healthmedicinet

Malaria antigens

Apical membrane antigen (AMA1) was from the Pichia pastoris expressed ectodomain of P. falciparum FVO strain comprised amino acids 25–545 9] (Donated by Dr Daniel Dodo, Noguchi Memorial Institute for Medical Research, University
of Ghana, Legon, Ghana).

MSP1_
₄₂
protein was from the C-terminal MSP1_
₄₂
amino acid sequence of the Uganda-Palo Alto (FUP) P. falciparum isolate (GenBank Accession No. M37213) expressed in Escherichia coli (Ec) system. The recombinant protein, EcMSP1_
₄₂
-FUP (Uganda-Palo Alto strain), represents the 33 kDa fragment from the 3D7 P. falciparum variant and the E-K-NG point mutations identified in the 19 kDa fragment within the
MSP1_
₄₂
native molecule 10].

CSP was a full-length protein expressed in an Escherichia coli system containing amino-acids Leu
¹⁹
to Ser
⁴¹¹
(Indian isolate, GenBankTM No: AAN87606) 11].

MSP1_
₄₂
and CPS were donated by Dr Patrick Duffy and Dr Richard Shimp from NIH/NIAID (National
Institutes of Health/National Institute of Allergy and Infectious Diseases).

Enzyme-linked immunosorbent assay (ELISA)

Three drops of blood were collected onto Whatman 3MM filter paper, which was sealed
and stored dry with desiccant at room temperature. Reconstituted sera were obtained
from filter paper bloods spots described previously 12], 13]. Sera were tested for anti-MSP1_
₄₂
IgG antibodies, anti-AMA1 IgG antibodies and anti-CSP antibodies by indirect ELISA.
Samples were also tested on freeze thawed P. falciparum schizont extract (concentration of 1 × 108/ml), which was coated onto ELISA plates
at 1/500.

Briefly, 96 well ELISA plates were coated with 100 µl/well of 0.1 ?l/well of MSP1_
₄₂
and CSP antigens and 0.026 ?l/well of AMA1 in coating buffer (1.59 g Na
₂
CO3, 2.93 g NaHCO3, 1 liter distilled water, pH 9.4). The plates were incubated overnight
at 4°C. After incubation, plates were washed at three times using PBS (5.7 g NaH
₂
PO
₄
, 16.7 g Na
₂
HPO
₄
, 85 g NaCl in 1 l distilled water) plus 0.05% Tween 20 (PBS/T) and blocked with 1%
(w/v) skimmed milk power in PBS/T for 1 h at 37°C. Eluates were removed from 4°C just
before use. After three more washes, eluates were diluted at a ration 1/100 in PBS/T
and added 200 µl in duplicate in a well plate.

For each plate three types of control were used: deep well without serum but with
a second antibody to measure the non-specific binding, pool of sera from patients
with P. falciparum malaria (positive control) and pool of sera from non-infected subjects (negative
control) from Copenhagen. Three washes were performed before incubation for 1 h at
37° with secondary antibody (100 µl of horseradish peroxidase-conjugated rabbit anti-human
IgG, SouthernBiotech
^®
). After incubation for 1 h at 37°C, plates were developed with TMB/E (Upstate
^®
, Chemicon
^®
et Linco
^®
, Millipore) as substrate for 30 min at room temperature in the absence of light and
the reaction was stopped by the addition of 50 µl/well of 2 M H
₂
SO
₄
. Optical density was read at 450 nm against a 620 nm with an ELISA TECAN SUNRISE
reader.

Haematological assessment

One drop of blood was collected from all participants for haemoglobin (Hb) level measurement
using Hemo-Cue machine (HemoCue
^®
Hb 301). Anaemia was defined as Hb concentration below 11 g/dl.

Statistical methods

After data collection, date entry work was performed using Excel software. Thereafter,
analysis was carried out using Stata software version IC 12 software.

For serological assessment, the optical density was obtained by subtracting the average
OD (Optical density) of duplicate wells from that of the corresponding blank wells.
Values were converted into arbitrary units (AUs), as follows 14]:

To assess the nutritional status, data were transferred into Epi Info 3.04 d. The
Z-scores for weight-for-age (underweight) and height-forage (stunting) were derived
using Epinut Anthropometry. Children with Z-scores below—2 standard deviation (SD) of the National Centre for Health Statistic
(NCHS), United States reference population median were considered to be malnourished.

Quantitative variables were described in terms of means, SD. Inter-group comparisons
were done using ANOVA test or Student t test where appropriate, otherwise non-parametric
tests such as Mann–Whitney or Kruskal–Wallis were used.

For descriptive data, percentage was used to each outcome. Antibodies seroprevalence
was calculated and expressed by percentage with their 95% confidence intervals. Proportions
were compared using Chi square test or the Fisher exact test (univariate analysis).
A stepwise logistic regression analysis was done to assess factors associated with
P. falciparum antibodies carriage. Significance level of the different tests was set at 5%.

Ethical considerations

The study was nested into a cluster-randomized trial 15] which had been approved by the Senegalese National Ethical Committee (Conseil National d’Ethique et de Recherche en Santé) and registered at the Pan African Clinical Trial Registry: registration number: PACTR201305000551876.
In the field, individual informed consent was required prior to each participant enrolment.
Community sensitization was done prior to the study to explain the planned investigations.