Prospective study of malaria in pregnancy, placental and congenital malaria in Northwest Colombia.

BACKGROUND: Pregnancy Associated Malaria (PAM) include malaria in pregnancy (MiP), placental malaria (PM), and congenital malaria (CM). The evidence available in Colombia on PAM focuses on one of the presentations (MiP, PM or CM), and no study longitudinally analyses the infection from the pregnant woman, passing through the placenta, until culminating in the newborn. This study determined the frequency of MiP, PM, and CM caused by Plasmodium vivax, Plasmodium falciparum, or mixed infections, according to Thick Blood Smear (TBS) and quantitative Polymerase Chain Reaction (qPCR). Identifying associated factors of PAM and clinical-epidemiological outcomes in northwestern Colombia. METHODS: Prospective study of 431 pregnant women, their placenta, and newborns registered in the data bank of the research Group "Salud y Comunidad César Uribe Piedrahíta" which collected information between 2014 and 2020 in endemic municipalities of the departments of Córdoba and Antioquia. The frequency of infection was determined with 95% confidence intervals. Comparisons were made with the Chi-square test, Student t-test, prevalence ratios, and control for confounding variables by log-binomial regression. RESULTS: The frequency of MiP was 22.3% (4.6% using TBS), PM 24.8% (1.4% using TBS), and CM 11.8% (0% using TBS). Using TBS predominated P. vivax. Using qPCR the proportions of P. vivax and P. falciparum were similar for MiP and PM, but P. falciparum predominated in CM. The frequency was higher in nulliparous, and women with previous malaria. The main clinical effects of PAM were anaemia, low birth weight, and abnormal APGAR score. CONCLUSIONS: The magnitude of infections was not detected with TBS because most cases were submicroscopic (TBS-negative, qPCR-positive). This confirmed the importance of improving the molecular detection of cases. PAM continue being underestimated in the country due to that in Colombia the control programme is based on TBS, despite its outcomes on maternal, and congenital health.

Ageing of Plasmodium falciparum malaria sporozoites alters their motility, infectivity and reduces immune activation in vitro.

BACKGROUND: Sporozoites (SPZ), the infective form of Plasmodium falciparum malaria, can be inoculated into the human host skin by Anopheline mosquitoes. These SPZ migrate at approximately 1 µm/s to find a blood vessel and travel to the liver where they infect hepatocytes and multiply. In the skin they are still low in number (50-100 SPZ) and vulnerable to immune attack by antibodies and skin macrophages. This is why whole SPZ and SPZ proteins are used as the basis for most malaria vaccines currently deployed and undergoing late clinical testing. Mosquitoes typically inoculate SPZ into a human host between 14 and 25 days after their previous infective blood meal. However, it is unknown whether residing time within the mosquito affects SPZ condition, infectivity or immunogenicity. This study aimed to unravel how the age of P. falciparum SPZ in salivary glands (14, 17, or 20 days post blood meal) affects their infectivity and the ensuing immune responses. METHODS: SPZ numbers, viability by live/dead staining, motility using dedicated sporozoite motility orienting and organizing tool software (SMOOT), and infectivity of HC-04.j7 liver cells at 14, 17 and 20 days after mosquito feeding have been investigated. In vitro co-culture assays with SPZ stimulated monocyte-derived macrophages (MoMɸ) and CD8+ T-cells, analysed by flow cytometry, were used to investigate immune responses. RESULTS: SPZ age did not result in different SPZ numbers or viability. However, a markedly different motility pattern, whereby motility decreased from 89% at day 14 to 80% at day 17 and 71% at day 20 was observed (p ≤ 0.0001). Similarly, infectivity of day 20 SPZ dropped to ~ 50% compared with day 14 SPZ (p = 0.004). MoMɸ were better able to take up day 14 SPZ than day 20 SPZ (from 7.6% to 4.1%, p = 0.03) and displayed an increased expression of pro-inflammatory CD80, IL-6 (p = 0.005), regulatory markers PDL1 (p = 0.02), IL-10 (p = 0.009) and cytokines upon phagocytosis of younger SPZ. Interestingly, co-culture of these cells with CD8+ T-cells revealed a decreased expression of activation marker CD137 and cytokine IFNγ compared to their day 20 counterparts. These findings suggest that older (day 17-20) P. falciparum SPZ are less infectious and have decreased immune regulatory potential. CONCLUSION: Overall, this data is a first step in enhancing the understanding of how mosquito residing time affects P. falciparum SPZ and could impact the understanding of the P. falciparum infectious reservoir and the potency of whole SPZ vaccines.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in mainland Tanzania, 2018.

BACKGROUND: The use of artemisinin-based combination therapy (ACT) is recommended by the World Health Organization for the treatment of uncomplicated falciparum malaria. Artemether-lumefantrine (AL) is the most widely adopted first-line ACT for uncomplicated malaria in sub-Saharan Africa (SSA), including mainland Tanzania, where it was introduced in December 2006. The WHO recommends regular assessment to monitor the efficacy of the first-line treatment specifically considering that artemisinin partial resistance was reported in Greater Mekong sub-region and has been confirmed in East Africa (Rwanda and Uganda). The main aim of this study was to assess the efficacy and safety of AL for the treatment of uncomplicated falciparum malaria in mainland Tanzania. METHODS: A single-arm prospective anti-malarial drug efficacy trial was conducted in Kibaha, Mlimba, Mkuzi, and Ujiji (in Pwani, Morogoro, Tanga, and Kigoma regions, respectively) in 2018. The sample size of 88 patients per site was determined based on WHO 2009 standard protocol. Participants were febrile patients (documented axillary temperature ≥ 37.5 °C and/or history of fever during the past 24 h) aged 6 months to 10 years. Patients received a 6-dose AL regimen by weight twice a day for 3 days. Clinical and parasitological parameters were monitored during 28 days of follow-up to evaluate the drug efficacy and safety. RESULTS: A total of 653 children were screened for uncomplicated malaria and 349 (53.7%) were enrolled between April and August 2018. Of the enrolled children, 345 (98.9%) completed the 28 days of follow-up or attained the treatment outcomes. There were no early treatment failures, but recurrent infections were higher in Mkuzi (35.2%) and Ujiji (23%). By Kaplan-Meier analysis of polymerase chain reaction (PCR) uncorrected adequate clinical and parasitological response (ACPR) ranged from 63.4% in Mkuzi to 85.9% in Mlimba, while PCR-corrected ACPR on day 28 varied from 97.6% in Ujiji to 100% in Mlimba. The drug was well tolerated; the commonly reported adverse events were cough, runny nose, and abdominal pain. No serious adverse event was reported. CONCLUSION: This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria. The high number of recurrent infections were mainly due to new infections, indicating the necessity of utilizing alternative artemisinin-based combinations, such as artesunate amodiaquine, which provide a significantly longer post-treatment prophylactic effect.

Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi.

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection.

Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

COVID-19 and malaria co-infection: a systematic review of clinical outcomes in endemic areas.

Background: COVID-19 and malaria cause significant morbidity and mortality globally. Co-infection of these diseases can worsen their impact on public health. This review aims to synthesize literature on the clinical outcomes of COVID-19 and malaria co-infection to develop effective prevention and treatment strategies. Methods: A comprehensive literature search was conducted using MeSH terms and keywords from the start of the COVID-19 pandemic to January 2023. The review included original articles on COVID-19 and malaria co-infection, evaluating their methodological quality and certainty of evidence. It was registered in PROSPERO (CRD42023393562). Results: Out of 1,596 screened articles, 19 met the inclusion criteria. These studies involved 2,810 patients, 618 of whom had COVID-19 and malaria co-infection. Plasmodium falciparum and vivax were identified as causative organisms in six studies. Hospital admission ranged from three to 18 days. Nine studies associated co-infection with severe disease, ICU admission, assisted ventilation, and related complications. One study reported 6% ICU admission, and mortality rates of 3%, 9.4%, and 40.4% were observed in four studies. Estimated crude mortality rates were 10.71 and 5.87 per 1,000 person-days for patients with and without concurrent malaria, respectively. Common co-morbidities included Diabetes mellitus, hypertension, cardiovascular diseases, and respiratory disorders. Conclusion: Most patients with COVID-19 and malaria co-infection experienced short-term hospitalization and mild to moderate disease severity. However, at presentation, co-morbidities and severe malaria were significantly associated with higher mortality or worse clinical outcomes. These findings emphasize the importance of early detection, prompt treatment, and close monitoring of patients with COVID-19 and malaria co-infection.

A Pfs48/45-based vaccine to block Plasmodium falciparum transmission: phase 1, open-label, clinical trial.

BACKGROUND: The stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans. METHODS: In this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18-55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants' serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes. RESULTS: Thirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity. CONCLUSIONS: R0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses. TRIAL REGISTRATION: The trial is registered with ClinicalTrials.gov under identifier NCT04862416.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

A phase IIa, randomized, double-blind, safety, immunogenicity and efficacy trial of Plasmodium falciparum vaccine antigens merozoite surface protein 1 and RTS,S formulated with AS02 adjuvant in healthy, malaria-naïve adults.

BACKGROUND: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment. METHODS: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI). RESULTS: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had âˆ¼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia. CONCLUSION: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection. CLINICALTRIALS: gov identifier: NCT01556945.

Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. METHODS: Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households' exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. RESULTS: Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75-100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9-100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). CONCLUSIONS: This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.

[Gastrointestinal symptoms may reflect complicated falciparum malaria]. / Gastrointestinale Symptome als Ausdruck einer komplizierten Malaria tropica.

HISTORY: A 42-year-old female presented with a two-day history of vomiting, diarrhea, fever and chills. Two weeks before she had returned to Germany from a Safari in Tanzania. She had disregarded the recommendation to take antimalarial chemoprophylaxis. CLINICAL FINDINGS AND DIAGNOSIS: The thin blood film showed Plasmodium falciparum-parasitized erythrocytes, and Plasmodium falciparum malaria was diagnosed. The full blood count showed thrombocytopenia and ultrasound imaging revealed splenomegaly. Initially the criteria for complicated malaria were not fulfilled. THERAPY AND COURSE: We started oral therapy with atovaquone/proguanil. The patient vomited the tablets twice. Therefore therapy was switched to intravenous artesunate. Subsequently, parasitemia dropped from 2.8 to 1.0 % within 22 hours. After 3 days of artesunate i. v., treatment could then be completed with oral atovaquone/proguanil, and the symptoms resolved. CONCLUSIONS: Patients with malaria and persistent vomiting should be treated intravenously and monitored closely, as severe gastrointestinal symptoms may reflect impending organ failure. We therefore propose including persistent vomiting in the list of criteria for complicated malaria.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine and prevalence of molecular markers of anti-malarial drug resistance in children in Togo in 2021.

BACKGROUND: Artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the currently recommended first- and second-line therapies for uncomplicated Plasmodium falciparum infections in Togo. This study assessed the efficacy of these combinations, the proportion of Day3-positive patients (D3 +), the proportion of molecular markers associated with P. falciparum resistance to anti-malarial drugs, and the variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single arm prospective study evaluating the efficacy of AL and DP was conducted at two sites (Kouvé and Anié) from September 2021 to January 2022. Eligible children were enrolled, randomly assigned to treatment at each site and followed up for 42 days after treatment initiation. The primary endpoint was polymerase chain reaction (PCR) adjusted adequate clinical and parasitological response (ACPR). At day 0, samples were analysed for mutations in the Pfkelch13, Pfcrt, Pfmdr-1, dhfr, dhps, and deletions in the hrp2/hrp3 genes. RESULTS: A total of 179 and 178 children were included in the AL and DP groups, respectively. After PCR correction, cure rates of patients treated with AL were 97.5% (91.4-99.7) at day 28 in Kouvé and 98.6% (92.4-100) in Anié, whereas 96.4% (CI 95%: 89.1-98.8) and 97.3% (CI 95%: 89.5-99.3) were observed at day 42 in Kouvé and Anié, respectively. The cure rates of patients treated with DP at day 42 were 98.9% (CI 95%: 92.1-99.8) in Kouvé and 100% in Anié. The proportion of patients with parasites on day 3 (D3 +) was 8.5% in AL and 2.6% in DP groups in Anié and 4.3% in AL and 2.1% DP groups in Kouvé. Of the 357 day 0 samples, 99.2% carried the Pfkelch13 wild-type allele. Two isolates carried nonsynonymous mutations not known to be associated with artemisinin partial resistance (ART-R) (A578S and A557S). Most samples carried the Pfcrt wild-type allele (97.2%). The most common Pfmdr-1 allele was the single mutant 184F (75.6%). Among dhfr/dhps mutations, the quintuple mutant haplotype N51I/C59R/S108N + 437G/540E, which is responsible for SP treatment failure in adults and children, was not detected. Single deletions in hrp2 and hrp3 genes were detected in 1/357 (0.3%) and 1/357 (0.3%), respectively. Dual hrp2/hrp3 deletions, which could affect the performances of HRP2-based RDTs, were not observed. CONCLUSION: The results of this study confirm that the AL and DP treatments are highly effective. The absence of the validated Pfkelch13 mutants in the study areas suggests the absence of ART -R, although a significant proportion of D3 + cases were found. The absence of dhfr/dhps quintuple or sextuple mutants (quintuple + 581G) supports the continued use of SP for IPTp during pregnancy and in combination with amodiaquine for seasonal malaria chemoprevention. TRIAL REGISTRATION: ACTRN12623000344695.

Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery.

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark ß-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (ß-D-galactoside) by a transgenic ß-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

Widespread pfhrp2/3 deletions and HRP2-based false-negative results in southern Ethiopia.

BACKGROUND: Rapid diagnostic tests (RDTs) play a significant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection RDTs are predominantly used to diagnose Plasmodium falciparum infection. However, the evolution and spread of P. falciparum parasite strains with deleted hrp2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2-detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis at selected health facilities in southern Ethiopia. METHODS: A multi-health facilities-based cross-sectional study was conducted on self-presenting febrile patients seeking treatment in southern Ethiopia from July to September 2022. A purposive sampling strategy was used to enroll patients with microscopically confirmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood film for microscopy and a RDT using the SD Bioline™ Malaria Pf/Pv Test. Dried blood spot samples were collected for further molecular analysis. DNA was extracted using gene aid kits and amplification was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confirm its deletion. The diagnostic performance of RDT was evaluated using PCR as the gold standard test for P. falciparum infections. RESULTS: Of 279 P. falciparum PCR-confirmed samples, 249 (89.2%) had successful msp-2 amplification, which was then genotyped for hrp2/3 gene deletions. The study revealed that pfhrp2/3 deletions were common in all health centres, and it was estimated that 144 patients (57.8%) across all health facilities had pfhrp2/3 deletions, leading to false-negative PfHRP2 RDT results. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%) of cases, respectively. The study findings revealed the prevalence of P. falciparum parasites lacking a single pfhrp2-/3-gene and that both genes varied across the study sites. This study also showed that the sensitivity of the SD Bioline PfHRP2-RDT test was 76.5% when PCR was used as the reference test. CONCLUSION: This study confirmed the existence of widespread pfhrp2/3- gene deletions, and their magnitude exceeded the WHO-recommended threshold (> 5%). False-negative RDT results resulting from deletions in Pfhrp2/3- affect a country's attempts at malaria control and elimination. Therefore, the adoption of non-HRP2-based RDTs as an alternative measure is required to avoid the consequences associated with the continued use of HRP-2-based RDTs, in the study area in particular and in Ethiopia in general.

Changes in the endemic-epidemic pattern of malaria in Colombia, 1978-2021.

BACKGROUND: Malaria is a major global public health issue with varying epidemiologies across countries. In Colombia, it is a priority endemic-epidemic event included in the national public health policy. However, evidence demonstrating nationwide variations in the disease behavior is limited. This study aimed to analyze changes in the levels and distribution of endemic-epidemic malaria transmission in the eco-epidemiological regions of Colombia from 1978 to 1999 and 2000 to 2021. METHODS: We conducted a comprehensive time-series study using official secondary data on malaria-associated morbidity and mortality in Colombia from 1978 to 2021. Temporal-spatial and population variables were analyzed, and the absolute and relative frequency measures of general and regional morbidity and mortality were estimated. RESULTS: We observed an 18% reduction in malaria endemic cases between the two study periods. The frequency and severity of the epidemic transmission of malaria varied less and were comparable across both periods. A shift was observed in the frequency of parasitic infections, with a tendency to match and increase infections by Plasmodium falciparum. The risk of malaria transmission varied significantly among the eco-epidemiological regions during both study periods. This study demonstrated a sustained decrease of 78% in malarial mortality. CONCLUSIONS: Although the endemic components of malaria decreased slightly between the two study periods, the epidemic pattern persisted. There were significant variations in the risk of transmission across the different eco-epidemiological regions. These findings underscore the importance of targeted public health interventions in reducing malarial morbidity and mortality rates in Colombia.

Malaria in a vulnerable population living in quilombo remnant communities in the Brazilian Amazon: a cross-sectional study from 2005-2020.

Quilombo remnant communities are areas officially recognized by the Brazilian government as historical communities founded by formerly enslaved individuals. These communities are mostly located in the endemic areas of malaria in the Brazilian Amazon. We retrospectively described the prevalence of malaria among individuals living in 32 recognized quilombo remnant communities in the Baiao and Oriximina municipalities located in the Para State. The number of malaria cases and the Annual Parasitic Incidence (API) recorded by the Brazilian malaria surveillance system (SIVEP-Malaria) from January 2005 to December 2020 were analyzed. We found that all communities registered at least one case over the 16-year period, the most frequent parasitic species being Plasmodium vivax (76.1%). During this period, 0.44% (4,470/1,008,714) of the malaria cases registered in Para State were reported in these quilombo remnant communities, with frequencies of 10.9% (856/7,859) in Baiao municipality and 39.1% (3,614/9,238) in Oriximina municipality, showing that individuals living in these rural communities are exposed to malaria. These data indicate that effective surveillance requires improved measures to identify malaria transmission among vulnerable populations living in quilombo remnant communities in the Brazilian Amazon.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in mainland Tanzania, 2019.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been a major contributor to the substantial reductions in global malaria morbidity and mortality over the last decade. In Tanzania, artemether-lumefantrine (AL) was introduced as the first-line treatment for uncomplicated Plasmodium falciparum malaria in 2006. The World Health Organization (WHO) recommends regular assessment and monitoring of the efficacy of the first-line treatment, specifically considering that artemisinin resistance has been confirmed in the Greater Mekong sub-region. This study's main aim was to assess the efficacy and safety of AL for treating uncomplicated P. falciparum malaria in Tanzania. METHODS: This was a single-arm prospective antimalarial drug efficacy trial conducted in four of the eight National Malaria Control Programme (NMCP) sentinel sites in 2019. The trial was carried out in outpatient health facilities in Karume-Mwanza region, Ipinda-Mbeya region, Simbo-Tabora region, and Nagaga-Mtwara region. Children aged six months to 10 years with microscopy confirmed uncomplicated P. falciparum malaria who met the inclusion criteria were recruited based on the WHO protocol. The children received AL (a 6-dose regimen of AL twice daily for three days). Clinical and parasitological parameters were monitored during follow-up over 28 days to evaluate drug efficacy. RESULTS: A total of 628 children were screened for uncomplicated malaria, and 349 (55.6%) were enrolled between May and September 2019. Of the enrolled children, 343 (98.3%) completed the 28-day follow-up or attained the treatment outcomes. There were no early treatment failures; recurrent infections during follow-up were common at two sites (Karume 29.5%; Simbo 18.2%). PCR-corrected adequate clinical and parasitological response (ACPR) by survival analysis to AL on day 28 of follow-up varied from 97.7% at Karume to 100% at Ipinda and Nagaga sites. The commonly reported adverse events were cough, skin pallor, and abdominal pain. The drug was well tolerated, and no serious adverse event was reported. CONCLUSION: This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria in Tanzania in 2019. The high recurrent infections were mainly due to new infections, highlighting the potential role of introducing alternative artemisinin-based combinations that offer improved post-treatment prophylaxis, such as artesunate-amodiaquine (ASAQ).

Randomized, placebo-controlled, double-blind phase I trial of co-administered pyronaridine and piperaquine in healthy adults of sub-Saharan origin.

Drug resistance to sulfadoxine-pyrimethamine and amodiaquine threatens the efficacy of malaria chemoprevention interventions in children and pregnant women. Combining pyronaridine (PYR) and piperaquine (PQP), both components of approved antimalarial therapies, has the potential to protect vulnerable populations from severe malaria. This randomized, double-blind, placebo-controlled (double-dummy), parallel-group, single site phase I study in healthy adult males or females of Black sub-Saharan African ancestry investigated the safety, tolerability, and pharmacokinetics of PYR + PQP (n = 15), PYR + placebo (n = 8), PQP + placebo (n = 8), and double placebo (n = 6) administered orally once daily for 3 days at the registered dose for the treatment of uncomplicated malaria. All participants completed the study. Forty-five adverse events were reported in 26 participants, most (41/45) were mild/moderate in severity, with no serious adverse events, deaths, or study withdrawals. Adverse events were reported in 66.7% (10/15) of participants administered PYR + PQP, 87.5% (7/8) with PYR + placebo, 50.0% (4/8) with PQP + placebo, and 83.3% (5/6) with placebo. For PYR containing regimens, five of 23 participants had asymptomatic transient increases in alanine and/or aspartate aminotransferase. With PQP containing regimens, four of 23 participants had mild Fridericia-corrected QT interval prolongation. Liver enzyme elevations and prolonged QTc interval were consistent with observations for PYR-artesunate and dihydroartemisinin-PQP, respectively, administered to healthy adults and malaria patients. Increases in PYR and PQP exposures were observed following co-administration versus placebo, with substantial interparticipant variability. The findings suggest that PYR + PQP may have potential in chemoprevention strategies. Further studies are needed in the target populations to assess chemoprotective efficacy and define the benefit-risk profile, with special considerations regarding hepatic and cardiac safety.