Rapid increase of Plasmodium falciparum dhfr/dhps resistant haplotypes, after the adoption of sulphadoxine-pyrimethamine as first line treatment in 2002, in southern Mozambique

<p>Abstract</p> <p>Background</p> <p>In late 2002, the health authorities of Mozambique implemented sulphadoxine-pyrimethamine (SP)/amodiaquine (AQ) as first-line treatment against uncomplicated falciparum malaria. In 2004, this has been altered to SP/artesunate in line with WHO recommendations of using Artemisinin Combination Therapies (ACTs), despite the fact that all the neighbouring countries have abandoned SP-drug combinations due to high levels of SP drug resistance. In the study area, one year prior to the change to SP/AQ, SP alone was used to treat uncomplicated malaria cases. The study described here investigated the immediate impact of the change to SP on the frequency of SP and CQ resistance-related haplotypes in the <it>Plasmodium falciparum </it>genes <it>Pfdhfr, Pfdhps </it>and <it>Pfcrt </it>before and a year after the introduction of SP.</p> <p>Methods</p> <p>Samples were collected during two cross sectional surveys in early 2002 and 2003 involving 796 and 692 children one year or older and adults randomly selected living in Maciana, an area located in Manhiça district, Southern Mozambique. Out of these, 171 and 173 <it>P. falciparum </it>positive samples were randomly selected to measure the frequency of resistance- related haplotypes in <it>Pfdhfr, Pfdhps </it>and <it>Pfcrt </it>based on results obtained by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)-ELISA.</p> <p>Results</p> <p>The frequency of the SP-resistance associated <it>Pfdhps </it>double mutant (SGEAA) haplotype increased significantly from 14% to 35% (P < 0.001), while the triple mutant <it>Pfdhfr </it>haplotype (CIRNI) remained high and only changed marginally from 46% to 53% (P = 0.405) after one year with SP as first-line treatment in the study area. Conversely, the combined <it>Pfdhfr/Pfdhps </it>quintuple mutant haplotype increased from 8% to 26% (P = 0.005). The frequency of the chloroquine resistance associated <it>Pfcrt</it>-CVIET haplotype was above 90% in both years.</p> <p>Conclusion</p> <p>These retrospective findings add to the general concern on the lifespan of the combination of SP/artesunate in Mozambique. The high frequency of quintuple <it>Pfdhfr</it>/<it>Pfdhps </it>haplotypes found here as early as 2002 most likely cause ideal conditions for the development of artesunate tolerance in the <it>P. falciparum </it>populations and may even endanger the sensitivity to the second-line drug, Coartem^®.</p

Drug-Resistant Polymorphisms and Copy Numbers in Plasmodium falciparum, Mozambique, 2015

One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases

Tracking SARS-CoV-2 introductions in Mozambique using pandemic-scale phylogenies: a retrospective observational study

9 hojas, 4 figuras, 1 tabla. Demographic data of the participants are available in the appendix (pp 40–42). Genome sequences are publicly available on GISAID. All scripts used for the analysis, and beta and delta subtree files are publicly available at https://gitlab.com/tbgenomicsunit/mozcovid. The study protocol (appendix pp 1–35) and clinical questionnaire (appendix pp 36–39) will be available with publicationBackground: 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. Methods: We did a retrospective, observational study in southern Mozambique. Patients from Manhiça presenting with respiratory symptoms were recruited, and those enrolled in clinical trials were excluded. Data were included from three sources: (1) a prospective hospital-based surveillance study (MozCOVID), recruiting patients living in Manhiça, attending the Manhiça district hospital, and fulfilling the criteria of suspected COVID-19 case according to WHO; (2) symptomatic and asymptomatic individuals with SARS-CoV-2 infection recruited by the National Surveillance system; and (3) sequences from SARS-CoV-2-infected Mozambican cases deposited on the Global Initiative on Sharing Avian Influenza Data database. Positive samples amenable for sequencing were analysed. We used Ultrafast Sample placement on Existing tRees to understand the dynamics of beta and delta waves, using available genomic data. This tool can reconstruct a phylogeny with millions of sequences by efficient sample placement in a tree. We reconstructed a phylogeny (~7·6 million sequences) adding new and publicly available beta and delta sequences. Findings: A total of 5793 patients were recruited between Nov 1, 2020, and Aug 31, 2021. During this time, 133 328 COVID-19 cases were reported in Mozambique. 280 good quality new SARS-CoV-2 sequences were obtained after the inclusion criteria were applied and an additional 652 beta (B.1.351) and delta (B.1.617.2) public sequences were included from Mozambique. We evaluated 373 beta and 559 delta sequences. We identified 187 beta introductions (including 295 sequences), divided in 42 transmission groups and 145 unique introductions, mostly from South Africa, between August, 2020 and July, 2021. For delta, we identified 220 introductions (including 494 sequences), with 49 transmission groups and 171 unique introductions, mostly from the UK, India, and South Africa, between April and November, 2021. Interpretation: The timing and origin of introductions suggests that movement restrictions effectively avoided introductions from non-African countries, but not from surrounding countries. Our results raise questions about the imbalance between the consequences of restrictions and health benefits. This new understanding of pandemic dynamics in Mozambique can be used to inform public health interventions to control the spread of new variants. Funding: European and Developing Countries Clinical Trials, European Research Council, Bill & Melinda Gates Foundation, and Agència de Gestió d'Ajuts Universitaris i de Recerca.This publication was produced by MozCovid which is part of the EDCTP2 programme supported by the EU (grant number RIA2020EF-3005-MozCOVID). The COVID-19 testing was supported by Emory Global Health Institute, University of Emory, through the CHAMPS Program funded by the Bill & Melinda Gates Foundation (under the grant OPP1126780 to Robert Breiman, subcontract SC00003286). This work was also supported by the European Research Council under the EU’s Horizon 2020 Research and Innovation Program grant (101001038; TB-RECONNECT), the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global), the Departament d’Universitats i Recerca de la Generalitat de Catalunya (AGAUR; 2021 SGR 01517), and the Ministerio de Ciencia e Innovación (Spanish Government) Project PID2019–104477RB-I00. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development. ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya (http://cerca.cat/en/suma/). We also acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the Centro de Excelencia Severo Ochoa 2019–2023 Program (CEX2018–000806-S).Peer reviewe

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website

Enhanced T cell activation in Plasmodium falciparum malaria-infected human immunodeficiency virus-1 patients from Mozambique

Submitted by Sandra Infurna ([email protected]) on 2017-05-16T12:13:15Z No. of bitstreams: 1 aldamaria_cruz_etal_IOC_2012.pdf: 514990 bytes, checksum: d75080e86e19b97881700b18bec471d5 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-05-16T12:19:41Z (GMT) No. of bitstreams: 1 aldamaria_cruz_etal_IOC_2012.pdf: 514990 bytes, checksum: d75080e86e19b97881700b18bec471d5 (MD5)Made available in DSpace on 2017-05-16T12:19:41Z (GMT). No. of bitstreams: 1 aldamaria_cruz_etal_IOC_2012.pdf: 514990 bytes, checksum: d75080e86e19b97881700b18bec471d5 (MD5) Previous issue date: 2012Ministério da Saúde. Instituto Nacional de Saúde. Maputo, Moçambique / Hospital Central de maputo. Laboratório de Biologia Molecular. Maputo, Moçambique.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Ministério da Saúde. Instituto Nacional de Saúde. Maputo, Moçambique / Hospital Central de maputo. Laboratório de Biologia Molecular. Maputo, Moçambique.Human immunodeficiency virus (HIV)-1 infection has an important impact on malaria. Plasmodium falciparum and HIV-1 co-infected patients (Pf/HIV) present with a high degree of anaemia, enhanced parasitaemia and decreased CD4+ T cell counts, which increase the risk of developing severe malaria. In addition, infection with either Pf or HIV-1 alone causes extensive immune activation. Our hypothesis was that lymphocyte activation is potentiated in Pf/HIV co-infected patients, consequently worsening their immunosuppressed state. To test this hypothesis, 22 Pf/HIV patients, 34 malaria patients, 29 HIV/AIDS patients and 10 healthy controls without malaria or HIV/acquired immune deficiency syndrome (AIDS) from Maputo/Mozambique were recruited for this study. As expected, anaemia was most prevalent in the Pf/HIV group. A significant variation in parasite density was observed in the Pf/HIV co-infected group (110-75,000 parasites/μL), although the median values were similar to those of the malaria only patients. The CD4+ T cell counts were significantly lower in the Pf/HIV group than in the HIV/AIDS only or malaria only patients. Lymphocyte activation was evaluated by the percentage of activation-associated molecules [CD38 expression on CD8+ and human leukocyte antigen-DR expression on CD3+ T cells]. The highest CD38 expression was detected in the Pf/HIV co-infected patients (median = 78.2%). The malaria only (median = 50%) and HIV/AIDS only (median = 52%) patients also exhibited elevated levels of these molecules, although the values were lower than those of the Pf/HIV co-infected cases. Our findings suggest that enhanced T-cell activation in co-infected patients can worsen the immune response to both diseases

Utilization of a local ‘Malaria Post’ indicates that carers from a village in Mozambique respond appropriately to malaria attacks

As malaria elimination becomes a possibility the focus of interventions changes from vector control to disease control. It is important that treatment occurs early during an infection in order for it to be efficacious, especially at the population level. The time between the onset of symptoms and treatment seeking is, therefore, crucial. Following a census and an oral autopsy survey of the inhabitants of Furvela, a village in southern Mozambique, a malaria post (MP) where malaria was diagnosed and treated was established in 2001. The time between the onset of symptoms and attendance at the MP was determined and compared to the severity of disease. A cross-sectional survey was also conducted, in 2007, to determine prevalence amongst 235 children aged between 6 months and 15 years of age. Malaria was hyperendemic in the village and was responsible for most deaths reported from the two years prior to the start of the project. In the prevalence survey 74% of two-to-four-year-old children had malaria parasites. The likelihood of being parasite positive was significantly higher in children living in houses with roofs made of traditional materials compared to those living in houses with tin roofs. At the start of the project only 12% of residents owned or used a mosquito net, most of which were not treated with insecticide. However, even before any formal intervention, malaria declined in the village between 2001 and 2007, but there was a rebound in later years. Nevertheless, the relative proportion of patients who had to be referred to the hospital declined significantly in the latter years of the project, and the incidence of both Plasmodium ovale and P. malariae also decreased significantly. Overall 16698 patients, the majority of which were under one year of age, attended the MP between 2001 and 2010. The proportion of patients with a positive slide for P. falciparum remained relatively constant throughout the study (mean 0.66 std. dev. 0.3) Most of the patients came from the village of Furvela, or its environs, but some came from the nearby town, ostensibly because of the good treatment they received. Infection rates increased up to the first three years of life to a peak incidence of 92% at 31 months. Children with fever had higher parasite densities than those without fever. Mothers generally bought their children to the MP on the second day of symptoms but on the first day if they had fever. Older patients, with lower density infections, delayed in coming for treatment. These patients may harbour sub-microscopic gametocytes which would help maintain transmission in the village. Mothers acted appropriately in their treatment seeking behaviour. The establishment of village-based MPs are an effective way of providing adequate diagnosis and treatment in villages such as Furvela.The Danish Bilharziasis Laboratory, Charlottenlund, Denmark subsequently called DBL Centre for Health Research and Development, Fredricksberg, Denmark.http://www.elsevier.com/locate/actatropica2022-06-23hj2021Zoology and Entomolog

Anti-malarial resistance in Mozambique: Absence of Plasmodium falciparum Kelch 13 (K13) propeller domain polymorphisms associated with resistance to artemisinins

Abstract Background Malaria remains one of the most serious public health problems in sub-Saharan Africa and Mozambique is the world's fourth largest contributor, with 4.7% of disease cases and 3.6% of total deaths due to malaria. Its control relies on the fight against the vector and treatment of confirmed cases with anti-malarial drugs. Molecular surveillance is an important tool for monitoring the spread of anti-malarial drug resistance. Methods A cross-sectional study recruited 450 participants with malaria infection detected by Rapid Diagnostic Tests, from three different study sites (Niassa, Manica and Maputo) between April and August 2021. Correspondent blood samples were collected on filter paper (Whatman® FTA® cards), parasite DNA extracted and pfk13 gene sequenced using Sanger method. SIFT software (Sorting Intolerant From Tolerant) was used, predict whether an amino acid substitution affects protein function. Results No pfkelch13-mediated artemisinin resistance gene mutation was detected in this study settings. However, non-synonymous mutations were detected at prevalence of 10.2%, 6% and 5% in Niassa, Manica and Maputo, respectively. Most (56.3%) of the reported non-synonymous mutations were due to substitution at the first base of the codon, 25% at the second base and 18.8% at the third base. Additionally, 50% of non-synonymous mutations showed a SIFTscore bellow cut off value of 0.05, therefore, they were predicted to be deleterious. Conclusion These results do not show an emergence of artemisinin resistance cases in Mozambique. However, the increased number of novel non-synonymous mutations highlights the relevance of increasing the number of studies focused on the molecular surveillance of artemisinin resistance markers, for its early detection