Developing, testing and implementing novel molecular diagnostic tests and procedures for malaria at the individual and population level in East and West Africa

Malaria is a global public health issue responsible for an estimated 219 million cases and
435’000 deaths in 2017. Although malaria control interventions have led to a significant decrease
in mortality over the past decade, no further progress in reducing global malaria burden
was achieved since 2016. Novel malaria control approaches, such as highly effective
vaccines and improved diagnostic tools are needed to prevent a resurgence of malaria as has
been seen in the 1970s.
The overall goal of this PhD thesis is to contribute to the development, validation and implementation
of novel molecular diagnostic tools in malaria endemic countries targeting all human
infective malaria species. This thesis was conducted within the framework of six clinical
trials evaluating the PfSPZ Vaccine, an experimental malaria vaccine based on the immunization
with radiation-attenuated whole, purified, cryopreserved, metabolically active sporozoites
in Tanzania and Equatorial Guinea. We used controlled human malaria infection (CHMI)
to assess vaccine efficacy in these cohorts. Compared to large field studies, CHMI studies
allow to test various vaccine regimen and dosing in smaller, well defined cohorts in a short
time period.
In part one, we aimed to closely monitor malaria parasites at the individual level by establishing
a robust laboratory qPCR-platform for molecular monitoring of asexual blood stage parasitaemia.
This platform was used to evaluate the efficacy of PfSPZ Vaccine by homologous
CHMI in Tanzanian and Equatorial Guinean adults. Part one was structured around four
manuscripts:
Manuscript 1-3: Safety, immunogenicity and efficacy of attenuated whole sporozoite
vaccines in Tanzanian and Equatorial Guinean volunteers.
In clinical trials conducted in malaria pre-exposed volunteers from Tanzania and Equatorial
Guinea, the PfSPZ Vaccine was found to be well tolerated and safe, even in high-risk populations
such as infants and HIV+ volunteers. The use of CHMI in malaria pre-exposed adults
has been proven to be highly effective. These trials revealed that the degree of immune response
and high level of protection seen in previous studies in malaria-naïve volunteers, was
not reproducible to the same level in Tanzanian and Equato-Guinean populations.
Manuscript 4: Controlled Human Malaria Infections in Tanzania and Equatorial Guinea:
The impact of malaria pre-exposure on diagnosis and multiplication rate of P. falciparum
parasites
We used the parasite multiplication rate (PMR), the fold change in number of parasites in
peripheral blood over one life cycle, to characterize the impact of malaria pre-exposure on the
outcome of CHMI. Compared to malaria-naïve German volunteers, the parasite’s ability to
replicate was significantly reduced in Tanzanian and Equato-Guinean volunteers when challenged
with the identical dose, strain and route of cryopreserved, purified P. falciparum sporozoites.
We used two different approaches to demonstrate the impact of malaria preexposure
on the multiplication rate of P. falciparum parasites. First, we showed that recently
drug-cleared parasitaemia from an ongoing P. falciparum field infection or P. falciparum and
P. malariae co-infection leads to a significantly reduced PMR. Second, PMR is significantly
reduced after two consecutive homologous CHMI conducted in the same volunteer at 7
months interval. This data indicates the rapid acquisition or recall of parasite growth reducing
immune effector mechanisms limiting the growth of asexual blood stages in vivo.
In part two, we aimed to adapt and extend molecular diagnostic tools for improvement of
malaria surveillance at the population level by developing novel high-throughput, fielddeployable
and highly sensitive tools and approaches. Part two was structured around five
manuscripts:
Manuscript 5: A multiplex qPCR approach for detection of pfhrp2 and pfhrp3 gene deletions
in multiple strain infections of Plasmodium falciparum
In response to the emerging threat of P. falciparum strains lacking pfhrp2 and/or pfhrp3
genes and therefore evading detection by PfHRP2-based RDTs, we developed a qPCR-based
assay well suited for high-throughput identification of pfhrp2 and pfhrp3 gene deletions in P.
falciparum isolates. The ability to detect “masked” pfhrp2/3 deletions is probably the most
interesting feature of our approach, because it will allow to study the epidemiology of
pfhrp2/3 deletions in malaria endemic regions where a high proportion of the population carries
multiple strain P. falciparum co-infections.
Manuscript 6: High Throughput Extraction and Analysis of Nucleic Acids from Rapid
Diagnostic Tests for Molecular Surveillance of Malaria
This manuscript describes our attempt to improve extraction of nucleic acids from used malaria
RDTs. We used an approach which enabled us to extract total nucleic acids, including
DNA and RNA from these RDT strips. This approach increased the overall sensitivity for the
detection of Plasmodium spp. parasites in the small volume of blood which is available on an
RDT stored at room temperature. We used samples collected during CHMI as a platform to
evaluate and compare the ability of our protocol to detect and quantify P. falciparum parasites
with microscopy and qPCR.
Manuscript 7: ELIMU-MDx: A Web-Based, Open-Source Platform for Storage, Management
and Analysis of Diagnostic qPCR Data
We developed a web-based and open-source software for storage, management and analysis
of diagnostic qPCR data. In response to the vast amount of qPCR data generated during a
series of CHMIs and other epidemiological studies conducted in Tanzania and Equatorial
Guinea, we decided to design and build this platform which facilities the larger scale analysis
and interpretation of diagnostic qPCR data.
Manuscript 8: Molecular monitoring of the diversity of human pathogenic malaria species
in blood donations on Bioko Island, Equatorial Guinea
In collaboration with the central blood bank in Malabo, Equatorial Guinea we analysed 200
blood donations for the presence of Plasmodium spp. parasites by a novel, multiplex qPCR
monitoring all human infective malaria species in a single reaction. We found that more than
one quarter of the blood donations contained malaria parasites and that 75% of P. falciparum
and 100% of P. malariae and P. ovale spp. infections were missed by routinely performed
RDT and microscopy.
Manuscript 9: Two cases of long-lasting, sub-microscopic Plasmodium malariae infections
in adults from Coastal Tanzania
In this report we describe two cases of P. malariae infections that were identified during a
study evaluating the safety, tolerability and efficacy of the PfSPZ Vaccine in Bagamoyo,
Tanzania. Since these two adult participants were enrolled into a clinical trial, we were provided
with a unique opportunity to study clinical manifestations of P. malariae over a follow
up period of four months.