Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.

Recent reports regarding the re-emergence of parasite sensitivity to chloroquine call for a new consideration of this drug as an interesting complementary tool in malaria elimination efforts, given its good safety profile and long half-life. A randomized (2:1), single-blind, placebo-controlled trial was conducted in Manhiça, Mozambique, to assess the in-vivo efficacy of chloroquine to clear plasmodium falciparum (Pf) asymptomatic infections. Primary study endpoint was the rate of adequate and parasitological response (ACPR) to therapy on day 28 (PCR-corrected). Day 0 isolates were analyzed to assess the presence of the PfCRT-76T CQ resistance marker. A total of 52 and 27 male adults were included in the CQ and Placebo group respectively. PCR-corrected ACPR was significantly higher in the CQ arm 89.4% (95%CI 80-98%) compared to the placebo (p < 0.001). CQ cleared 49/50 infections within the first 72 h while placebo cleared 12/26 (LRT p < 0.001). The PfCRT-76T mutation was present only in one out of 108 (0.9%) samples at baseline, well below the 84% prevalence found in 1999 in the same area. This study presents preliminary evidence of a return of chloroquine sensitivity in Mozambican Pf isolates, and calls for its further evaluation in community-based malaria elimination efforts, in combination with other effective anti-malarials.

Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or tolerance that are affected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune responses are responsible for the symptoms and pathological alterations during disease and are expected to change rapidly upon malaria exposure or cessation.

Aedes-borne arboviruses have emerged as an important public health problem worldwide and, in Mozambique, the number of cases and its geographical spread have been growing. However, information on the occurrence, distribution and ecology of Aedes aegypti and Ae. albopictus mosquitoes remain poorly known in the country.

Health care workers (HCW) are a high-risk population to acquire SARS-CoV-2 infection from patients or other fellow HCW. This study aims at estimating the seroprevalence against SARS-CoV-2 in a random sample of HCW from a large hospital in Spain. Of the 578 participants recruited from 28 March to 9 April 2020, 54 (9.3%, 95% CI: 7.1-12.0) were seropositive for IgM and/or IgG and/or IgA against SARS-CoV-2. The cumulative prevalence of SARS-CoV-2 infection (presence of antibodies or past or current positive rRT-PCR) was 11.2% (65/578, 95% CI: 8.8-14.1). Among those with evidence of past or current infection, 40.0% (26/65) had not been previously diagnosed with COVID-19. Here we report a relatively low seroprevalence of antibodies among HCW at the peak of the COVID-19 epidemic in Spain. A large proportion of HCW with past or present infection had not been previously diagnosed with COVID-19, which calls for active periodic rRT-PCR testing in hospital settings.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Since the announcement of the coronavirus disease (COVID-19) pandemic in January 30th 2020, 68 countries reported to the World Health Organization that they were experiencing disruptions in malaria diagnosis and treatment. This situation had the potential to lead to delays in diagnosis and treatment, which could result in an increase in severe cases and deaths. This analysis was based on findings from a field visit, carried out between June 30th and July 1st, 2020, to a warehouse, to two health facilities, and a meeting with a community health worker, and an descriptive epidemiologic data analysis of health information system (HIS) to evaluate trends of the number of people tested for malaria and number of malaria cases reported, by comparing data from 2018, 2019 and 2020 for the period between January and May. The two health facilities and the warehouse had about two months of stock of antimalarial drugs, and patients with malaria symptoms were being tested for malaria at the COVID-19 screening site. The HIS data showed that the number of reported malaria cases decreased by 3.0% (177.646/172.246) in April, and 7.0% (173.188/161.812) in May, when comparing 2019 and 2020 data. People tested for malaria in community increased by 39.0% (190.370/264.730), between 2019 and 2020. The COVID-19 may have had a negative impact on the diagnosis and treatment of malaria in health facility (HF). The decrease in people tested for malaria in the health facilities may have overwhelmed the activities of the community.

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.

Malaria eradication remains the long-term vision of the World Health Organization (WHO). However, whether malaria elimination is feasible in areas of stable transmission in sub-Saharan Africa with currently available tools remains a subject of debate. This study aimed to evaluate a multiphased malaria elimination project to interrupt Plasmodium falciparum malaria transmission in a rural district of southern Mozambique.

From the start of the SARS-CoV-2 outbreak, global sequencing efforts have generated an unprecedented amount of genomic data. Nonetheless, unequal sampling between high-income and low-income countries hinders the implementation of genomic surveillance systems at the global and local level. Filling the knowledge gaps of genomic information and understanding pandemic dynamics in low-income countries is essential for public health decision making and to prepare for future pandemics. In this context, we aimed to discover the timing and origin of SARS-CoV-2 variant introductions in Mozambique, taking advantage of pandemic-scale phylogenies.

The impact of the age of first Plasmodium falciparum infection on the rate of acquisition of immunity to malaria and on the immune correlates of protection has proven difficult to elucidate. A randomized, double-blind, placebo-controlled trial using monthly chemoprophylaxis with sulphadoxine-pyrimethamine plus artesunate was conducted to modify the age of first P. falciparum erythrocytic exposure in infancy and assess antibodies and malaria risk over two years.

As malaria cases increase in some of the highest burden countries, more strategic deployment of new and proven interventions must be evaluated to meet global malaria reduction goals.

Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated.

This study aimed to capture the acceptability prior to, during and after the implementation of the first year of MDA rounds conducted under the Magude project, a malaria elimination project in southern Mozambique.

Social network structure is a fundamental determinant of human health, from infectious to chronic diseases. However, quantitative and unbiased approaches to measuring social network structure are lacking. We hypothesized that genetic relatedness of oral commensal bacteria could be used to infer social contact between humans, just as genetic relatedness of pathogens can be used to determine transmission chains of pathogens. We used a traditional, questionnaire survey-based method to characterize the contact network of the School of Public Health at a large research university. We then collected saliva from a subset of individuals to analyze their oral microflora using a modified deep sequencing multilocus sequence typing (MLST) procedure. We examined micro-evolutionary changes in the S. viridans group to uncover transmission patterns reflecting social network structure. We amplified seven housekeeping gene loci from the Streptococcus viridans group, a group of ubiquitous commensal bacteria, and sequenced the PCR products using next-generation sequencing. By comparing the generated S. viridans reads between pairs of individuals, we reconstructed the social network of the sampled individuals and compared it to the network derived from the questionnaire survey-based method. The genetic relatedness significantly (p-value < 0.001) correlated with social distance in the questionnaire-based network, and the reconstructed network closely matched the network derived from the questionnaire survey-based method. Oral commensal bacterial are thus likely transmitted through routine physical contact or shared environment. Their genetic relatedness can be used to represent a combination of social contact and shared physical space, therefore reconstructing networks of contact. This study provides the first step in developing a method to measure direct social contact based on commensal organism genotyping, potentially capable of unmasking hidden social networks that contribute to pathogen transmission.

Poor knowledge on the afebrile Plasmodium falciparum biology limits elimination approaches to target asymptomatic malaria. Therefore, the association of parasite factors involved in cytoadhesion, parasite multiplication and gametocyte maturation with afebrile malaria was assessed.

Recent anti-malarial resistance monitoring in Angola has shown efficacy of artemether-lumefantrine (AL) in certain sites approaching the key 90% lower limit of efficacy recommended for artemisinin-based combination therapy. In addition, a controversial case of malaria unresponsive to artemisinins was reported in a patient infected in Lunda Sul Province in 2013.

Lingering post-treatment parasite antigen in blood complicates malaria diagnosis through antigen detection. Characterization of antigen clearance dynamics is important for interpretation of positive antigen detection tests.

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions.