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Moonmilk-type deposits exemplify carbonated Martian analogues existing in the subsurface of Earth, an endokarstic speleothem with a possible biochemical origin composed principally by carbonates, mainly huntite and dolomite. In this work, samples of moonmilk located in Nerja Cave (southern Spain) have been studied by LIBS with the aim of identifying carbon of biogenic origin by establishing a relationship between a molecular emission indicator, CN signal, and the organic carbon content. The characterization of this kind of carbonate deposit with a multiple mineralogical composition has been completed using scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and X-ray diffraction techniques for qualitative and semi-quantitative analysis. The information attained from LIBS regarding energy thresholds and time-resolved kinetics of CN emissions provides useful insight into the identification of different molecular emitters, namely organic and inorganic CN, depending on the laser irradiance and time settings conditions. These promising results are of application in the search and identification of biosignatures in upcoming planetary missions with astrobiological purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Monitoring antimalarial efficacy is important to detect the emergence of parasite drug resistance. Angola conducts in vivo therapeutic efficacy studies (TESs) every 2 years in its fixed sentinel sites in Benguela, Lunda Sul, and Zaire provinces. Children with uncomplicated Plasmodium falciparum malaria were treated with artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), dihydroartemisinin-piperaquine (DP), or artesunate-pyronaridine (ASPY) and followed for 28 (AL and ASAQ) or 42 days (DP and ASPY) to assess clinical and parasitological response to treatment. Two drugs were sequentially assessed in each site in February-July 2021. The primary indicator was the Kaplan-Meier estimate of the PCR-corrected efficacy at the end of the follow-up period. A total of 622 patients were enrolled in the study and 590 (95%) participants reached a study endpoint. By day 3, ≥98% of participants were slide-negative in all study sites and arms. After PCR correction, day 28 AL efficacy was 88.0% (95% CI: 82%-95%) in Zaire and 94.7% (95% CI: 90%-99%) in Lunda Sul. For ASAQ, day 28 efficacy was 92.0% (95% CI: 87%-98%) in Zaire and 100% in Lunda Sul. Corrected day 42 efficacy was 99.6% (95% CI: 99%-100%) for ASPY and 98.3% (95% CI: 96%-100%) for DP in Benguela. High day 3 clearance rates suggest no clinical evidence of artemisinin resistance. This was the fourth of five rounds of TES in Angola showing a corrected AL efficacy <90% in a site. For Zaire, AL has had an efficacy <90% in 2013, 2015, and 2021. ASAQ, DP, and ASPY are appropriate choices as artemisinin-based combination therapies in Angola., Competing Interests: The authors declare no conflict of interest.

Introduction: The study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting., Methods: In the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR&#95;fit) dose-response curves was the variable used to quantify the cellular response towards TKIs., Results: Spheroids derived from ROS1 G2032R cells were significantly more resistant to repotrectinib (AOC fold change = - 7.33), lorlatinib (AOC fold change = - 6.17), ceritinib (AOC fold change = - 2.8) and entrectinib (AOC fold change = - 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = - 2.35), entrectinib (AOC fold change = - 2.44) and ceritinib (AOC fold change = - 2.12) in targeting the ROS1 G2032R mutation. ROS1 L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = - 2.19) and entrectinib (spheroid AOC fold change = - 1.98). ROS1 S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = - 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose-response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells., Conclusion: In this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations., (© 2024. The Author(s).)

Background and Aims: The acute tropical infectious disease known as yellow fever (YF) is caused by an arbovirus and is characterized by fever, jaundice, hemorrhage, headache, muscle pain, nausea, vomiting, and fatigue. Angola experienced a yellow fever virus (YFV) outbreak that was documented in December 2015. However, little is known about the outcome of this outbreak. We aimed to demonstrate epidemic features and lessons learned during the YF epidemic in Angola., Methods: A total of 4618 blood samples from suspected YF cases were sent to the Instituto Nacional de Investigação em Saúde (INIS), a national referral and public health laboratory, between December 5, 2015, and December 23, 2016. Sample analyses were conducted using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assays. Blood samples were sent from 16 out of the 18 provinces of Angola., Results: We detected 884 (19.1%) cases that were positive for ELISA, which were confirmed by RT-PCR assay. Considering the positive cases, the incidence among male patients was around three times higher ( n  = 223; 10.9%) than in female patients ( n  = 59; 2.6%) in the 20-29 age group, followed by the age group 10-19 with n  = 211 (6.8%) in males versus n  = 108 (3.3%) in females; and the age group 30-39 had n  = 68 (4.8%) in males versus n  = 28 (1.8%) in females. The other groups had an incidence below 3.0%. The case fatality ratio for YF was in young adults in the age group 20-29 with n  = 39 cases, followed by the age group 10-19 with n  = 16 cases, and finally the age group 0-9 with n  = 13 cases. The other age groups had several deaths by YF below 10 cases., Conclusions: This study demonstrates features of the YF epidemic that occurred in Angola. Also, it demonstrates that YF causes deaths in young people but is preventable by high vaccine coverage. Thus, public health laboratory surveillance must be strengthened to reduce the possibility of emerging and re-emerging human infections., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Aims: Evaluate the impact of an intervention program in non-adherent patients with ulcerative colitis., Methods: Parallel controlled randomized clinical trial (1:1), approved by the ethics committee (No. 3.068.511/2018) and registered at The Brazilian Clinical Trials Registry (No. RBR-79dn4k). Non-adherent ulcerative colitis patients according to the Morisky-Green-Levine-test were included. Recruitment began in August 2019 until August 2020, with 6-month follow-up. All participants received standard usual care, and additionally the intervention group received educational (video, educational leaflet, verbal guidance) and behavioral interventions (therapeutic scheme, motivational and reminder type short message services). Researchers were blinded for allocation prior to data collection at Visits 1 and 2 (0 and 6 months). Primary outcome: 180-day adherence rate, with relative risk 95%CI. Secondary outcome: 180-day quality of life according to SF-36 domains, using Student's t test. Variables with p<0.20 were selected for regression. Analysis included data from August/2019 to May/2021., Results: Forty-six and 49 participants were allocated in control and intervention groups, respectively. Two were excluded due to intervention refusal, and 4 and 6 were lost to follow-up in control and intervention groups. There was no post-intervention adherence rate difference, even after adjustment for type of non-adherence (unintentional/both/intentional) as confounder, or if considered as adherent the intervention group participants lost in follow-up. Interventions promoted better quality of life scores even after multivariate analysis for "Pain", when adjusted for ulcerative colitis severity, sex, and marital status (β = 18.352, p = 0.004), "Vitality", when adjusted for ulcerative colitis severity (β = 10.568, p = 0.015) and "Emotional Aspects", when adjusted for disease severity, income, and education (β = 24.907, p = 0.041)., Conclusions: The intervention program was not able to produce a significant medication adherence rate difference between comparative groups, however, there was a significant improvement in quality of life. Study limitations may include: sample size calculated to identify differences of 30%, leading to a possible insufficient power; non blinded participants, exposing the results to the risk of performance bias; outcomes based on self-reported data., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: GENOILE SANTANA has received payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events. Other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Araucaria angustifolia is a critically endangered species and its distribution can be affected by an increase in temperature. In this study, we evaluated the effects of heat stress (30°C) on Araucaria angustifolia cell lines responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The viability of both cell lines was reduced by heat stress and mitochondria were the organelles most affected. Heat stress for 24h increased the reactive oxygen species (ROS) levels in SE1 cells, followed by a reduction at 48 and 72h. In SE6 cells, an increase occurred after 24 and 48h of stress, returning to control levels at 72h. H2 O2 levels were increased after 24h for both SE1 and SE6 cells, being higher for SE6. Interestingly, at 48 and 72h, H2 O2 levels decreased in SE1 cells, while in SE6, the values returned to the control levels. The respiration of SE6 cells in the presence of oxidisable substrates was inhibited by heat stress, in agreement with the high lipid peroxidation levels. The AaSERK1 gene was identified in both cultures, with greater expression in the SE1 line. Heat stress for 24 and 48h increased gene expression only in this cell line. The activity of peroxidase, superoxide dismutase and enzymes of the glutathione/ascorbate cycle was increased in both cell lines subjected to heat stress. Catalase activity was increased only in SE6 cells at 72h of exposure. These results show that responsive SE1 cells can modulate ROS levels more efficiently than SE6 when these cells are stressed by heat. This ability may be related to the maturation capacity of these cells.

In this work, a geological sample of great astrobiological interest was studied through analytical techniques that are currently operating in situ on Mars and others that will operate in the near future. The sample analyzed consisted of an oncoid, which is a type of microbialite, collected in the Salar Carachi Pampa, Argentina. The main peculiarity of microbialites is that they are organo-sedimentary deposits formed by the in situ fixation and precipitation of calcium carbonate due to the growth and metabolic activities of microorganisms. For this reason, the Carachi Pampa oncoid was selected as a Martian analog for astrobiogeochemistry study. In this sense, the sample was characterized by means of the PIXL-like, SuperCam-like and SHERLOC-like instruments, which represent instruments on board the NASA Perseverance rover, and by means of RLS-like and MOMA-like instruments, which represent instruments on board the future ESA Rosalind Franklin rover. It was possible to verify that the most important conclusions and discoveries have been obtained from the combination of the results. Likewise, it was also shown that Perseverance rover-like remote-sensing instruments allowed a first detailed characterization of the biogeochemistry of the Martian surface. With this first characterization, areas of interest for in-depth analysis with Rosalind Franklin-like instruments could be identified. Therefore, from a first remote-sensing elemental identification (PIXL-like instrument), followed by a remote-sensing molecular characterization (SuperCam and SHERLOC-like instruments) and ending with an in-depth microscopic analysis (RLS and MOMA-like instruments), a wide variety of compounds were found. On the one hand, the expected minerals were carbonates, such as aragonite, calcite and high-magnesium calcite. On the other hand, unexpected compounds consisted of minerals related to the Martian/terrestrial surface (feldspars, pyroxenes, hematite) and organic compounds related to the past biological activity related to the oncoid (kerogen, lipid biomarkers and carotenes). Considering samples resembling microbialites have already been found on Mars and that one of the main objectives of the missions is to identify traces of past life, the study of microbialites is a potential way to find biosignatures protected from the inhospitable Martian environment. In addition, it should be noted that in this work, further conclusions have been obtained through the study of the results as a whole, which could also be carried out on Mars., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jennifer Huidobro reports article publishing charges was provided by University of the Basque Country. Jennifer Huidobro reports equipment, drugs, or supplies was provided by University of the Basque Country., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase ( pfdhfr ) and dihydropteroate synthase ( pfdhps ) genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools. The N51I, C59R, and S108N pfdhfr mutations and A437G pfdhps mutations were present at high proportions in all provinces (weighted allele frequencies, 62% to 100%). The K540E pfdhps mutation was present at lower proportions (10% to 14%). The A581G pfdhps mutation was only observed in Zaire, at a 4.6% estimated prevalence. The I431V and A613S mutations were also only observed in Zaire, at a prevalence of 2.8% to 2.9%. The most common (27% to 66%) reconstructed haplotype in all three provinces was the canonical quadruple pfdhfr pfdhps mutant. The canonical quintuple mutant was absent in Lunda Sul and Benguela and present in 7.9% of samples in Zaire. A single canonical sextuple (2.6%) mutant was observed in Zaire Province. Proportions of the pfdhps K540E and A581G mutations were well below the World Health Organization thresholds for meaningful SP resistance (prevalence of 95% for K540E and 10% for A581G). Samples from therapeutic efficacy studies represent a convenient source of samples for monitoring SP resistance markers.

EFSA was asked for a scientific opinion on the risks to public health related to the presence of N -nitrosamines ( N -NAs) in food. The risk assessment was confined to those 10 carcinogenic N -NAs occurring in food (TCNAs), i.e. NDMA, NMEA, NDEA, NDPA, NDBA, NMA, NSAR, NMOR, NPIP and NPYR. N -NAs are genotoxic and induce liver tumours in rodents. The in vivo data available to derive potency factors are limited, and therefore, equal potency of TCNAs was assumed. The lower confidence limit of the benchmark dose at 10% (BMDL 10 ) was 10 μg/kg body weight (bw) per day, derived from the incidence of rat liver tumours (benign and malignant) induced by NDEA and used in a margin of exposure (MOE) approach. Analytical results on the occurrence of N -NAs were extracted from the EFSA occurrence database (n = 2,817) and the literature (n = 4,003). Occurrence data were available for five food categories across TCNAs. Dietary exposure was assessed for two scenarios, excluding (scenario 1) and including (scenario 2) cooked unprocessed meat and fish. TCNAs exposure ranged from 0 to 208.9 ng/kg bw per day across surveys, age groups and scenarios. 'Meat and meat products' is the main food category contributing to TCNA exposure. MOEs ranged from 3,337 to 48 at the P95 exposure excluding some infant surveys with P95 exposure equal to zero. Two major uncertainties were (i) the high number of left censored data and (ii) the lack of data on important food categories. The CONTAM Panel concluded that the MOE for TCNAs at the P95 exposure is highly likely (98-100% certain) to be less than 10,000 for all age groups, which raises a health concern., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Undernutrition, anemia, and intestinal parasitic infections are public health problems in Angola, especially in pre-school children. We analyzed binary data from a longitudinal four-arm randomized parallel trial conducted in Bengo Province, northern Angola, over the course of two years, with seven follow-up assessments to explore the effects of four interventions (deworming and a test-and-treat approach for intestinal parasites, at both the individual and household levels) on wasting and stunting, and to understand their indirect benefits for anemia, malaria, diarrhea, and vomiting. A total of 121 children with intestinal parasitic infections received baseline treatment, and were allocated to the four arms (1:1:1:1). Using continuous outcome variables of height-for-age (HAZ) and weight-for-height (WHZ) statistical approaches did not reveal a clear benefit of any particular arm (Pathogens 2021, 10, 309). Next, HAZ and WHZ were transformed into binary variables of stunting and wasting, respectively, considering their mild-to-severe (Z-score < −1) and moderate-to-severe degrees (Z-score < −2). Original clinical data (on anemia, diarrhea, vomiting, and malaria) were also analyzed. From a binary longitudinal analysis with different dependence structures, using the R package bild, fitted models revealed the potential benefit of a test-and-treat approach at the individual level for wasting compared with annual albendazole at the individual level, especially considering mild-to-severe forms (ORadj = 0.27; p = 0.007). All arms showed similar effects on stunting, compared with annual albendazole, at a 5% significance level. Time and age at baseline presented favorable effects in the percentage of stunting using both severity degrees. Results showed a decreased chance of having anemia and diarrhea over time, although with no significant differences between arms. Data from longitudinal studies are essential to study the direct and indirect effects of interventions, such as deworming, and to explore additional approaches aiming at better understanding the temporal structure of nutrition and health outcomes in children.

In the present paper, confined dry Cu nanoaerosols of controlled particle size are inspected under a time-resolved LIBS scheme to explore the effect of laser-particulate matter interaction upon the detection capability of airborne nanoparticulate material. Optically catapulted streams probed showed linear intensity vs mass correlation and similar signal stability which is linked to the seeding effect caused by smaller particles yielding hotter, albeit shorter plasmas. Seeding effect is demonstrated by hyperspectral time-resolved aerosol inspection, which exposes both, the interaction between multiple plasma nuclei and the discrete nature of the laser-particle interaction. Observed population/exhaustion cycles at the focal volume of the inspection laser explained the uncertainty values characteristic of LIBS inspection of aerosols. A thorough inspection of the emission in time evidenced a significantly different evolution of the intensity profile for commonly monitored Cu lines owed not only to the nature of the monitored transit and pulse energy, but also to particle size. These results suggest that the experimental settings for quantitative ultrafine aerosol inspection need to be tuned according to the target particle size and the particle density of the aerosol as seeding effects facilitates signal saturation, therefore this effect simultaneously contributes to and detracts from the analytical performance of LIBS on nanometric aerosols., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Malnutrition and intestinal parasites continue to have serious impacts on growth and cognitive development of children in Angola. A longitudinal four-arm randomized parallel trial was conducted to investigate if deworming with a single annual dose of albendazole (annual-ALB) or a four-monthly test-and-treat (4TT) intestinal parasites approach at individual or household levels improve nutritional outcomes of pre-school children in Bengo province. Children with intestinal parasites (n = 121) were randomly assigned (1:1:1:1) to arm A1: annual-ALB*individual level; A2: annual-ALB*household level; A3: 4TT*individual; and A4: 4TT*household level. At baseline, 4, 8, 12, 16, 20, and 24 months of follow-up, growth was assessed by height, weight, height-for-age, weight-for-height, weight-for-age, and mid-upper arm circumference. Intention-to-treat analysis was done using non-parametric approach, mixed effect models, and generalized estimating equations (GEE). Initially, 57% and 26% of the children were infected by Giardia lamblia and Ascaris lumbricoides , respectively. This study did not show that a 4TT intestinal parasites approach results on better growth outcomes of children (height, weight, HAZ, WAZ, WHZ and MUACZ) when compared with annual ALB, with exception of height and WHZ using GEE model at 5% level. Positive temporal effects on most nutrition outcomes were observed. Implementing a longitudinal study in a poor setting is challenging and larger sample sizes and 'pure and clean' data are difficult to obtain. Nevertheless, learned lessons from this intensive study may contribute to future scientific research and to tailor multidisciplinary approaches to minimize malnutrition and infections in resource-poor countries.

Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated Plasmodium falciparum infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers. Samples from treatment failures were genotyped for the pfk13 , pfcrt , and pfmdr1 genes. Day 3 clearance rates were ≥95% in all arms. Uncorrected day 28 Kaplan-Meier efficacy estimates ranged from 84.2 to 90.1% for the AL arms and 84.7 to 100% for the ASAQ arms. Corrected day 28 estimates were 87.6% (95% confidence interval [CI], 81 to 95%) for the AL arm in Lunda Sul, 92.2% (95% CI, 87 to 98%) for AL in Zaire, 95.6% (95% CI, 91 to 100%) for ASAQ in Zaire, 98.4% (95% CI, 96 to 100%) for AL in Benguela, and 100% for ASAQ in Benguela and Lunda Sul. All 103 analyzed samples had wild-type pfk13 sequences. The 76T pfcrt allele was found in most (92%; 11/12) ASAQ late-failure samples but in only 16% (4/25) of AL failure samples. The N86 pfmdr1 allele was found in 97% (34/35) of treatment failures. The AL efficacy in Lunda Sul was below the 90% World Health Organization threshold, the third time in four rounds that this threshold was crossed for an AL arm in Angola. In contrast, the observed ASAQ efficacy has not been below 95% to date in Angola, including this latest round., (Copyright © 2021 Dimbu et al.)

Immunosenescence is marked by a systemic process named inflammaging along with a series of defects in the immunological activity that results in poor responses to infectious agents and to vaccination. Inflammaging, a state of low-grade chronic inflammation, usually leads to chronic inflammatory diseases and frailty in the elderly. However, some elderly escape from frailty and reach advanced age free of the consequences of inflammaging. This process has been called immunological remodeling, and it is the hallmark of healthy aging as described in the studies of centenarians in Italy. The biological markers of healthy aging are still a matter of debate, and the studies on the topic have focused on inflammatory versus remodeling processes and molecules. The sub-clinical inflammatory status associated with aging might be a deleterious event for populations living in countries where chronic infectious diseases are not prevalent. Nevertheless, in other parts of the world where they are, two possibilities may occur. Inflammatory responses may have a protective effect against these infectious agents. At the same time, the long-term consequences of protective immune responses during chronic infections may result in accelerated immunosenescence in these individuals. Therefore, the biological markers of healthy aging can vary according to environmental, cultural, and geographical settings that reflect worldwide, and in a non-biased, non-westernized perspective, the changes that we experience regarding our contacts with microorganisms and the outcomes of such contacts., (Copyright © 2020 Batista, Calvo-Fortes, Silveira-Nunes, Camatta, Speziali, Turroni, Teixeira-Carvalho, Martins-Filho, Neretti, Maioli, Santos, Brigidi, Franceschi and Faria.)

Background: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear., Methods: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels., Results: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss., Conclusions: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

Background: Malaria is one of the main causes of death in Angola, particularly among children under 5 years of age. An essential means to improve the situation is with strong malaria case management; this includes diagnosing suspected patients with a confirmatory test, either with a rapid diagnostic test (RDT) or microscopy, prompt and correct treatment with artemisinin-based combination therapy (ACT), and proper case registration (track). In 2011, the United States President's Malaria Initiative (PMI) launched a country-wide programme to improve malaria case management through the provision of regular training and supervision at different levels of health care provision. An evaluation of malaria testing, treatment and registration practices in eight provinces, and at health facilities of various capacities, across Angola was conducted to assess progress of the national programme implementation., Methods: A retrospective assessment analysed data collected during supervision visits to health facilities conducted between 2012 and 2016 in 8 provinces in Angola. The supervision tool used data collected for malaria knowledge, testing, treatment and case registration practices among health workers as well as health facilities stock outs from different levels of health care delivery. Contingency tables with Pearson chi-squared (χ 2 ) tests were used to identify factors associated with "knowledge", "test", "treat" and "track." Multivariable logistic regression models were used to assess factors associated with the defined outcomes., Results: A total of 7156 supervisions were conducted between September 2012 and July 2016. The overall knowledge, testing, treatment and tracking practices among health care workers (HCWs) increased significantly from 2013 to 2016. Health care workers in 2016 were 3.3 times (95% CI: 2.7-3.9) as likely to have a higher knowledge about malaria case management as in 2013 (p < 0.01), 7.4 (95% CI: 6.1-9.0) times as likely to test more suspected cases (p < 0.01), 10.9 (95% CI: 8.6-13.6) times as likely to treat more confirmed cases (p < 0.01) and 3.7 (95% CI: 3.2-4.4) times as likely to report more accurately in the same period (p < 0.01)., Discussion: Improvements demonstrated in knowledge about malaria case management, testing with RDT and treatment with artemisinin-based combinations among HCWs is likely associated with malaria case management trainings and supportive supervisions. Gaps in testing and treatment practices are associated with RDT and ACT medicines stock outs in health facilities. Tracking of malaria cases still poses a major challenge, despite training and supervision. Hospitals consistently performed better compared to other health facilities against all parameters assessed; likely due to a better profile of HCWs., Conclusion: Significant progress in malaria case management in eight provinces Angola was achieved in the period of 2013-2016. Continued training and supportive supervision is essential to sustain gains and close existing gaps in malaria case management and reporting in Angola.

This report describes a survey of RNA and DNA viruses carried out in adult mosquitoes from Angola, raised under laboratory conditions from field-collected immature forms. This viral genomic survey was performed using different sets of primers targeting groups of arboviruses with a considerable impact on human health, including flaviviruses, alphaviruses, and phleboviruses. Furthermore, the viral survey that was performed also included detection of densoviruses. The obtained results did not reveal the presence of recognizable pathogenic arboviruses but allowed the identification of insect-specific flaviviruses from two genetic lineages and a single lineage of brevidensoviruses. These viruses, collectivelly detected in Anopheles sp. and Culex pipiens s.l. mosquitoes, were most probably transmitted vertically., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The extensive use of insecticides for vector control has led to the development of insecticide resistance in Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies., Competing Interests: The authors have declared that no competing interests exist.

Background: 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., Results: We used a bead-based serological assay to measure lactate dehydrogenase (LDH), aldolase (Aldo), and histidine-rich protein 2 (HRP2) levels in 196 children with Plasmodium falciparum malaria treated with effective antimalarials and followed for 28 to 42 days as part of therapeutic efficacy studies in Angola. Compared to pre-treatment levels, antigen concentrations two days after treatment declined by 99.7% for LDH, 96.3% for Aldo, and 54.6% for HRP2. After Day 2, assuming a first-order kinetics clearance model, half-lives of the antigens were 1.8 days (95% CI: 1.5-2.3) for LDH, 3.2 days (95% CI: 3.0-3.4) for Aldo, and 4.8 days (95% CI: 4.7-4.9) for HRP2., Conclusions: LDH and Aldo show substantially different clearance rates than HRP2, and their presence is largely indicative of active infection.

Low-density malaria infections are a source of human morbidity in endemic settings and potentially contribute to ongoing malaria transmission. Conventional rapid diagnostic tests (RDTs) were designed to detect clinically relevant parasite and antigen levels, but it is largely unknown what proportion of parasite (and antigen positive) infections are missed by conventional RDTs. Furthermore, RDTs can also provide false positives from lingering histidine-rich protein 2 (HRP2) antigenemia from a past infection. We analyzed 207 samples from Angolan outpatients with a bead-based HRP2 antigen assay and by qRT-PCR for the presence of parasite nucleic acids. Among patients HRP2 positive but negative by conventional RDT, the rate of quantitative reverse transcription-PCR (qRT-PCR) positivity was 45% (95% CI: 35-56%), with a median parasitemia of 3.4 parasites/µL (interquartile range: 0.14-4.8). Only 15% (7-26%) of HRP2-negative samples were found to have parasite nucleic acids. A substantial proportion of persons with blood HRP2 antigen concentrations not detected by the conventional RDT were found to have evidence of active infection, but at low parasite density levels.

The density of malaria parasites is a key determinant of whether an infected individual develops fever. While the pyrogenic threshold for malaria parasite density has been well studied, there are no analogous data on the antigen levels associated with fever during infection. Samples from 797 afebrile and 457 febrile outpatients from two provinces in Angola with known concentrations of histidine-rich protein 2 (HRP2), aldolase, and lactate dehydrogenase (LDH) antigens were analyzed by Bayesian latent class modeling to attribute malarial etiology to the fevers and to estimate the sensitivity and specificity of different antigen thresholds for detection of malaria fevers. Among patients with aldolase or LDH levels detectable with a bead-based assay, the concentrations of these two antigens did not differ between afebrile and febrile patients. In contrast, the concentrations of HRP2 were substantially higher in febrile HRP2-positive patients than in afebrile HRP2-positive patients. When HRP2 concentrations were considered, the malaria-attributable fractions of fever cases were 0.092 in Huambo Province and 0.39 in Uíge Province. Diagnostic tests detecting HRP2 with limits of detection (LODs) in the range of 3,000 to 10,000 pg/µl would provide ideal sensitivity and specificity for determination of malarial etiology among febrile persons., (Copyright © 2019 American Society for Microbiology.)

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis., Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed., Results: Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3., Conclusions: These are the first reports of Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density P. falciparum, non-falciparum, and Pfhrp2/3-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

Background: The Angolan government recommends three artemisinin-based combinations for the treatment of uncomplicated Plasmodium falciparum malaria: artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Due to the threat of emerging anti-malarial drug resistance, it is important to periodically monitor the efficacy of artemisinin-based combination therapy (ACT). This study evaluated these medications' therapeutic efficacy in Benguela, Lunda Sul, and Zaire Provinces., Methods: Enrollment occurred between March and July 2017. Study participants were children with P. falciparum monoinfection from each provincial capital. Participants received a 3-day course of a quality-assured artemisinin-based combination and were monitored for 28 (AL and ASAQ arms) or 42 days (DP arm). Each ACT was assessed in two provinces. The primary study endpoints were: (1) follow-up without complications and (2) failure to respond to treatment or development of recurrent P. falciparum infection. Parasites from each patient experiencing recurrent infection were genotyped to differentiate new infection from recrudescence of persistent parasitaemia. These parasites were also analysed for molecular markers associated with ACT resistance., Results: Of 608 children enrolled in the study, 540 (89%) reached a primary study endpoint. Parasitaemia was cleared within 3 days of medication administration in all participants, and no early treatment failures were observed. After exclusion of reinfections, the corrected efficacy of AL was 96% (91-100%, 95% confidence interval) in Zaire and 97% (93-100%) in Lunda Sul. The corrected efficacy of ASAQ was 100% (97-100%) in Benguela and 93% (88-99%) in Zaire. The corrected efficacy of DP was 100% (96-100%) in Benguela and 100% in Lunda Sul. No mutations associated with artemisinin resistance were identified in the pfk13 gene in the 38 cases of recurrent P. falciparum infection. All 33 treatment failures in the AL and ASAQ arms carried pfmdr1 or pfcrt mutations associated with lumefantrine and amodiaquine resistance, respectively, on day of failure., Conclusions: AL, ASAQ, and DP continue to be efficacious against P. falciparum malaria in these provinces of Angola. Rapid parasite clearance and the absence of genetic evidence of artemisinin resistance are consistent with full susceptibility to artemisinin derivatives. Periodic monitoring of in vivo drug efficacy remains a priority routine activity for Angola.

Background: Artemisinin-based combination therapy is the first-line anti-malarial treatment for uncomplicated Plasmodium falciparum infection in Angola. To date, the prevalence of polymorphisms in the pfk13 gene, associated with artemisinin resistance, and pfmdr1, associated with lumefantrine resistance, have not been systematically studied in Angola., Methods: DNA was isolated from pretreatment and late treatment failure dried blood spots collected during the 2015 round of therapeutic efficacy studies in Benguela, Lunda Sul, and Zaire Provinces in Angola. The pfk13 propeller domain and pfmdr1 gene were sequenced and analysed for polymorphisms. Pfmdr1 copy number variation was assessed using a real-time PCR method. The association between pfmdr1 and pfk13 mutations and treatment failure was investigated., Results: The majority of pretreatment (99%, 466/469) and all late treatment failure (100%, 50/50) samples were wild type for pfk13. Three of the pretreatment samples (1%) carried the A578S mutation commonly observed in Africa and not associated with artemisinin resistance. All 543 pretreatment and day of late treatment failure samples successfully analysed for pfmdr1 copy number variation carried one copy of pfmdr1. The NYD haplotype was the predominant pfmdr1 haplotype, present in 63% (308/491) of pretreatment samples, followed by NFD, which was present in 32% (157/491) of pretreatment samples. The pfmdr1 N86 allele was overrepresented in day of late treatment failure samples from participants receiving artemether-lumefantrine (p value 0.03)., Conclusions: The pretreatment parasites in patients participating in therapeutic efficacy studies in 2015 in Angola's three sentinel sites showed genetic evidence of susceptibility to artemisinins, consistent with clinical outcome data showing greater than 99% day 3 clearance rates. The lack of increased pfmdr1 copy number is consistent with previous reports from sub-Saharan Africa. Although pfmdr1 NYD and NFD haplotypes were overrepresented in artemether-lumefantrine late treatment failure samples, their role as markers of resistance was unclear given that these haplotypes were also present in the majority of successfully treated patients in the artemether-lumefantrine treatment arms.

Background: The response to antimalarial treatment is assessed using serial microscopy. New techniques for accurate measurement of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen have allowed for monitoring of the antigen concentration over time, offering a potential alternative for assessing treatment response., Methods: Posttreatment HRP2 concentrations were measured in samples obtained longitudinally from 537 individuals with P. falciparum malaria who were participating in efficacy trials in Angola, Tanzania, and Senegal. The HRP2 half-life was estimated using a first-order kinetics clearance model. The association between the HRP2 concentration 3 days after treatment and recrudescence of infection was assessed., Results: Despite substantial variation in HRP2 concentrations among participants at baseline, concentrations consistently showed a first-order exponential decline. The median half-life of HRP2 was estimated to be 4.5 days (interquartile range [IQR], 3.3-6.6 days) in Angola, 4.7 days (IQR, 4.0-5.9 days) in Tanzania, and 3.0 days (IQR, 2.1-4.5 days) in Senegal. The day 3 HRP2 concentration was predictive of eventual recrudescence, with an area under the receiver operating characteristic curve of 0.86 (95% confidence interval, .73-.99)., Conclusions: Consistent HRP2 clearance dynamics following successful antimalarial treatment imply a common underlying mechanism of biological clearance. Patients who ultimately did not respond to treatment did not exhibit this same pattern of clearance, even in the absence of other indications of inadequate response to treatment., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Most malaria testing is by rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein 2 (HRP2). Recently, several RDT manufacturers have developed highly sensitive RDTs (hsRDTs), promising a limit of detection (LOD) orders of magnitude lower than conventional RDTs. To model the added utility of hsRDTs, HRP2 concentration in Angolan outpatients was measured quantitatively using an ultrasensitive bead-based assay. The distribution of HRP2 concentration was bimodal in both afebrile and febrile patients. The conventional RDT was able to detect 81% of all HRP2-positive febrile patients and 52-77% of HRP2-positive afebrile patients. The added utility of hsRDTs was estimated to be greater in afebrile patients, where an hsRDT with a LOD of 200 pg/mL would detect an additional 50-60% of HRP2-positive persons compared with a conventional RDT with a LOD of 3,000 pg/mL. In febrile patients, the hsRDT would detect an additional 10-20% of cases. Conventional RDTs already capture the vast majority of symptomatic HRP2-positive individuals, and hsRDTs would have to reach a sufficiently low LOD approaching 200 pg/mL to provide added utility in identifying HRP2-positive, asymptomatic individuals.

Microscopic blood smear examinations done in health centers of Angola demonstrated a large overdiagnosis of malaria cases with an average rate of errors as high as 85%. Overall 83% of patients who received Coartem ® had an inappropriate treatment. Overestimated malaria diagnosis was noticed even when specific symptoms were part of the clinical observation, antimalarial treatments being subsequently given. Then, malaria overdiagnosis has three main consequences, (i) the lack of data reliability is of great concern, impeding epidemiological records and evaluation of the actual influence of operations as scheduled by the National Malaria Control Programme; (ii) the large misuse of antimalarial drug can increase the selective pressure for resistant strain and can make a false consideration of drug resistant P. falciparum crisis; and (iii) the need of strengthening national health centers in term of human, with training in microscopy, and equipment resources to improve malaria diagnosis with a large scale use of rapid diagnostic tests associated with thick blood smears, backed up by a "quality control" developed by the national health authorities. Monitoring of malaria cases was done in three Angolan health centers of Alto Liro (Lobito town) and neighbor villages of Cambambi and Asseque (Benguéla Province) to evaluate the real burden of malaria. Carriers of Plasmodium among patients of newly-borne to 14 years old, with or without fever, were analyzed and compared to presumptive malaria cases diagnosed in these health centers. Presumptive malaria cases were diagnosed six times more than the positive thick blood smears done on the same children. In Alto Liro health center, the percentage of diagnosis error reached 98%, while in Cambambi and Asseque it was of 79% and 78% respectively. The percentage of confirmed malaria cases was significantly higher during the dry (20.2%) than the rainy (13.2%) season. These observations in three peripheral health centers confirmed what has already been noticed in other malaria endemic regions, and highlight the need for an accurate evaluation of the Malaria control programme implemented in Angola., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Malaria accounts for the largest portion of healthcare demand in Angola. A pillar of malaria control in Angola is the appropriate management of malaria illness, including testing of suspect cases with rapid diagnostic tests (RDTs) and treatment of confirmed cases with artemisinin-based combination therapy (ACT). Periodic systematic evaluations of malaria case management are recommended to measure health facility readiness and adherence to national case management guidelines., Methods: Cross-sectional health facility surveys were performed in low-transmission Huambo and high-transmission Uíge Provinces in early 2016. In each province, 45 health facilities were randomly selected from among all public health facilities stratified by level of care. Survey teams performed inventories of malaria commodities and conducted exit interviews and re-examinations, including RDT testing, of a random selection of all patients completing outpatient consultations. Key health facility readiness and case management indicators were calculated adjusting for the cluster sampling design and utilization., Results: Availability of RDTs or microscopy on the day of the survey was 71% (54-83) in Huambo and 85% (67-94) in Uíge. At least one unit dose pack of one formulation of an ACT (usually artemether-lumefantrine) was available in 83% (66-92) of health facilities in Huambo and 79% (61-90) of health facilities in Uíge. Testing rates of suspect malaria cases in Huambo were 30% (23-38) versus 69% (53-81) in Uíge. Overall, 28% (13-49) of patients with uncomplicated malaria, as determined during the re-examination, were appropriately treated with an ACT with the correct dose in Huambo, compared to 60% (42-75) in Uíge. Incorrect case management of suspect malaria cases was associated with lack of healthcare worker training in Huambo and ACT stock-outs in Uíge., Conclusions: The results reveal important differences between provinces. Despite similar availability of testing and ACT, testing and treatment rates were lower in Huambo compared to Uíge. A majority of true malaria cases seeking care in health facilities in Huambo were not appropriately treated with anti-malarials, highlighting the importance of continued training and supervision of healthcare workers in malaria case management, particularly in areas with decreased malaria transmission.

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., Competing Interests: The authors have declared that no competing interests exist.

Background: 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., Methods: During January-June 2015, investigators monitored the clinical and parasitological response of children with uncomplicated Plasmodium falciparum infection treated with AL, artesunate-amodiaquine (ASAQ), or dihydroartemisinin-piperaquine (DP). The study comprised two treatment arms in each of three provinces: Benguela (AL, ASAQ), Zaire (AL, DP), and Lunda Sul (ASAQ, DP). Samples from treatment failures were analysed for molecular markers of resistance for artemisinin (K13) and lumefantrine (pfmdr1)., Results: A total of 467 children reached a study endpoint. Fifty-four treatment failures were observed: four early treatment failures, 40 re-infections and ten recrudescences. Excluding re-infections, the 28-day microsatellite-corrected efficacy was 96.3% (95% CI 91-100) for AL in Benguela, 99.9% (95-100) for ASAQ in Benguela, 88.1% (81-95) for AL in Zaire, and 100% for ASAQ in Lunda Sul. For DP, the 42-day corrected efficacy was 98.8% (96-100) in Zaire and 100% in Lunda Sul. All treatment failures were wild type for K13, but all AL treatment failures had pfmdr1 haplotypes associated with decreased lumefantrine susceptibility., Conclusions: No evidence was found to corroborate the specific allegation of artemisinin resistance in Lunda Sul. The efficacy below 90% of AL in Zaire matches findings from 2013 from the same site. Further monitoring, particularly including measurement of lumefantrine blood levels, is recommended.

Background: Artemisinin-based combination therapies (ACTs) against malaria are subsidized in many African countries, but the impact of subsidy programs in reducing the sales of concomitantly available antimalarial monotherapies is poorly defined., Methods: Data from The MENTOR initiative, that introduced subsidized artemether-lumefantrine (sAL) in the private sector of Huambo province, Angola, were used. The main response variable was represented by sales of sAL and of monotherapies, measured as number of treatment courses. Sales in private pharmacies of sAL and four antimalarial monotherapies between 2009 and 2013 were organized in four time-periods, and analyzed using generalized linear models for repeated measures. A secondary analysis evaluated changes in relative market share., Results: We analyzed data from 34 pharmacies at four time points, taken from a larger survey that involved 165 pharmacies between June 2009 and March 2013. The sAL, following its introduction, became the dominant antimalarial treatment in the private sector, usually exceeding the total sales of all antimalarial monotherapies combined (1480/2800 total treatment courses, 52.8% of all sales in March 2013). Sales of monotherapies decreased significantly, but did not stop, representing 36.7% (1028/2800) of sales at the end of the survey., Conclusions: Subsidized ACTs can attain rapidly a high relative market share. Their introduction reduced, but did not eliminate the demand for less effective monotherapies, that might favor parasite resistance., (© The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Angola is a sub-Saharan country in southern Africa highly affected by diarrhoeal disease with limited epidemiological data regarding etiologic agents. This study was performed during 2012-2013, prior to rotavirus vaccine introduction, with the objective to detect and characterize the rotavirus strains circulating in four provinces of the country: Huambo, Luanda, Zaire, and Cabinda. A high rotavirus detection rate (35%, 117/334) was observed. G1 was the most common G-genotype (83.6%), whereas P[8] (50.9%) followed by P[6] (38.8%) were the most common P-types. G1P[8] was identified as the predominant combination (50%), followed by the unusual G1P[6] (29.3%). Strains such G2P[4], G8P[6], G9P[6], and G12P[6] were also found in lower frequencies (5.2-1.7%). The P[6] strains did not cluster in the phylogenetic trees according to their geographic origin or even the corresponding G-genotype, suggesting a limited number of recent introductions and extensive reassortment events. Our results represent the first report on rotavirus genotype profiles in Angola, showing a wide circulation of the unusual genotype G1P[6], and underline the importance of RV surveillance after the vaccine introduction. J. Med. Virol. 88:1511-1520, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively., Methods: Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing., Results: In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted., Conclusion: pfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

Premenopausal women are known to show lower incidence of cardiovascular disease than men. During myocardial infarction (MI), homeostatic responses are activated, including the sympathetic autonomic nervous system and the rennin-angiotensin-aldosterone system, which is related to the fluid and electrolyte balance, both aiming to maintain cardiac output. This study sought to perform a serial evaluation of sexual dimorphism in cardiac autonomic control and fluid and electrolyte balance during the development of MI-induced heart failure in rats. Experimental MI was induced in male (M) and female (F) adult (7-9 weeks of age) Wistar rats. The animals were placed in metabolic cages to assess fluid intake and urine volume 1 and 4 weeks after inducing MI (male myocardial infarction (MMI) and female myocardial infarction (FMI) groups). They subsequently underwent echocardiographic evaluation and spectral analysis of heart rate variability. After completing each protocol, the animals were killed for postmortem evaluation and histology. The MMI group showed earlier and more intense cardiac morphological and functional changes than the FMI group, although the extent of MI did not differ between groups (P > 0.05). The MMI group showed higher sympathetic modulation and sodium and water retention than the FMI group (P < 0.05), which may partly explain both the echocardiographic and pathological findings. Females subjected to infarction seem to show attenuation of sympathetic modulation, more favourable fluid and electrolyte balances, and better preserved cardiac function compared to males subjected to the same infarction model., (© 2016 John Wiley & Sons Australia, Ltd.)

Introduction: Intestinal parasites are responsible for morbidity in children worldwide, especially in low income countries. In the present study we determine the prevalence of intestinal parasites and explore its association with anemia and stunting in school-aged children., Methods: A cross-sectional study was conducted from September to October 2010 enrolling 328 children attending the primary school in Lubango, the second largest city after the capital Luanda. Stool samples were collected for parasite detection through microscopy and molecular identification of Entamoeba histolytica and Entamoeba dispar. Stunting was assessed using the z-scores of height for age and hemoglobin concentration was determined using a portable hemoglobin analyzing system., Results: The global prevalence of pathogenic intestinal parasites was 44.2%, the most common being Ascaris lumbricoides (22.0%), Giardia lamblia (20.1%) and Hymenolepis nana (8.8%). Molecular detection revealed that 13.1% of the children carried E. dispar and 0.3% were infected with E. histolytica. The prevalence of stunting (mild to severe) was 41.5%. Stunting was more frequent in older children (p = 0.006, OR = 1.886), while anemia was more frequent in younger children (p = 0.005, OR = 2.210). The prevalence of anemia was 21.6%, and we found a significant association with infection by H. nana (p = 0.031, OR = 2.449)., Conclusions: This is one of the few published studies reporting intestinal parasites infection, nutritional status and anemia in children from Angola. Furthermore, the present work highlights the importance of regular intestinal parasites screening in children.

From 2003 to 2007, entomological surveys were conducted in Lobito town (Benguela Province, Angola) to determine which Anopheles species were present and to identify the vectors responsible for malaria transmission in areas where workers of the Sonamet Company live. Two types of surveys were conducted: (1) time and space surveys in the low and upper parts of Lobito during the rainy and dry periods; (2) a two-year longitudinal study in Sonamet workers' houses provided with long-lasting insecticide-treated nets (LLIN), "PermaNet," along with the neighboring community. Both species, An. coluzzii (M molecular form) and An. gambiae (S molecular form), were collected. Anopheles coluzzii was predominant during the dry season in the low part of Lobito where larvae develop in natural ponds and temporary pools. However, during the rainy season, An. gambiae was found in higher proportions in the upper part of the town where larvae were collected in domestic water tanks built near houses. Anopheles melas and An. listeri were captured in higher numbers during the dry season and in the low part of Lobito where larvae develop in stagnant brackish water pools. The infectivity rates of An. gambiae s.l. varied from 0.90% to 3.41%., (© 2015 The Society for Vector Ecology.)

Evaluation of vector control is crucial for improving malaria containment and, according to World Health Organization, new complementary indicators would be very valuable. In this study the IgG response to the Anopheles-specific cE5 salivary protein was tested as a tool to evaluate the efficacy of insecticide-treated nets in reducing human exposure to malaria vectors. Sera collected during a longitudinal study carried out in Angola, and including entomological and parasitological data, were used to assess the IgG response to the Anopheles gambiae cE5 in both children and adults, before and after the application of insecticide-treated nets. Seasonal fluctuation of specific IgG antibody levels according to exposure was only found in children (up to ≈ 14 years old) whose anti-cE5 IgG response dropped after bed nets installation. These results were fully consistent with previous findings obtained with the same set of sera and indicating a substantial reduction of human-vector contact shortly after nets implementation. Overall, children IgG response to the cE5 protein appeared a very sensitive biomarker, which allowed for the detection of even weak exposure to Anopheles bites, indicating it may represent a reliable additional tool to evaluate the efficacy of vector control interventions., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: The malaria situation has been worsening in Angola, partly due to armed conflict until the recent past and drug-resistant Plasmodium falciparum. Malaria transmission is heterogeneous within the country, and data on drug-resistant malaria in different parts of the country are incomplete. The aim of the present study was to evaluate resistance to 4-aminoquinolines and antifolate drugs in P. falciparum isolates collected in Benguela province, central Angola, using molecular markers., Methods: Fingerprick capillary blood was collected from asymptomatic children aged less than 15 years old during a household survey in and around Balombo town in 2010-2011. Samples were screened for P. falciparum by nested PCR. Molecular markers (P. falciparum dihydrofolate reductase [pfdhfr], P. falciparum dihydropteroate synthase [pfdhps], P. falciparum chloroquine resistance transporter [pfcrt], and P. falciparum multidrug-resistance gene 1 [pfmdr1]) were sequenced to determine the key codons associated with drug resistance., Results: A total of 60 blood samples were positive for P. falciparum. Most isolates with successful PCR amplification had mutant pfdhfr alleles, with either double mutant AICNI (69%) or triple mutant AIRNI (21%) haplotypes. A16V, S108T, and I164L substitutions were not found. Many of the isolates were carriers of either SGKAA (60%) or AGKAA (27%) pfdhps haplotype. K540E substitution was absent. There were only two pfcrt haplotypes: wild-type CVMNK (11%) and mutant CVIET (89%). Wild-type pfmdr1 NYSND haplotype was found in 19% of the isolates, whereas single mutant pfmdr1 YYSND and NFSND haplotypes occurred in 48% and 11%, respectively. Double mutant pfmdr1 haplotypes (YFSND and YYSNY) occurred rarely., Conclusions: The results suggest that the high prevalence of mutant pfcrt CVIET haplotype is in agreement with low clinical efficacy of chloroquine observed in earlier studies and that the double pfdhfr mutant AICNI and single pfdhps mutant SGKAA are currently the predominant haplotypes associated with antifolate resistance in Benguela province. The hallmark of clinical resistance observed in East Africa, i.e. triple pfdhfr mutant haplotype (AIRNI) and double pfdhps mutant haplotype (SGEAA), was absent. These molecular findings need to be further evaluated in parallel with clinical studies, in particular with the efficacy of intermittent preventive treatment using sulphadoxine-pyrimethamine in pregnant women and artesunate-amodiaquine for uncomplicated malaria.

The development of resistance to antimalarials is a major challenge for global malaria control. Artemisinin-based combination therapies, the newest class of antimalarials, are used worldwide but there have been reports of artemisinin resistance in Southeast Asia. In February through May 2013, we conducted open-label, nonrandomized therapeutic efficacy studies of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) in Zaire and Uíge Provinces in northern Angola. The parasitological and clinical responses to treatment in children with uncomplicated Plasmodium falciparum monoinfection were measured over 28 days, and the main outcome was a PCR-corrected adequate clinical and parasitological response (ACPR) proportion on day 28. Parasites from treatment failures were analyzed for the presence of putative molecular markers of resistance to lumefantrine and artemisinins, including the recently identified mutations in the K13 propeller gene. In the 320 children finishing the study, 25 treatment failures were observed: 24 in the AL arms and 1 in the DP arm. The PCR-corrected ACPR proportions on day 28 for AL were 88% (95% confidence interval [CI], 78 to 95%) in Zaire and 97% (91 to 100%) in Uíge. For DP, the proportions were 100% (95 to 100%) in Zaire, and 100% (96 to 100%) in Uíge. None of the treatment failures had molecular evidence of artemisinin resistance. In contrast, 91% of AL late-treatment failures had markers associated with lumefantrine resistance on the day of failure. The absence of molecular markers for artemisinin resistance and the observed efficacies of both drug combinations suggest no evidence of artemisinin resistance in northern Angola. There is evidence of increased lumefantrine resistance in Zaire, which should continue to be monitored., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: In Angola, malaria is an endemic disease having a major impact on the economy. The WHO recommends testing for all suspected malaria cases, to avoid the presumptive treatment of this disease. In malaria endemic regions laboratory technicians must be very comfortable with microscopy, the golden standard for malaria diagnosis, to avoid the incorrect diagnosis. The improper use of medication promotes drug resistance and undesirable side effects. The present study aims to assess the impact of a three-day refresher course on the knowledge of technicians, quality of blood smears preparation and accuracy of microscopy malaria diagnosis, using qPCR as reference method., Methods: This study was implemented in laboratories from three hospitals in different provinces of Angola: Bengo, Benguela and Luanda. In each laboratory samples were collected before and after the training course (slide with thin and thick blood smears, a dried blood spot and a form). The impact of the intervention was evaluated through a written test, the quality of slide preparation and the performance of microscopy., Results: It was found a significant increase on the written test median score, from 52.5% to 65.0%. A total of 973 slides were analysed to evaluate the quality of thick and thin blood smears. Considering all laboratories there was a significant increase in quality of thick and thin blood smears. To determine the performance of microscopy using qPCR as the reference method we used 1,028 samples. Benguela presented the highest values for specificity, 92.9% and 98.8% pre and post-course, respectively and for sensitivity the best pre-course was Benguela (75.9%) and post-course Luanda (75.0%). However, no significant increase in sensitivity and specificity after the training course was registered in any laboratory analysed., Discussion: The findings of this study support the need of continuous refresher training for microscopists and other laboratory staff. The laboratories should have a quality control programme to supervise the diagnosis and also to assess the periodicity of new training. However, other variables needed to be considered to have a correct malaria diagnosis, such as adequate equipment and reagents for staining and visualization, good working conditions, motivated and qualified personnel.

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

Human antibody (Ab) response to Anopheles whole saliva, used as biomarker of Anopheles exposure, was investigated over a period of two years (2008-2009), in children between 2 to 9 years old, before and after the introduction of three different malaria vector control methods; deltamethrin treated long lasting impregnated nets (LLIN) and insecticide treated plastic sheeting (ITPS)--Zero Fly®) (ITPS-ZF), deltamethrin impregnated Durable (Wall) Lining (ITPS-DL--Zerovector®) alone, and indoor residual spraying (IRS) with lambdacyhalothrin alone. These different vector control methods resulted in considerable decreases in all three entomological (82.4%), parasitological (54.8%) and immunological criteria analyzed. The highest reductions in the number of Anopheles collected and number of positive blood smears, respectively 82.1% and 58.3%, were found in Capango and Canjala where LLIN and ITPS-ZF were implemented. The immunological data based on the level of anti-saliva IgG Ab in children of all villages dropped significantly from 2008 to 2009, except in Chissequele. These results indicated that these three vector control methods significantly reduced malaria infections amongst the children studied and IRS significantly reduced the human-Anopheles contact. The number of Anopheles, positive blood smears, and the levels of anti-saliva IgG Ab were most reduced when LLIN and ITPS-ZF were used in combination, compared to the use of one vector control method alone, either ITPS-DL or IRS. Therefore, as a combination of two vector control methods is significantly more effective than one control method only, this control strategy should be further developed at a more global scale.

Background: Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola., Methods: Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes., Results: The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples)., Conclusion: This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola.

Background: The inherited, low-penetrance arginine-to-histidine substitution at codon 337 (R337H) of the tumor protein 53 gene (TP53) is clustered in southeast Brazil (estimated frequency, 0.3%). Although its tumorigenic effect initially appeared to be tissue-specific, recent evidence suggests its association with a broader range of tumors. Therefore, the authors of this report investigated the spectrum of pediatric malignancies associated with the TP53 R337H mutation at a single referral institution in southeast Brazil., Methods: Genomic DNA samples from 493 children with malignancies were screened for the R337H mutation. Available tumor samples from carriers were investigated for loss of heterozygosity (LOH) and nuclear p53 accumulation. Clinical data were obtained from medical records., Results: Sixty-five of 70 patients (93%) with adrenocortical tumors (ACTs), 9 of 13 patients (69%) with choroid plexus carcinoma (CPC), and 3 of 41 patients (7.3%) with osteosarcoma carried the mutation. The proportion of CPC to choroid plexus papilloma (CPP) was much higher than that reported elsewhere. Osteosarcoma in carriers had a significantly poorer outcome (P = .02). The mutation was not identified in patients who had acute lymphoblastic leukemia (ALL) (n = 187), recurrent ALL (n = 49), acute myeloid leukemia (n = 44), lymphoma (n = 30), non-CPC central nervous system tumors (n = 26), Ewing sarcoma (n = 25), or rhabdomyosarcoma (n = 8). Among the tumors that were available for analysis, LOH with retention of the mutant allele was confirmed in 21 of 21 ACTs, in 2 of 2 CPCs, and in 2 of 3 osteosarcomas that were positive for R337H. CPCs and osteosarcomas that were positive for R337H had marked nuclear accumulation of p53., Conclusions: The current findings demonstrated compellingly that the TP53 R337H mutation is associated not only with ACT but also with CPC and, to a lesser extent, with osteosarcoma, both of which are core-component tumors of the Li-Fraumeni syndrome., (2010 American Cancer Society.)

Background: Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to Plasmodium falciparum infection are pregnant women and children under five years of age. The use of an intermittent preventive treatment (IPT) with sulphadoxine/pyrimethamine (SP) in pregnant women was introduced in Angola in 2006 by the National Malaria Control Programme, and currently this strategy has been considered to be used for children malaria control. Considering the previous wide use of SP combination in Angola, together to the reported cases of SP treatment failure it is crucial the evaluation of the prevalence of five mutations in pfdhfr and pfdhps genes associated to P. falciparum resistance to SP before the introduction of S/P IPT in children., Methods: The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in pfdhfr and pfdhps genes was carried out in 452 P. falciparum blood samples by PCR RFLP., Results: For pfdhfr gene, 90,3% of the samples carried the mutation 51I, with 7.5% of mixed infections; 51% carried wild type allele 59C, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 108N. Concerning, pfdhps gene, 83,1% were mutant type 437G with 11% mixed infections , while 87% of the studied isolates were wild type for codon 540., Discussion: This is the first representative epidemiological study of the whole Angola country on the prevalence of the genotypes associated with SP chemoresistance. A high frequency of individual mutations in both genes (51I and 108N in pfdhfr, and 437G in pfdhps) was found, besides a low prevalence of the quintuple mutation., Conclusion: The data showed that the implementation IPT using SP in children needs to be reviewed.

Laser-induced breakdown spectroscopy (LIBS) has been used to identify the differences or similarities between crude oil and fuel residues. Firstly, a man portable LIBS analyzer was used for the on-site environmental control and analysis of the oil spill from The Prestige. An exhaustive analysis of crude oil and oil spill residues (collected during the field campaign in the Galician Coast) was performed in the laboratory. Characteristics elements in petroleum such as C, H, N, O, Mg, Na, Fe and V were detected. In addition, contributions from Ca, Si and Al in the composition of residues have been found. The use of intensity ratios of line and band emissions in the original fuel (crude oil) and in the aged residues allowed a better characterization of the samples than the simple use of peak intensities. The chemical composition between the crude oil and the fuel residues was found completely different. As well, a statistical method was employed in order to discriminate residues. Although significant differences were observed, no conclusions in terms of age and provenance could be reached due to the unknowledgment in the origin of the samples., (Copyright © 2010 Elsevier B.V. All rights reserved.)

To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of Anopheles gambiae salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to Anopheles salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to Anopheles. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact.

For the fight against malaria, the World Health Organization (WHO) has emphasized the need for indicators to evaluate the efficacy of vector-control strategies. This study investigates a potential immunological marker, based on human antibody responses to Anopheles saliva, as a new indicator to evaluate the efficacy of insecticide-treated nets (ITNs). Parasitological, entomological, and immunological assessments were carried out in children and adults from a malaria-endemic region of Angola before and after the introduction of ITNs. Immunoglobulin G (IgG) levels to An. gambiae saliva were positively associated with the intensity of An. gambiae exposure and malaria infection. A significant decrease in the anti-saliva IgG response was observed after the introduction of ITNs, and this was associated with a drop in parasite load. This study represents the first stage in the development of a new indicator to evaluate the efficacy of malaria vector-control strategies, which could apply in other arthropod vector-borne diseases.