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ORIGINAL ARTICLE
Corresponding Author: I.B. Abiodun
Department of Biochemistry and Nutrition, Nigerian Institute of Medical Research, Lagos, Nigeria
Tel: +234 1 8023357646, Fax: +234 1 8504327, E-mail address: bamwal@yahoo.com
Plasmodium falciparum Merozoite Surface Protein-1 Polymorphisms among
Asymptomatic Sickle Cell Anemia Patients in Nigeria
Iwalokun Bamidele Abiodun1, Afolabi Oluwadun2, Aina Olugbenga Ayoola3,
and Iwalokun Senapon Olusola4
1 Molecular Biology and Biotechnology Division, Nigerian Institute of Medical Research, Lagos, Nigeria
2 Department of Medical Microbiology and Parasitoogy, Olabisi Onabanjo University, Sagamu-Ogun State
3 Maternal and Child Complex, Isolo General Hospital, Nigeria
4 Olorunda Primary Health Centre, Badagry, Lagos, Nigeria
Received: 17 May 2014; Accepted: 24 Dec. 2014
Abstract- Asymptomatic malaria (ASM) has been implicated in the development of hemolytic crisis in
infected sickle cell anemia (SCA) patients worldwide. This study surveyed steady state SCA Nigerian
patients for ASM to investigate the influence of malaria prevention behaviors and age on parasitaemia and
multiplicity of infection (MOI). A total of 78 steady SCA patients aged 5 – 27 years on routine care at three
health facilities in Lagos were investigated for ASM by light microscopy and PCR with a multiplicity of
infection determined by genotyping block 2 of merozoite surface protein 1 (msp1) gene of Plasmodium
falciparum (P. falciparum). Use of malaria prevention measures was captured using a semi-structured
questionnaire. The prevalence rates of ASM (due to Pf only) by microscopy and PCR were found to be 27.3%
and 47.4% respectively (P < 0.05) with a Mean + SEM parasite density of 2238.4 + 464.3 parasites/uL. Five
distinct msp1 genotypes [K1 (2), MAD20 (2), RO33 (1)] were detected and significant (P<0.05) disparity in
allele frequencies (K1, 91.8%, MAD20, 32.4%; RO33, 18.9%) was found. The overall MOI was 1.43 and
37.8% of infections were polyclonal (P<0.05). ASM was associated with non-use of preventive measures and
occurred in 62.1% of SCA patients aged < 10y with lower MOI of 1.3 compared to 38.1% in older patients
with a higher MOI of 1.5 (P<0.05). We conclude that PCR improved the diagnosis of ASM among Nigerian
SCA patients with infections being of low complexity and associated with non-use of preventive
interventions and R033 msp1 allele selection.
© 2015 Tehran University of Medical Sciences. All rights reserved.
Acta Med Iran 2016;54(1):44-53.
Keywords: Asymptomatic malaria; Plasmodium falciparum; Parasite diversity; Multiplicity of infection;
Chemoprophylaxis
Introduction
Despite the yearly improvement in the global malaria
prevention and control efforts since 2000, malaria
remains a major scourge in Sub-Saharan Africa, causing
morbidity and mortality in children under 5 years,
pregnant women and immunosuppressed populations
like sickle cell anemia (SCA) patients (1). In 2010, an
estimated 305,800 newborns with SCA were reported
globally with 75% of these births occurring in Africa
(2). Due to its perennial transmission in many African
countries, particularly in the West Africa sub-region,
where SCA is more prevalent, malaria remains a major
cause of severe anemia, crisis and deaths among SCA
patients (1). It has been reported that between 50% and
80% of SCA patients die annually and malaria remains a
major cause of these deaths (2). Asymptomatic malaria
(ASM) is well known as a common manifestation of
plasmodium infection in malaria endemic countries in
Africa irrespective of the level of transmission (3,4).
The concerning aspect of ASM is the role it plays in the
development of clinical malaria and other consequences
such as anemia, thrombocytopenia, low birth weight and
cognitive impairment as well as making carriers serve as
hotpops of malaria transmission (5,6,7,8). The passive
surveillance system of national control programmes and
I.B. Abiodun, et al.
Acta Medica Iranica, Vol. 54, No. 1 (2016) 45
post-treatment gametocytaemia induced by anti-malarial
drugs have been implicated as potential causes of many
ASM cases that are often undetected and untreated in
many malaria endemic countries (8). Meanwhile, in
consideration of its importance, ASM is seen as
reflecting a state of immunological tolerance that is
mediated by two types of anti-malarial immunity:
clinical immunity, which prevent symptoms and anti-
parasitic immunity, which conditions parasitaemia to a
lower density, thereby providing protection against
uncomplicated and severe malaria (9). The multiplicity
of infection (MOI) defined as the number of parasite
clones per infection has been used to explain partial
immunity conferred by ASM in African children living
in areas of high and low or unstable malaria
transmission (4,9,10). Many investigators have linked
MOI with protection in high transmission areas and
clinical malaria susceptibility in low transmission areas;
age and exposure in children have important roles in the
acquisition of malarial immunity (9,10). Generally, the
prevalence of ASM has been shown to vary in different
geographical areas and high-risk population in Africa.
Bousemma et al., (11), Nkoghe et al., (12), and Kimbi et
al., (13) reported ASM
Prevalence rates of 73.4%, 1.7-8.7% and 60 -100%
among Kenyan, Gabonese and Cameroonian children.
An ASM prevalence rate of 24% by microscopy was
also recently reported by Douamba et al., among
pregnant women from Burkina Faso. In Nigeria, ASM
has also been reported by many investigators (14).
Ojuroungbe et al., reported an ASM prevalence rate of
25.6% among children aged 4 – 15 years in Osogbo,
south West Nigeria, linking its occurrence with
helminthic co-infection (15). In another study, Uneke et
al., found a correlation between ASM, anemia and low
birth weight among pregnant women in southeast
Nigeria (16). However, despite a concern of its
occurrence, there has been a dearth of information
regarding the burden, risk factors and consequences of
ASM in SCA patients in Nigeria. Kotila et al., reported a
prevalence rate of 24% by microscopy among
asymptomatic sickle cell adults in Ibadan, south West
Nigeria (17). Although this study is of relevance as it
validated SCA patients as potential sources of malaria
transmission during the dry season in the study area
coupled with a potential limitation of chemoprophylaxis
in averting ASM, two important limitations were found.
First, the study included patients with sickle cell
haemoglobin C disease whose course of crisis is lower
in frequency and magnitude compared to SCA patients
(10% of cases) and the second is the inclusion of
patients who received malaria treatment in the month
preceding the study. The latter may lead to an
overestimation of ASM among the patients studied.
Apart from chemoprophylaxis, other malaria prevention
methods have not been evaluated as risk factors for
ASM in the SCA population and there is a lack of data
on the genetic diversity of parasite population involved
in ASM in this cohort. Information on risk factors and
complexity of infection would be needed for designing
appropriate preventive interventions against ASM and
for generating baseline data for use in surveillance of
ASM SCA patients at risk of clinical malaria in Nigeria.
The purpose of this study was to determine the
prevalence of ASM by microscopy and PCR and
investigate its association with malaria prevention
practices among a cohort of steady state SCA patients in
Lagos. The complexity of ASM infection based on msp1
block 2 polymorphisms in this cohort was also
investigated.
Materials and Methods
Study design and setting.
This was a cross-sectional study of steady state SCA
patients aged 5 – 27 years on routine care at three health
facilities in Lagos between February – April, 2009 in
Lagos. The health facilities, which provide routine care
services for SCA patients were Lagos State University
Teaching Hospital (LASUTH), Maternal and Child
Health Complex (MCHC), General Hospital Ikorodu
and Massey Street Children hospital (MSCH). Lagos is
located on latitude 6.50 north of the equator with a mean
rainfall of 1538 mm and average minimum and
maximum temperatures are put at 22.7oC and 30.7oC
per annum (18). Malaria is transmitted throughout the
year in the state with high intensity of transmission
experienced during the rainy season from May – July,
October and November and low transmission during the
dry season from December – February and during the
short period of no rain (August –September). An
estimated 17.5 million people live in Lagos and all the
inhabitants are at risk of malaria infection (18,19).
Female anopheles mosquitoes belonging to the
Anopheles gambie complex remains the main vector of
transmission, while P. falciparum is responsible for over
95% of reported malaria cases in the state (19). Malaria
prevalence survey of 2010 reported parasite rates of
6.9% and 3.5% in children and adults. Use of net the
previous night was 26.5% in children and 17% of the
general population. Post LLIN campaign survey in 2012
revealed a net retention was 88.1% in the state (18).
Plasmodium falciparum msp1 polymorphism
46 Acta Medica Iranica, Vol. 54, No. 1 (2016)
Sampling and Patients’ selection
The studied SCA patients were selected by
convenient sampling as they present for routine care at
MCHC, LASUTH and MSCH during the study period.
Informed consent was obtained from each patient prior
to enrollment. For minors, consent was obtained from
their caregivers. The purpose, procedures, benefits,
risks, and confidentiality components of the study were
explained to the patients in the course of seeking their
consents. Patients excluded from the study included
those who declined consent, patients with fever, history
of fever or fever related symptoms such as a headache
and vomiting in the previous 72 h at presentation,
patients who received malaria treatment in the previous
8 weeks and patients in crises as previously described
(18). The study protocol was approved by the ethics
committee of Hospital Management Board, Lagos state.
After enrollment, the SCA status of each patient was
confirmed by a positive sickling test and s-haemoglobin
genotyping at pH 8.6 by acetate cellulose
electrophoresis compared with the control HbAA,
HbAS, HbSS and HbSC (20). The patients’ case files
were reviewed for age, sex, and number of blood
transfusion in the previous six months. This was
followed by the administration of a pre-tested semi-
structured questionnaire on each patient to obtain
information on the intake of anti-malarial
chemoprophylaxis (daily for proguanil or in the last 7
days for pyrimethamine) and sleeping under LLIN the
previous night as well as using an insecticide spray. The
minimum number of patients for this study was
calculated to be 76 using the proportion method of
randomization that was based on the recent mean
parasite prevalence rate of 5.2% (p) in the state with
90% statistical power at 95% confidence interval
(z=1.96) and an error rate (d) of 5%. Patients diagnosed
with asymptomatic malaria by microscopy or PCR were
treated with arthemether-lumefantrine according to the
national malaria treatment guidelines for children and
adults (21).
Laboratory investigations
Haematology and microscopy
Venous blood sample (2 mL) was collected from
each patient into sequestrin bottle and used for
haemoglobin determination and total leukocyte count
using standard methods (22). Diagnosis of asymptomatic
falciparum malaria was performed by light microscopy
and PCR as described by Snounou et al., (23). For the
microscopic test, two grease-free slides, each
comprising thin (3 uL of whole blood) and thick (15 uL
of whole blood) were prepared per sample. The thin film
was fixed in methanol, and both films were stained with
3% Giemsa stain for 45 min after drying. Both films
were then examined under oil immersion (x 1000
magnification) for the detection of malaria parasite by
species using the thin film and counting of the asexual
erythrocytic stage of P. falciparum using the thick film.
The slides were also examined for the presence of
gametocytes. This was done by two trained
microscopists who worked independently. Parasites
were counted against 300 leukocytes by two and
parasitaemia was measured as an average of two counts.
Parasitaemia was measured as a number of parasites per
uL of blood-based on the number leukocytes counted
per uL of blood for each patient. A slide was considered
to be negative after examining 100 high power fields
without parasites. No P. falciparum positive slides with
discordant in count > 20% was observed and no
gametocytes were seen. All the asexual stage parasites
detected were those of Plasmodium falciparum.
Molecular methods
For PCR, parasite DNA was first extracted from 400
uL of whole blood samples using the Qiagen blood
DNA extraction kit (Qiagen Hilden, Germany) and
following the protocol provided by the manufacturer.
The prepared DNA samples were assessed for yield and
purity and then stored at -20oC until use. Each extracted
parasite DNA was then used to genotype block 2 allele
families of msp1 by nested PCR using primers designed
by Snounou et al., (23) (Table 1). The primary PCR
reaction was a 25 uL reaction mixture comprising 1X of
PCR buffer, 1.5mM MgCl2, 125uM of dNTPs, 30
picomoles of each primer, 75 – 100 ng of DNA and 1.25
units of Taq polymerase. All reagents were from
Biomers (Biomers, Germany). Amplification was done
in a Techne thermocycler (TC 312) under the following
conditions: initial denaturation at 940C then 3 cycles of
940C for 1 min, 580C for 2 min and 720C for 2 min,
followed by 25 cycles of denaturation at 940C for 1 min,
primer annealing at 580C for 2 min and extension at
720C for 2 min and finally a single annealing step at
580C for 2 min and an extension step at 720C for 5 min.
The secondary PCR reaction was also a 25 uL reaction,
using 3 uL of the primary PCR product as the DNA
template with 2 mM of MgCl2, 30 picomole each of
allele specific primers for K1, MAD20 and R033 (Table
1), 200 uM of dNTPs and 1.25 units of Taq polymerase
in 1 X PCR buffer. The cycling conditions for the
secondary PCR were an initial denaturation at 950C for
10 min, followed by 40 cycles of denaturation at 940C
I.B. Abiodun, et al.
Acta Medica Iranica, Vol. 54, No. 1 (2016) 47
for 30 sec, primer annealing at 580C for 30 sec and
extension at 720C for 1 min and, a final extension at
720C for 10 min. The resulting PCR products were
electrophoresed on 3% agarose gel pre-stained with
ethidium bromide (0.5 ug/mL), and DNA bands were
visualized under a UV transilluminator. The molecular
sizes of the visualized DNA bands were extrapolated
based on the mobility of 50 bp DNA markers
(Fermentas, Germany).
Table 1. Sequences of primers used to amplify the MSP-1 genes of P. falciparum
isolates recovered from the asymptomatic sickle cell anemia patients.
S/N PCR
REACTION PRIMER PRIMER SEQUENCE
1 Primary PCR MSP1-OF 5- -CTAGAAGCTTTAGAAGATGCAGTATTG-3-
MSP1-OR 5- -CTTAAATAGTATTCTAATTCAAGTGGATCA-3-
2 Secondary
PCR
MSP1-IKF 5- -AAATGAAGAAGAAATTACTACAAAAGGTGC-3-
MSP1-IKR 5- -GCTTGCATCAGCTGGAGGGCTTGCACCAGA-3-
MSP1-IMF 5- -AAATGAAGGAACAAGTGGAACAGCTGTTAC-3-
MSP1-IMR 5- -ATCTGAAGGATTTGTACGTCTTGAATTACC-3-
MSP1-IROF 5- -TAAAGGATGGAGCAAATACTCAAGTTGTTG-3-
MSP1-IROR 5- -CATCTGAAGGATTTGCAGCACCTGGAGATC-3-
Definitions and Data management
Asymptomatic malaria (ASM) was defined as the
presence of asexual stage of P. falciparum in the whole
blood (microscopy) or detection of one or more msp1
allele bands from DNA samples by electrophoresis after
PCR amplification. Isolates with one msp1 alleles were
considered to elicit monoclonal infections while those
with two or more msp1 alleles were considered as
polyclonal infections. The multiplicity of infection
(MOI) was determined by calculation of the total
number of msp1 alleles seen divided by the number of
msp1 positive samples. Msp1 polymorphism was
determined as the number of distinct K1, MAD20 and
R033 alleles recovered on the basis of size differences.
The frequency of a msp1 allele (i.e. allele frequency)
was expressed as a percentage of the total number of
allele DNA bands detected in msp1 positive samples.
The number of msp1 genotypes was defined as the
numbers of distinct types of msp1 block 2 alleles
detected. Infection type was defined by clonality (i.e.
monoclonal vs. polyclonal) or by allele type. For each
infection type, parasite density was computed only for
cases with parasitaemia by microscopy since
quantitative PCR was not done. Data were double
entered into Microsoft excel and Microsoft access
worksheets, cleaned and validated before analyses.
Continuous variables such as haemoglobin (Hb), total
leukocytes (WBC) and parasitaemia were computed as a
mean + standard error of mean (SEM) while categorical
variables such as sex, residence and use of
chemoprophylaxis were computed as numbers and
percentages (%). Parasite infection variables were
compared between < 10 years and 11 and above age
groups. Univariate analysis was performed to evaluate
the association between ASM or P. falciparum infection
parameters and other covariates such as age, sex and
malaria prevention practice indicators (i.e.
chemoprophylaxis, LLIN and insecticide spray use
rates). This was done using student’s t-test and chi-
square test or Fischer exact test for mean and proportion.
Multiple allele group comparison for parasitaemia was
done using two-tailed one-way analysis of variance
(ANOVA) and Turkey-Kramer posthoc test. Outcomes
of analyses with P-value < 0.05 was regarded as
significant. Analyses were done using Statistical
package for social science version 15.0 (SPSS 15.0).
Results
Data presented in Table 2 summarizes the general
characteristics of the studied steady state SCA patients.
A total of 78 steady-state SCA patients with a mean age
of 14.2 years and comprising 59% females were
enrolled. Among the patients screened for ASM, 37
(47.4%) were positive by PCR while 17 (21.8%) were
positive by microscopy (P<0.05) (Tables 2&3).
Although no significant (P>0.05) disparity in mean age
was observed between ASM positive and ASM negative
patients, age<10 years, rural residence, and not sleeping
under LLIN or spraying insecticide spray the previous
night were significantly (P = 0.0026 – 0.046) associated
with ASM. Mean haemoglobin level of 8.4 +0.1 g/dl
among ASM patients was also significantly (P<0.05)
lower than 9.2 + 0.3 g/dl in ASM negative patients. The
disparity in parasite density between age<10 y and 11
years and above was not significant (P>0.05). On the
whole, 14.1% of the SCA with ASM elicited
parasitaemia in the range of 500 – 5000 parasites/uL.
Plasmodium falciparum msp1 polymorphism
48 Acta Medica Iranica, Vol. 54, No. 1 (2016)
The percentages of SCA aged > 10 years and > 11 years
with ASM at this density range were 17.2% and 12.2%,
respectively (P >0.05), while the overall MOI observed
for the ASM was 1.43 (Table 3), one to three distinct
parasite infections per sample were seen with age 11
years and above associated with higher MOI compared
with infection in infection in the < 10 y age group. (1.5
vs. 1.3; P<0.05) (Table 3).
Further analysis showed that 37.8% of the ASM
infections were polyclonal (P<0.05) and was associated
with higher parasite density compared to monoclonal
infections (3850 vs. 1480 parasites/uL; P<0.05) (Figure
1). Allele pattern of infection revealed the occurrence of
the 200 bp KI allele in 78.4% of ASM cases seen,
followed by MAD20 180 bp (24.3%), RO33 180 bp
(18.9%) and MAD20 220 bp (8.1%). No parasites were
seen by microscopy for the KI 250 msp1 allele. The
observed disparity in the occurrence of these alleles was
also significant (P<0.05) but density disparity was not
significant (Figure 1). The non-usage of
chemoprophylaxis, LLIN and insecticide spray was also
associated with significant (P<0.05) increase in MOI
(1.1 – 1.3 vs. 1.3 – 1.6) with further selection of RO33
P. falciparum strains due to chemoprophylaxis non-use
(Table 4).
Table 2. General characteristics of the SCA patients: comparison of variables based on
ASM positivity by PCR
Variable Description
Total ASM
positive
ASM
negative P-Value
N = 78 N = 37 N = 41
Mean age Mean + SEM, years 14.2 +
0.6 13.3 + 0.9 15 + 0.8 > 0.05
Age < 10 years n (%) 29 (37.2) 18 (23.1) 11 (14.1) 0.046
Sex Female, n(%) 46 (59) 21 (56.7) 25 (61) 0.71
Residence Rural, n (%) 19 (24.4) 14 (37.8) 5 (12.2) 0.009
Sleep under LLIN the
previous night No, n (%) 43 (55.1) 27 (73) 16 (39) 0.0026
Use of chemoprophylaxis No, n (%) 10 (12.8) 7 (18.9) 3 (7.3) 0.13
Use of insecticide spray the
previous night No, n (%) 36 (46.2) 22 (59.5) 14 (34.1) 0.025
Blood transfusion in the
previous 6 months Yes, n (%) 6 (7.7) 3 (8.1) 3 (7.3) 0.9
Crisis in the previous 6
months Yes, n (%) 10 (12.8) 6 (16.2) 4 (9.7) 0.39
Haemoglobin Mean + SEM, g/dl 8.8 +
0.08 8.4 + 0.1 9.2 + 0.3 < 0.001
Leukocyte count Mean + SEM, Cells x
103/mm3
11.6 +
0.1 11.8 + 0.2 11.4 + 1.0 >0.05
Bolded P-Values are significant.
Table 3. Characteristics and pattern of Plasmodium falciparum parasitaemia according to age among the
asymptomatic SCA patients
P. falciparum infection variables Total < 10 y 11 y and above P-Value
N = 78 N = 29 N = 49
Parasite rate by microscopy, n (%) 17 (21.8) 8 (27.6) 9 (18.4) 0.34
Parasite rate by PCR, n (%) 37 (47.4)* 18 (62.1) 19 (38.8) 0.046
Parasite density, parasites/uL, mean + SD 2238.8 + 464.3 1820 + 558.5 2611.1 + 731.4 0.41
@Parasite density,
parasites/uL, range, n
(%)
< 500 4 (5.1) 3 (10.3) 1 (2.0)
500 – 5000 11 (14.1) 5 (17.2) 6 (12.2) 0.21
> 5000 2 (2.6) 0 (0) 2 (4.1)
MOI 1.43 1.33 1.53 0.01
Msp1 genotrypes 5 3 5 ND
Allele frequency, n(%)
K1 34 (91.8)** 16 (88.8) 18 (84.7)
MAD20 12 (32.4) 6 (33.3) 6 (31.6) 0.24
RO33 7 (18.9) 1 (5.6) 6 (31.6)
@Parasite density was determined only for 18 samples that showed parasitaemia by microscopy. *P<0.05 (Parasite rate by PCR vs. parasite
rate by microscopy); **P<0.05 (K1 alleles vs. other alleles)
I.B. Abiodun, et al.
Acta Medica Iranica, Vol. 54, No. 1 (2016) 49
Table 4. Association between P. falciparum infection variables and malaria prevention practice
indicators among the SCA patients
Parameter Parasitedensity,parasites/uL MOI Msp1 block2 alleles
Mean + SEM K1 MAD20 RO33
Residence
Rural (N = 19) 2850 + 819.7b 1.4 17 (89.5) 6 (31.6) 5 (26.3)
Urban (N = 18) 1820 + 907.6 1.5 13 (72.2) 6 (33.3) 1 (5.6)
Chemoprophylaxis
Yes (n = 29) 1604.6 + 387.9a 1.3 27 (93.1) 10 (34.5) 3 (10.3)
No (n = 8) 3300 + 1038 1.6* 7 (87.5) 2 (25) 4 (50)a
LLIN
Yes (n = 10) 1480 + 445.2a 1.1 9 (90) 2 (20) 0 (0)
No (n = 27) 3630 + 791.1 1.6** 13 (48.1) 5 (18.5) 2 (7.4)
Insecticide spray
Yes (n = 15) 1570 + 365.1a 1.2 13 (86.7) 5 (33.3) 2 (13.3)
No (n = 22) 3580 + 425.7 1.5* 21 (95.5) 7 (31.8) 5 (22.7)
Data are mean + SEM and number (%). The disparity between mean values was evaluated by student’s t-test and proportions by Chi-
square (2) or Fischer Exact test. P-value < 0.05 was considered to be significant. *P<0.01; **P<0.001; aP<0.05
0
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Polyclonal Monoclonal K1(200bp) K1 (250bp) MAD20 (180bp) MAD20(220bp) RO33(150bp)
Infection type
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[14 (37.8%)]*
[23(62.2%)]
[29(78.4%)]*
(5)
[9(24.3%)]
[3(8.1%)]
[7(18.9%)]
Figure 1. Plasmodium falciparum parsitaemia and infection dynamics among the asymptomatic sickle cell araemia patient
Each bar represents mean parasite density of cases with parasitaemia by light microscopy. Figures in parasitaemia represent total number of
microscopic and sub-microscopic asymptomatic infections detected. *P<0.01 (polyclonal vs. monoctonal infections). Student’s t-test; *P<0.05 (allele
infection parasitaemia comparison); *P<0.05 (proportion between alleles and between polyclonal and monoclonal).
Discussion
The steady state of SCA has been described as a state
of sub-clinical chronic inflammation in which affected
patients remains susceptible to infectious diseases,
including malaria (24,25). In a malaria endemic Lagos,
this study found 47.4% of the steady SCA patients study
to harbor 5 distinct clones of P. falciparum strains
asymptomatically when PCR was used for diagnosis and
21.8% ASM rate when light microscopy was employed.
Association of ASM with further reduction in
haemoglobin level and its greater occurrence among
patients of rural residence and who were not sleeping
under LLIN and using insecticide spray. This study
confirms the greater sensitivity of PCR over microscopy
in diagnosing ASM and reveals a higher rate of ASM
Plasmodium falciparum msp1 polymorphism
50 Acta Medica Iranica, Vol. 54, No. 1 (2016)
among Nigerian patients with SCA than previously
reported. The observed 21.8% ASM rate among our
SCA patients is similar to the 25.6% rate reported by
Ojuroungbe et al., (15) among children aged 4 – 15
years and 24% reported by Kotila et al., (17) among
asymptomatic adults, though both investigators did not
provide information on parasite diversity associated with
the ASM cases seen. Although in the work of Ojurougbe
et al., (15), haemoglobin S status of the infected children
was provided, African children heterozygous for the
haemoglobin S gene has been shown to habor P.
falciparum strains asymptomatically at an equal
frequency to children homozygous for the sickle gene
(26). However, our finding implies that 25.9% of more
cases of ASM will be detected if PCR instead of
microscopy is used for diagnosing ASM, giving a better
picture of ASM burden in SCA population. This is also
applicable to non-SCA patients with ASM irrespective
of age in Nigeria. Therefore, in the context of malaria
elimination as recently adopted by the national malaria
control programme, using PCR for malaria diagnosis
would enable identification and treatment of a higher
number of people, including SCA patients. This would
provide tremendous benefits in reducing transmission to
a significant level and enhance the feasibility of
eliminating malaria from Nigeria. The relevance of PCR
in ASM diagnosis has also been demonstrated by
Ntounmi et al., (26), who reported P falciparum ASM
prevalence rate of 61% among Gabonese children with
different haemoglobin variants. In terms of species
composition, only P. falciparum trophozoites were
detected in this study. This is contrary to the findings of
Danquah et al., (27).
The workers detected P. falciparum, P malariae and
P. ovale by PCR at 74.5%, 9.7% and 5.5% in 56% of
2108 asymptomatic Ghanaian children. This disparity
can be attributed to the differences in sample size and
malaria transmission patterns of the study areas. In this
study, sampling was done between the end of dry season
and in the middle of rainy when the transmission is not
at its peak in Lagos. Studies conducted during this
period have consistently demonstrated the predominance
of P. falciparum (28, 29). However, in this study,
gametocytes were not seen by microscopy to inform the
infectiousness of SCA subjects who harbored parasites.
This may be because the molecular approach of
detecting sub-microscopic gametocytes (30) was not
adopted in this study. Anemia has been reported as a
common consequence of ASM, our observed lower Hb
among parasite carriers compared to non-carriers
alluded to this fact. Our finding seems to align with
work of Olaniyi and Arinola (25) who reported elevation
in C-reactive protein but the reduction in transferrin and
haptoglobin levels in steady state SCA patients with P.
falciparum parasitaemia, suggesting enhanced
inflammatory response and decreased erythropoietic
activity. In this study, the occurrence of ASM was
associated with non-use of LLIN and insecticide spray.
Several studies have identified the non-use of these
preventive measures as risk factors for clinical malaria
in Nigerian patients like other malaria endemic areas
outside Nigeria. Therefore, our finding indicates that
risk factors associated with clinical malaria also promote
the occurrence of ASM among SCA patients in the
country. Although evidence from the clinical trial has
revealed preventive benefits of chemoprophylaxis in
reducing the frequency of crisis and blood transfusion
and conferring protection against clinical malaria,
concerns are mounting regarding protection against
asymptomatic malaria (31). This study also did not find
an association between the use of chemoprophylaxis
with pyrimethamine or proguanil and the occurrence of
ASM in our SCA cohort and thus agrees with the
finding of Kotila et al., (17). Meanwhile, mutations
occurring in P. falciparum dihydrofolate reductase and
cytochrome oxidase genes have been linked to the
therapeutic failure of pyrimethamine and sulphadoxine-
pyrimethamine in several epidemiological studies,
including those done in Nigeria (32-34). These markers
were not investigated in this study.
However, for the first time and similar to other
malaria prevention measures studied, not using
chemoprophylaxis was found to significantly correlate
with a higher multiplicity of infection (MOI) coupled
with a specific selection of RO33 allele among Nigerian
SCA patients with ASM. In this study, the observed
MOI of 1.43 among our ASM cases that was
characterized by 1-3 distinct parasite infection per
sample implies the low complexity of infection. This can
be explained by the higher rate of monoclonal infections
seen in 62.2% of ASM cases detected by PCR. Our MOI
of 1.43 is similar to the 1.1 reported by Olaseinde et al.,
(35) for malaria cases detected recently in Ogun states,
south west Nigeria but lower than the MOI range of 3 –
4 reported by Happi et al., (36) prior to treatment policy
change of 2005 in Nigeria. Whether or not the MOI
observed in this study reflects a changing epidemiology
of malaria in Lagos or other factors such asymptomatic
manifestation of malaria and the higher pyrogenic
threshold for fever during the rainy season will require
an investigation of malaria transmission and mosquito
infectivity or in-breeding in the study area. MOI has also
I.B. Abiodun, et al.
Acta Medica Iranica, Vol. 54, No. 1 (2016) 51
been found useful in understanding parasite diversity
and mechanisms of anti-malarial immunity, both of
which have values in planning control intervention and
designing malaria vaccines. In this study, block 2
domain of msp1 was genotyped being the domain under
the strongest diversifying selection and involving apart
from K1 and MAD20, an additional RO33 allele family
absent in the other 16 domains of the erythrocyte
invasion protein coding gene (37). In this study, 5
distinct msp1 genotypes, comprising 2 K1 alleles of
sizes 200 bp and 250 bp, 2MAD20 alleles of sizes 180
bp and 220 bp and 1 RO33 allele of size 150 bp. Despite
the similar low complexity of infection reported by
Olaseinde et al., (35), 8 distinct msp1 genotypes,
comprising 4K1, 3 MAD20 and 1RO33 were found.
This again suggests that the selection of msp1 alleles for
P. falciparum infections vary between geographical
areas and may be due to the difference in malaria
transmission pattern and impact of control interventions.
However, this study reveals a consistency in the
predominance and presence of several polymorphisms
of the K1 alleles among field isolates of P falciparum in
south West Nigeria (35 – 37). The specific selection of
the RO33 allele due to non-use of chemoprophylaxis
will require further studies for a meaningful
interpretation. This is because results have been
inconsistent, regarding the influence of the RO33 allele
on clinical malaria susceptibility. In a Camerounian
study, RO33/K1 co-infection was found to be strongly
associated with fever and anemia irrespective of age in
infected patients (38).
On the contrary, al-Yaman et al., (39) found
asymptomatic infection due to the RO33 allele to be
associated with reduced risk of clinical infection in a
highly endemic area of Papua New Guinea. However,
contrary to the significant allele-dependent variations in
parasitaemia observed by these workers (38,39), we
found an in-significant difference in parasitaemia
elicited by parasite population belonging to different
alleles (singly or in combination) among our SCA
cohort with ASM. This disparity may be attributed to the
differences in sample size, geographical location and
study designs between the two studies.
However, it is important to note that this study has
some limitations that will be addressed in future studies.
First is the need for quantitative PCR to enable
quantitation of sub-microscopic parasitaemia and
molecular approach for detecting and quantitating sub-
microscopic gametocytaemia to enable conclusion on
the infectiousness of steady state SCA patients with
ASM in Lagos and other regions of the country. Lastly,
the small sample size and cross-sectional design of this
study prevent adjustment of variables such as rural
residence and sex for multivariate analysis to reveal
independent determinants of ASM in the current study
cohort. We were also unable to monitor the evolution of
the observed parasite diversity as the course of infection
becomes symptomatic. Such information has value in
malaria vaccine design. Therefore, longitudinal and
large sample size studies in Lagos and other regions of
the country is sought.
Nevertheless, despite the above limitations, this
study has revealed commonality in risk factors
associated with clinical and asymptomatic malaria in the
country and has thus highlighted the need to scale up
behavioral change communication intervention in the
country in the context of scaling up for impact (SUFI)
by targeting universal access to information, education
and communication (IEC) materials in Nigeria as a
whole.
In conclusion, the findings from this study indicate
that ASM infection appears to be more common than
previously reported among Nigerian SCA patients with
infection characterized by low complexity, K1 allele
dominance and potential selection of the RO33 allele in
the absence of chemoprophylaxis.
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