/ml

1.5
(1.67)

Arntzen et al., 1995; Austgulen et al., 1993

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RESULTS/DISCUSSION (4)

Influence of malaria in pregnancy

p
<0.00006
p=
0.005

37.48
(32.42)

0.73
(4.87)

9.90
(33.43)

Median
(IQR)
ng/ml

5.95
(4.00)

Davison et al., 2006....

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RESULTS/DISCUSSION (5)

Influence of malaria in pregnancy

p
=0.557
p
=0.298

37.48
(32.42)

1.80
(13.16)

9.90
(33.43)

Median
(IQR)
ng/ml

15.55
(41.57)

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RESULTS/DISCUSSION (6)

Association between peripheral parasitaemia and urine levels

of:

sTNF-R1

sTNF-R2

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RESULTS/DISCUSSION (7)

Association between peripheral parasitaemia and urine sTNF-

R1 and sTNF-R2 levels.

Parasitaemia (p/l)
n = 23
(only Pg+ Mal+)

Parasitaemia (p/l)
n = 33
(Pg Mal and Pg Mal )
+

Urine sTNF-R2 (ng/ml)

Spearman rs = 0.784

Spearman rs = 0.816

p < 0.001

p < 0.001

Spearman rs = 0.730

Spearman rs = 0.742

p < 0.001

p < 0.001

partial r = 0.556

partial r = 0.634

p = 0.001

p < 0.001

Parasitaemia (p/ul)
Adjusted for body

Urine sTNF-R1(ng/ml)

temperature.

PCV: sTNF-R1: rs = - 0.207, p = 0.095; sTNF-R2: rs = - 0.341, p = 0.005

Body temperature: sTNF-R1: rs = 0.370, p = 0.002; sTNF-R2: rs = 0.420, p < 0.001

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ROC curve
Ideal Test:
Perfect
Discrimination
AUC = 1

Intermediate
Discrimination
1 > AUC > 0.5

True positive Rate

(Sensitivity)

RESULTS/DISCUSSION (8)

Performance of sTNF-R1 and sTNF-R2 assays:

No
Discrimination.
Useless
AUC = 0.5
False Positive Rate (1 Specificity)
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RESULTS/DISCUSSION (9)

Performances of the sTNF-R1 and sTNF-R2 assays

82.6%,87%
65%, 94.5%

95.7%,21.7%
26.3%, 94.5%

78.3%,100%
100%,94%

56.5%,95.7%
79.3%,88.3%

AUC
R2=0.892
R1=0.741

Kabyemela et al., 2008 (R1 = 0.635, R2 = 0.731)

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p=0.002

CONCLUSIONS (1)

Malaria in pregnancy is accompanied by significant

increases in the levels of soluble TNF- receptors 1

and 2 in urine.

Peripheral parasitaemia positively and significantly

correlates with urine levels of both sTNF-R1 and

sTNF-R2 even in the absence of fever.

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CONCLUSIONS (2)

Singly, urine sTNF-R2 is potentially useful as

biomarker for malaria screening during pregnancy.

The sTNF-R1 assay alone is less accurate but could

be used combination with the sTNF-R2 assay to

achieve a better global performance.

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RECOMMENDATIONS (1)

Evaluate the influence of sub-microscopic

parasitaemia on the urine levels of sTNF-R1 and

sTNF-R2

Evaluate the relative influence of malaria over other

pathologies on the urine levels of sTNF-R1 and

sTNF-R2 during pregnancy.

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RECOMMENDATIONS (2)

Carryout validation studies for the use of soluble

receptors of TNF- in urine-based screening malaria

in pregnancy.

Envisage associating intermittent screening for

malaria with Intermittent Preventive Treatment in

the control of malaria in pregnancy

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Rsum

25

MERCI POUR VOTRE

AIMABLE ATTENTION

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Special Thanks
To the:
Study participants for accepting to provide their
specimen.
Staff of the Immunology Laboratory, BTC UY1, for the

technical support
NIH for the grant through Pr Rose Leke.
FMBS UY1 for the academic and professional training.
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