Potential application of the haematology analyser XN-31 prototype for field malaria surveillance in Kenya.

BACKGROUND: Simple and accurate diagnosis is a key component of malaria control programmes. Microscopy is the current gold standard, however it requires extensive training and the results largely rely on the skill of the microscopists. Malaria rapid diagnostic tests (RDT) can be performed with minimal training and offer timely diagnosis, but results are not quantitative. Moreover, some Plasmodium falciparum parasites have evolved and can no longer be detected by existing RDT. Developed by the Sysmex Corporation, the XN-31 prototype (XN-31p) is an automated haematology analyser capable of detecting Plasmodium-infected erythrocytes and providing species differentiation and stage specific parasite counts in venous blood samples without any preparation in approximately one minute. However, factors such as stable electricity supply in a temperature-controlled room, cost of the instrument and its initial set-up, and need for proprietary reagents limit the utility of the XN-31p across rural settings. To overcome some of these limitations, a hub and spoke diagnosis model was designed, in which peripheral health facilities were linked to a central hospital where detection of Plasmodium infections by the XN-31p would take place. To explore the feasibility of this concept, the applicability of capillary blood samples with the XN-31p was evaluated with respect to the effect of sample storage time and temperature on the stability of results. METHODS: Paired capillary and venous blood samples were collected from 169 malaria-suspected outpatients in Homa Bay County Referral Hospital, Kenya. Malaria infections were diagnosed with the XN-31p, microscopy, RDT, and PCR. Capillary blood samples were remeasured on the XN-31p after 24 h of storage at either room (15-25 °C) or chilled temperatures (2-8 °C). RESULTS: Identical results in malaria diagnosis were observed between venous and capillary blood samples processed immediately after collection with the XN-31p. Relative to PCR, the sensitivity and specificity of the XN-31p with capillary blood samples were 0.857 and 1.000, respectively. Short-term storage of capillary blood samples at chilled temperatures had no adverse impact on parasitaemia and complete blood counts (CBC) measured by the XN-31p. CONCLUSION: These results demonstrate the potential of the XN-31p to improve routine malaria diagnosis across remote settings using a hub and spoke model.

Improvement of malaria diagnostic system based on acridine orange staining.

BACKGROUND: Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. METHODS: The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. RESULTS: Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. CONCLUSIONS: Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Evaluation of a financial incentive intervention on malaria prevalence among the residents in Lake Victoria basin, Kenya: study protocol for a cluster-randomized controlled trial.

BACKGROUND: In the Lake Victoria basin of western Kenya, malaria remains highly endemic despite high coverage of interventions such as mass distribution of long-lasting insecticidal nets (LLIN), indoor residual spraying (IRS) programs, and improvement of availability and accessibility of rapid diagnostic tests (RDT) and artemisinin-based combination therapy (ACT) at community healthcare facilities. We hypothesize that one major cause of the residual transmission is the lack of motivation among residents for malaria prevention and early treatment. METHODS: This study will aim to develop a demand-side policy tool to encourage local residents' active malaria prevention and early treatment-seeking behaviors. We examine the causal impact of a financial incentive intervention complemented with malaria education to residents in malaria-prone areas. A cluster-randomized controlled trial is designed to assess the effect of the financial incentive intervention on reducing malaria prevalence in residents of Suba South in Homa Bay County, Kenya. The intervention includes two components. The first component is the introduction of a financial incentive scheme tied to negative RDT results for malaria infection among the target population. This study is an attempt to promote behavioral changes in the residents by providing them with monetary incentives. The project has two different forms of incentive schemes. One is a conditional cash transfer (CCT) that offers a small reward (200 Ksh) for non-infected subjects during the follow-up survey, and the other is a lottery incentive scheme (LIS) that gives a lottery with a 10% chance of winning a large reward (2000 Ksh) instead of the small reward. The second component is a knowledge enhancement with animated tablet-based malaria educational material (EDU) developed by the research team. It complements the incentive scheme by providing the appropriate knowledge to the residents for malaria elimination. We evaluate the intervention's impact on the residents' malaria prevalence using a cluster-randomized control trial. DISCUSSION: A policy tool to encourage active malaria prevention and early treatment to residents in Suba South, examined in this trial, may benefit other malaria-endemic counties and be incorporated as part of Kenya's national malaria elimination strategy. TRIAL REGISTRATION: UMIN000047728. Registered on 29th July 2022.

Tracing the origins of Plasmodium vivax resurgence after malaria elimination on Aneityum Island in Vanuatu.

BACKGROUND: Five years after successful malaria elimination, Aneityum Island in Vanuatu experienced an outbreak of Plasmodium vivax of unknown origin in 2002. Epidemiological investigations revealed several potential sources of P. vivax. We aimed to identify the genetic origin of P. vivax responsible for the resurgence. METHODS: Five P. vivax microsatellite markers were genotyped using DNA extracted from archived blood samples. A total of 69 samples from four P. vivax populations was included: 29 from the outbreak in 2002, seven from Aneityum in 1999 and 2000, 18 from visitors to Aneityum in 2000, and 15 from nearby Tanna Island in 2002. A neighbour-joining phylogenetic tree was constructed to elucidate the relationships among P. vivax isolates. STRUCTURE and principal component analysis were used to assess patterns of genetic structure. RESULTS: Here we show distinct genetic origins of P. vivax during the outbreak on Aneityum. While the origin of most P. vivax lineages found during the outbreak remains unidentified, limited genetic diversity among these lineages is consistent with a rapid expansion from a recent common ancestor. Contemporaneous P. vivax from neighboring Tanna and potential relapse of P. vivax acquired from other islands in 1999 and 2000 are also identified as minor contributors to the outbreak. CONCLUSIONS: Multiple reintroductions of P. vivax after elimination highlight the high receptivity and vulnerability to malaria resurgence in island settings of Vanuatu, despite robust surveillance and high community compliance to control measures.

Plasmodium vivax is one of several parasite species that cause malaria. On Aneityum Island in Vanuatu, malaria had been eliminated in 1997, but an outbreak was reported in 2002 despite protective measures still being in place. Here, we analysed DNA of parasites from the outbreak to understand its origin, since parasites of different origins will have slight differences in their DNA. Most parasites had similar DNA suggesting they had a recent shared common ancestor whose origin remains unidentified. From this analysis we were also able to find a minority of parasites that likely came from Tanna in 2002, while another small group of parasites may have originated from parasites imported to Aneityum in 1999 or 2000. This illustrates the difficulty of maintaining a malaria-free status in resource-limited areas and the threat of imported malaria to elimination efforts.

Drug resistance profiling of asymptomatic and low-density Plasmodium falciparum malaria infections on Ngodhe island, Kenya, using custom dual-indexing next-generation sequencing.

Malaria control initiatives require rapid and reliable methods for the detection and monitoring of molecular markers associated with antimalarial drug resistance in Plasmodium falciparum parasites. Ngodhe island, Kenya, presents a unique malaria profile, with lower P. falciparum incidence rates than the surrounding region, and a high proportion of sub-microscopic and low-density infections. Here, using custom dual-indexing and Illumina next generation sequencing, we generate resistance profiles on seventy asymptomatic and low-density P. falciparum infections from a mass drug administration program implemented on Ngodhe island between 2015 and 2016. Our assay encompasses established molecular markers on the Pfcrt, Pfmdr1, Pfdhps, Pfdhfr, and Pfk13 genes. Resistance markers for sulfadoxine-pyrimethamine were identified at high frequencies, including a quintuple mutant haplotype (Pfdhfr/Pfdhps: N51I, C59R, S108N/A437G, K540E) identified in 62.2% of isolates. The Pfdhps K540E biomarker, used to inform decision making for intermittent preventative treatment in pregnancy, was identified in 79.2% of isolates. Several variants on Pfmdr1, associated with reduced susceptibility to quinolones and lumefantrine, were also identified (Y184F 47.1%; D1246Y 16.0%; N86 98%). Overall, we have presented a low-cost and extendable approach that can provide timely genetic profiles to inform clinical and surveillance activities, especially in settings with abundant low-density infections, seeking malaria elimination.

Evaluation of the protective efficacy of Olyset®Plus ceiling net on reducing malaria prevalence in children in Lake Victoria Basin, Kenya: study protocol for a cluster-randomized controlled trial.

BACKGROUND: In the Lake Victoria Basin of western Kenya, malaria remains highly endemic despite high coverage of interventions such as insecticide-impregnated long-lasting insecticidal nets (LLIN). The malaria-protective effect of LLINs is hampered by insecticide resistance in Anopheles vectors and its repurposing by the community. Ceiling nets and LLIN with synergist piperonyl butoxide (PBO-LLIN) are novel tools that can overcome the problems of behavioral variation of net use and metabolic resistance to insecticide, respectively. The two have been shown to reduce malaria prevalence when used independently. Integration of these two tools (i.e., ceiling nets made with PBO-LLIN or Olyset®Plus ceiling nets) appears promising in further reducing the malaria burden. METHODS: A cluster-randomized controlled trial is designed to assess the effect of Olyset®Plus ceiling nets on reducing malaria prevalence in children on Mfangano Island in Homa Bay County, where malaria transmission is moderate. Olyset®Plus ceiling nets will be installed in 1315 residential structures. Malaria parasitological, entomological, and serological indicators will be measured for 12 months to compare the effectiveness of this new intervention against conventional LLIN in the control arm. DISCUSSION: Wider adoption of Olyset®Plus ceiling nets to complement existing interventions may benefit other malaria-endemic counties and be incorporated as part of Kenya's national malaria elimination strategy. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000045079. Registered on 4 August 2021.

Interaction Between the Complement System and Infectious Agents - A Potential Mechanistic Link to Neurodegeneration and Dementia.

As part of the innate immune system, complement plays a critical role in the elimination of pathogens and mobilization of cellular immune responses. In the central nervous system (CNS), many complement proteins are locally produced and regulate nervous system development and physiological processes such as neural plasticity. However, aberrant complement activation has been implicated in neurodegeneration, including Alzheimer's disease. There is a growing list of pathogens that have been shown to interact with the complement system in the brain but the short- and long-term consequences of infection-induced complement activation for neuronal functioning are largely elusive. Available evidence suggests that the infection-induced complement activation could be protective or harmful, depending on the context. Here we summarize how various infectious agents, including bacteria (e.g., Streptococcus spp.), viruses (e.g., HIV and measles virus), fungi (e.g., Candida spp.), parasites (e.g., Toxoplasma gondii and Plasmodium spp.), and prion proteins activate and manipulate the complement system in the CNS. We also discuss the potential mechanisms by which the interaction between the infectious agents and the complement system can play a role in neurodegeneration and dementia.

Characterizing the genomic variation and population dynamics of Plasmodium falciparum malaria parasites in and around Lake Victoria, Kenya.

Characterising the genomic variation and population dynamics of Plasmodium falciparum parasites in high transmission regions of Sub-Saharan Africa is crucial to the long-term efficacy of regional malaria elimination campaigns and eradication. Whole-genome sequencing (WGS) technologies can contribute towards understanding the epidemiology and structural variation landscape of P. falciparum populations, including those within the Lake Victoria basin, a region of intense transmission. Here we provide a baseline assessment of the genomic diversity of P. falciparum isolates in the Lake region of Kenya, which has sparse genetic data. Lake region isolates are placed within the context of African-wide populations using Illumina WGS data and population genomic analyses. Our analysis revealed that P. falciparum isolates from Lake Victoria form a cluster within the East African parasite population. These isolates also appear to have distinct ancestral origins, containing genome-wide signatures from both Central and East African lineages. Known drug resistance biomarkers were observed at similar frequencies to those of East African parasite populations, including the S160N/T mutation in the pfap2mu gene, which has been associated with delayed clearance by artemisinin-based combination therapy. Overall, our work provides a first assessment of P. falciparum genetic diversity within the Lake Victoria basin, a region targeting malaria elimination.

Plasmodium falciparum SURFIN4.1 forms an intermediate complex with PTEX components and Pf113 during export to the red blood cell.

Plasmodium falciparum malaria parasites export several hundred proteins to the cytoplasm of infected red blood cells (RBCs) to modify the cell environment suitable for their growth. A Plasmodium translocon of exported proteins (PTEX) is necessary for both soluble and integral membrane proteins to cross the parasitophorous vacuole (PV) membrane surrounding the parasite inside the RBC. However, the molecular composition of the translocation complex for integral membrane proteins is not fully characterized, especially at the parasite plasma membrane. To examine the translocation complex, here we used mini-SURFIN4.1, consisting of a short N-terminal region, a transmembrane region, and a cytoplasmic region of an exported integral membrane protein SURFIN4.1. We found that mini-SURFIN4.1 forms a translocation intermediate complex with core PTEX components, EXP2, HSP101, and PTEX150. We also found that several proteins are exposed to the PV space, including Pf113, an uncharacterized PTEX-associated protein. We determined that Pf113 localizes in dense granules at the merozoite stage and on the parasite periphery after RBC invasion. Using an inducible translocon-clogged mini-SURFIN4.1, we found that a stable translocation intermediate complex forms at the parasite plasma membrane and contains EXP2 and a processed form of Pf113. These results suggest a potential role of Pf113 for the translocation step of mini-SURFIN4.1, providing further insights into the translocation mechanisms for parasite integral membrane proteins.

Multiple charged amino acids of Plasmodium falciparum SURFIN4.1 N-terminal region are important for efficient export to the red blood cell.

Plasmodium falciparum, an obligate intracellular protozoan parasite which causes the severe form of human malaria, exports numerous proteins to the infected red blood cell that are important for its survival and of severe pathological effect to its host. These proteins and their export mechanisms are candidates for drug and vaccine development, and among them is the Plasmodium SURFIN family of proteins. Previously we showed that the N-terminal region along with the sequence surrounding the transmembrane domain of SURFIN4.1 is essential for its export to Maurer's clefts in the red blood cell cytoplasm. We proposed that this region is recognized by a machinery responsible for protein translocation across the parasitophorous vacuole membrane surrounding the parasite. To understand the export mechanism further, we utilized a fluorescent protein-tagged mini-SURFIN4.1 consisting of the minimum essential components for export. Alanine scanning of all charged amino acids within the N-terminal region revealed that replacement of 3 glutamic acid and 2 lysine residues significantly impairs the export efficiency of this protein across the parasitophorous vacuole membrane. In addition, N-terminally Myc-tagged mini-SURFIN4.1 and mini-SURFIN4.2 with similar architectures were detected with anti-Myc antibody at Maurer's clefts, indicating that elements required for export to Maurer's clefts are conserved between SURFIN4.1 and SURFIN4.2, and that N-terminal sequences of these SURFIN members are not cleaved during export. Our results implicate a conserved nature of SURFIN export to the red blood cell, particularly an important role of multiple glutamic acid and lysine residues in the SURFIN N-terminal region.

Malaria resurgence after significant reduction by mass drug administration on Ngodhe Island, Kenya.

Although WHO recommends mass drug administration (MDA) for malaria elimination, further evidence is required for understanding the obstacles for the optimum implementation of MDA. Just before the long rain in 2016, two rounds of MDA with artemisinin/piperaquine (Artequick) and low-dose primaquine were conducted with a 35-day interval for the entire population of Ngodhe Island (~500 inhabitants) in Lake Victoria, Kenya, which is surrounded by areas with moderate and high transmission. With approximately 90% compliance, Plasmodium prevalence decreased from 3% to 0% by microscopy and from 10% to 2% by PCR. However, prevalence rebounded to 9% by PCR two months after conclusion of MDA. Besides the remained local transmission, parasite importation caused by human movement likely contributed to the resurgence. Analyses of 419 arrivals to Ngodhe between July 2016 and September 2017 revealed Plasmodium prevalence of 4.6% and 16.0% by microscopy and PCR, respectively. Risk factors for infection among arrivals included age (0 to 5 and 11 to 15 years), and travelers from Siaya County, located to the north of Ngodhe Island. Parasite importation caused by human movement is one of major obstacles to sustain malaria elimination, suggesting the importance of cross-regional initiatives together with local vector control.

The Cytoplasmic Region of Plasmodium falciparum SURFIN4.2 Is Required for Transport from Maurer's Clefts to the Red Blood Cell Surface.

BACKGROUND: Plasmodium, the causative agent of malaria, exports many proteins to the surface of the infected red blood cell (iRBC) in order to modify it toward a structure more suitable for parasite development and survival. One such exported protein, SURFIN4.2, from the parasite of human malignant malaria, P. falciparum, was identified in the trypsin-cleaved protein fraction from the iRBC surface, and is thereby inferred to be exposed on the iRBC surface. SURFIN4.2 also localize to Maurer's clefts-parasite-derived membranous structures established in the RBC cytoplasm and tethered to the RBC membrane-and their role in trafficking suggests that they are a pathway for SURFIN4.2 transport to the iRBC surface. It has not been determined the participation of protein domains and motifs within SURFIN4.2 in transport from Maurer's clefts to the iRBC surface; and herein we examined if the SURFIN4.2 intracellular region containing tryptophan-rich (WR) domain is required for its exposure on the iRBC surface. RESULTS: We generated two transgenic parasite lines which express modified SURFIN4.2, with or without a part of the intracellular region. Both recombinant SURFIN4.2 proteins were exported to Maurer's clefts. However, only SURFIN4.2 possessing the intracellular region was efficiently cleaved by surface treatment of iRBC with proteinase K. CONCLUSIONS: These results indicate that SURFIN4.2 is exposed on the iRBC surface and that the intracellular region containing WR domain plays a role on the transport from Maurer's clefts to the iRBC membrane.