Malaria Elimination in Ethiopia: Relevance of advanced Molecular/Diagnostic Tools in Epidemiological Studies
No Thumbnail Available
Date
2019-11
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Addis Abeba University
Abstract
Background:
Malaria is still among the major diseases of public health importance in Ethiopia. Ethiopia presents
a diversified ecological situation, resulting in a highly variable eco-epidemiology of malaria.
Following the scale-up of antimalarial interventions in the past two decades, malaria burden has
significantly declined leading to the National Malaria Control Program (NMCP) in Ethiopia to reembark
on
a strategy for step by step nationwide malaria elimination. Learning from the failed
malaria elimination endeavors of the 1960s, achieving such an ambitious target, given the
complicated eco-epidemiology of malaria in Ethiopia may require several inputs and evidences.
Measures of malaria burden and transmission dynamics using conventional diagnostic methods
[microscopy and Rapid Diagnostic tests (RDTs)] may be incomplete, particularly, in low and
seasonal transmission settings, where few infections are detected. Unlike most parts of Africa, P.
falciparum and P. vivax co-exist in Ethiopia. Malaria elimination requires determining the actual
burden, distribution as well as detection and cleaning of all forms of malaria infection. The
knowledge gap in the occurrence, prevalence and distribution of Glucose-6-Phosphate
Dehydrogenase (G6PD) deficiency has been a limiting factor for radical cure of relapsing malaria
and transmission interruption. The group of drugs within 8-aminoquinolines, such as Primaquine
and Tafenaquine, are the only available treatment of relapsing malaria. Eight-aminoquinolines can
induce severe hemolysis in G6PD deficient individuals. The study used advanced tools to
investigate the epidemiological risk factors relevant for malaria elimination.
Objective:
This study used serology and molecular methods to describe the malaria (Plasmodium spp) burden
and distribution, as well as to determine G6PD deficiency prevalence and allelic types, in order to
produce reliable evidence for malaria elimination in Ethiopia.
Methodology:
Dried blood spot (DBS) samples collected in 2011 and 2015 as part of the national household
Malaria Indicator Surveys (MIS) were used. The Ethiopian Malaria Indicator Surveys (EMISs) utilized a multi-stage cross sectional surveys representating the various malaria epidemiological
settings in Ethiopia. EMIS-2015 samples were investigated using bead-based multiplex assays for
IgG antibodies for six Plasmodium spp antigens: four human malaria species-specific merozoite
surface protein 1 19kD antigens (MSP-1) and apical membrane antigen 1 (AMA-1) for P.
falciparum and P. vivax. Seroprevalence was estimated by age group, elevation, and administrative
regions. Seroconversion rates were estimated using a reversible catalytic model fitted with
maximum likelihood method. Sub samples of EMIS-2015 from three administrative regions
(Amhara, Tigray and Benishangul Gumuz regions) were screened by Microscopy, RDTs and
nested Polymerase Chain Reaction (nPCR) for malaria parasites and results were compared to
determine prevalence of subpatent infections. A randomly selected subset of samples from EMIS2011
were genotyped by polymerase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) technique and three common G6PD genotype variants: G6PD*A (A376G),
G6PD*A- (G202A) and Mediterranean (C563T) were investigated.
Data were analysed using Stata 13 (College Station, USA). Serology data were generated from a
multiplex instrument as Mean Floruescent Intensity minus background (MFI-bg). To dichotomize
seropositivity, log-transformed MFI-bg values were fitted to a two-component Finite Mixture
Model (FMM) by the FMM procedure with normal distribution and maximum likelihood
estimation outputs. A seropositivity cutoff value was determined by the mean MFI-bg value of the
first (assumed seronegative) component plus three standard deviations. Plasmodium falciparum
and P. vivax seropositivity were defined as being positive for either or both MSP-1 and AMA-1
antigens. Species specific MSP-1 antigens were used for P. malariae and P. ovale seropositivity.
EMISs sampling weights were used to ensure the representativeness of the samples tested to the
study population. Adjustments were made by region, elevation, and age group. Linear and multiple
logistic regression models with 95% confidence intervals (CI) were employed to determine the
association of risk factors with Plasmodium spp infection. Differences in distributions were
evaluated using Chi square (χ2) test with P value < 0.05 considered significant. Spatial analysis
and geographical mapping were done using QGIS and ArcGIS softwares.
Main Findings:
National seroprevalence for antibodies to P. falciparum was 32.1% (95% CI: 29.8-34.4) and 25.0%
(95% CI: 22.7-27.3) for P. vivax. Estimated seroprevalence for P. malariae and P. ovale were
8.6% (95% CI: 7.6-9.7) and 3.1% (95% CI: 2.5-3.8), respectively. Seroprevalence estimates were
significantly higher at lower elevations (<2000 m) compared to higher elevations (2000-2500 m),
for P. falciparum [Adjusted Odds Ratio (aOR) 4.4 (95% CI: 2.7 -7.0), p<0.01].], although evidence
was weak for P. vivax [aOR 1.5 (95% CI: 0.9-2.3), p>0.05]]. Among administrative regions, P.
falciparum seroprevalence ranged from 11.0% (95% CI: 8.8-13.7) in the Somali to 65.0% (95%
CI: 58.0-71.4) in Gambela Region. Seroprevalence for P. vivax ranged from 4.0% (95% CI: 2.66.2)
in the Somali to 36.7% (95% CI: 30.0-44.1) in Amhara Region. Models fitted to measure
seroconversion rates showed variation nationally and by elevation, region, antigen type and within
species.
Malaria DNA screening using nPCR from three regions (Amhara, Tigray and Benishangul Gumuz)
detected 3.3% (95% CI: 2.7-4.1) positive cases. P. falciparum accounted for 3.1% (95% CI: .53.8),
P.
vivax
0.4%
(95%
CI:
0.2-0.7),
mixed
(P.
falciparum
and
P.
vivax)
0.1%
(95%
CI:
0.0-0.4),
mixed
(P.
falciparum
and
P.
malariae)
0.1%
(95%
CI:
0.0-0.3).
nPCR
detected
nearly
three-fold
more
positives
compared
to
microscopy.
Sixty
one
percent
of
the
nPCR
positive
cases
were
from
Benishangul
Gumuz
Region.
The
G6PD genotyping
study
showed
the
more
severe
G6PD deficiency
allelic
types,
G202A
(A-)
and
C563T (Mediterranean), to be absent in the current study. A national prevalence of 8.1%
G6PD*A (A376G) mutation variants was observed with regional variation, with highest
prevalence observed in Tigray Region (13.7%) to none in Harari. Of the G6PD*A (A376G), 31%
were hemizygous males and 62.1% and 6.8% were heterozygous and homozygous females,
respectively.
Conclusion and Recommendations:
The current study used multiplex serology and serological markers to report the malaria exposure
burden and transmission intensity of the four human malaria species. The study documented
heterogeneity in malaria burden and transmission over different elevations, administrative regions,
and age groups. Malaria exposure was by far higher compared to the active infection reported by
microscopy and RDTs. P. falciparum sero prevalence increases with decreasing elevation, whereas
P. vivax slightly increases with elevation in the study setting up to 2,500 m; showing P. vivax is
more prevalent in highlands compared to P. falciparum. Variation was observed on the spatial
distribution and dynamics of transmission over the regions. The northwestern part of the country
is carrying the largest burden of malaria compared to the east. Among the regions, Gambela and
Benishangul-Gumuz had the highest burden of malaria transmission. The current study
documented the presence of P. malariae and P. ovale in all administrative regions. Given that P.
ovale possesses a hypnozoite stage, its control and elimination requires programmatic attention.
The seroprevalence results may be used as baseline data for the future malaria elimination efforts
and may help the NMPCP in tailoring intervention approaches.
The current study documented a considerable proportion of subpatent Plasmodium spp infections
undetected by microscopy. Such subpatent infections are potentially infective to mosquitoes,
contributing for malaria transmission in addition to their debilitating chronic effect on the
individuals affected. Efficient malaria elimination efforts have to address the impact of subpatent
infection on transmission and health.
In this study, the more severe variants G6PD *A- (G202A) and Mediterranean (C563T) mutations
were not observed. The G6PD *A (A376G) mutation observed is a mild variant resulting in close
to normal (85%) enzyme activity of a non-deficient person, without significant clinical
manifestations of G6PD deficiency related hemolysis. This study investigated three of the most
important and potentially expected mutation types in the study area among the hundreds of known
G6PD variants worldwide. Although the study cannot definitively conclude the absence of any
clinically important G6PD deficiency, it suggests a low risk of hemolysis, and confirms the utility
of the recently adopted Primaquine treatment without prior G6PD testing. The risks and benefits
of Primaquine radical cure without G6PD testing may need to be further assessed in Ethiopia as
the P. vivax and P. ovale case management is evolving and may incorporate higher dose and shorter
course regimens of Primaquine and Tafenoquine.
In summary, the current work used advanced serological and molecular diagnostic tools to produce
evidence to the epidemiological factors that may be relevant for malaria elimination. It also
emphasized the need for assessing and introducing advanced diagnostic techniques, such as PCR
and multiplex serology to provide releable evidences required towards malaria elimination in
Ethiopia.
Description
Keywords
Malaria Elimination, G6PD deficiency, Asymptomatic Infection, Subpatent infection, Seroprevalence, Multiplex serology, Microscopy, RDT, nPCR, Ethiopia