Irene Molina-De la Fuente

The routine diagnostic method used for clinical samples suspected of filarial infection in our laboratory is the Filaria‐real time‐PCR (F‐RT‐PCR). The drawback of this method is the need for melting temperature analysis and PCR products' electrophoresis to identify the filarial species. Therefore, the aim of this study was to design a quantitative real‐time PCR (qPCR) assay using filarial‐specific hydrolysis probes, targeting 18S rRNA and ITS1 genes, allowing the simultaneous diagnosis of loiasis, mansonellosis and other human filariasis without the need of electrophoresis or melting temperature analysis. To achieve this objective, three filarial probes (Fil‐Hum‐GT1, Loa‐Hum‐GT2 and Mp‐Hum‐GT2) were designed, optimised and validated for integration into a single qPCR assay, named filarial‐multiplexed probe‐quantitative PCR (F‐mp‐qPCR). For the optimisation and validation of the F‐mp‐qPCR method, a total of 304 clinical samples as dried blood spot were used with their corresponding thick blood smears stained by Giemsa 3%. The detection limit of the Fil‐Hum‐GT1, Loa‐Hum‐GT2 and Mp‐Hum‐GT2 probes was 0.05, 0.5 and 3 mF/mL, respectively. The most sensitive and specific probe was the general filarial probe Fil‐Hum‐GT1, with a sensitivity of 92.0% to detect L. loa, 88.6% to detect M. perstans and 85.7% to detect mixed infections, with 100% specificity. Agreement with microscopy was excellent for the Fil‐Hum‐GT1 probe. In contrast, the Mp‐Hum‐GT2 probe showed the lowest performance, with a sensitivity of 81.8% to detect M. perstans, decreasing to 42.9% for mixed infections. Although the developed method did not prove to be significantly more sensitive than microscopy, this novel method is faster and easier to perform compared to microscopy and is very useful for screening large population groups in a context of medium‐to‐low human filariasis transmission.

: Artemisinin-combined treatments are the recommended first-line treatment of Plasmodium falciparum malaria, but they are being threatened by emerging artemisinin resistance. Mutations in pfk13 are the principal molecular marker for artemisinin resistance. This study characterizes the presence of mutations in pfk13 in P. falciparum in Western Equatoria State, South Sudan. We analyzed 468 samples from patients with symptomatic malaria and found 15 mutations (8 nonsynonymous and 7 synonymous). Each mutation appeared only once, and none were validated or candidate markers of artemisinin resistance. However, some mutations were in the same or following position of validated and candidate resistance markers, suggesting instability of the gene that could lead to resistance. The R561L nonsynonymous mutation was found in the same position as the R561H validated mutation. Moreover, the A578S mutation, which is widespread in Africa, was also reported in this study. We found a high diversity of other pfk13 mutations in low frequency. Therefore, routine molecular surveillance of resistance markers is highly recommended to promptly detect the emergence of resistance-related mutations and to limit their spread.

Eritrea is the first African country to switch away from exclusive use of HRP2-based RDTs for the detection of P. falciparum due to high prevalence of pfhrp2 / 3- deleted P. falciparum parasites causing false-negative RDT results. While heavy reliance on malaria RDTs played a significant role in the rapid expansion of pfhrp2/3 -deleted parasites in Eritrea, we hypothesize that the use of antimalarial (artesunate-amodiaquine) may have also contributed to their spread. We conducted a retrospective investigation of mutations in the propeller domain of the P. falciparum kelch13 gene in samples collected in 2016 (n=50) from the Northern Red Sea Zone before the RDT switch away from HRP2-RDTs and in samples collected in 2018-2020 (n=587) from the Gash Barka, Anseba and Debub Zones after the RDT switch. No mutations were identified in the 2016 samples. However, in 2018-2019 samples, we detected five different single non-synonymous mutations. The most prevalent mutation was pfk13 R622I, which was detected in samples collected from all health centres, with an overall prevalence of 11.9% (ranging from 5.9% to 28%). Parasites carrying the R622I mutation have diverse microsatellite marker haplotypes, indicating that they had evolved multiple times from different genetic backgrounds. The prevalence pfk13 R622I was significantly higher in single pfhrp3 -deleted parasites (18.0%) compared to parasites without pfhrp2/3 deletions (6.2%) and dual pfhrp2/3 -deleted parasites (9.0%), suggesting association between the pfk13 R622I mutation and the pfhrp2/3 deletions in Eritrea. Continuous monitoring the trends in pfhrp2/3 and pfk13 mutants is needed to inform effective malaria management strategies in Eritrea.

Objective Description of tuberculosis (TB)-focused point-of-care ultrasound (POCUS) findings for children with presumptive TB. Design Cross-sectional study (July 2019 to April 2020). Setting Simão Mendes hospital in Bissau, a setting with high TB, HIV, and malnutrition burdens. Participants Patients aged between 6 months and 15 years with presumptive TB. Interventions Participants underwent clinical, laboratory and unblinded clinician-performed POCUS assessments, to assess subpleural nodules (SUNs), lung consolidation, pleural and pericardial effusion, abdominal lymphadenopathy, focal splenic and hepatic lesions and ascites. Presence of any sign prompted a POCUS positive result. Ultrasound images and clips were evaluated by expert reviewers and, in case of discordance, by a second reviewer. Children were categorised as confirmed TB (microbiological diagnosis), unconfirmed TB (clinical diagnosis) or unlikely TB. Ultrasound findings were analysed per TB category and risk factor: HIV co-infection, malnutrition and age. Results A total of 139 children were enrolled, with 62 (45%) women and 55 (40%) aged Conclusions We found a high prevalence of POCUS signs in children with TB compared with children with unlikely TB. POCUS-positivity was dependent on nutritional status but not on HIV status or age. TB-focused POCUS could potentially play a supportive role in the diagnosis of TB in children. Trial registration number NCT05364593.

Background: Although rapid diagnostic tests (RDTs) play a key role in malaria-control strategies, their efficacy has been threatened by deletion and genetic variability of the genes pfhrp2/3. This study aims to characterize the deletion, genetic patterns and diversity of these genes and their implication for malaria RDT effectiveness, as well as their genetic evolution in the Amhara region of Ethiopia. Methods: The study included 354 isolates from symptomatic patients from the Amhara region of Ethiopia who tested positive by microscopy. Exon 1–2 and exon 2 of genes pfhrp2 and -3 were amplified, and exon 2 was sequenced to analyse the genetic diversity, phylogenetic relationship and epitope availability. Results: The deletion frequency in exon 1–2 and exon 2 was 22 and 4.6% for pfhrp2, and 68 and 18% for pfhrp3, respectively. Double deletion frequency for pfhrp2 and pfhrp3 was 1.4%. High genetic diversity, lack of clustering by phylogenetic analysis and evidence of positive selection suggested a diversifying selection for both genes. The amino-acid sequences, classified into different haplotypes, varied widely in terms of frequency of repeats, with novel amino-acid changes. Aminoacidic repetition type 2 and type 7 were the most frequent in all the sequences. The most frequent epitopes among protein sequences were those recognized by MAbs 3A4 and C1-13. Conclusion: Deletions and high amino acidic variation in pfhrp2 and pfhrp3 suggest their possible impact on RDT use in the Amhara region, and the high genetic diversity of these genes could be associated with a diversifying selection in Ethiopia. Surveillance of these genes is, therefore, essential to ensure the effectiveness of public health interventions in this region. Graphical

Background: Malaria is one of the deadliest disease in the world, particularly in Africa. The resistance to antimalarial drugs is one of the most important problems to global malaria control. The study assess the evolution of the different resistance markers over time and the possible influence of the interventions and treatment changes that have been made in Equatorial Guinea.Methods: 1223 biological samples distributed from 1999 to 2019 were included in the study. The screening of the mutations in pfdhfr, pfdhps, pfmdr1 and pfcrt genes were carried out by nested PCR and RFLPs, and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations Results: the haplotypes partially and fully resistant (pfdhfr + pfdhps) increase over time. In 2019, the fully resistant is increasing although super resistant remains lower. There is also a continued decline over time in pfmdr1 and pfcrt gene mutations. Since 2008, when ACTs were introduced in the country, the number of mutations detected in pfk13 has been increasing over time, with more mutations being observed in 2019. In this year, were detected 2 synonymous and 5 non-synonymous mutations, although are not related to resistance to ACTs. In addition, the non-synonymous A578S mutation, the most frequent on the African continent, was detected in 2013, although it has not been detected in the following years.Conclusions: The withdrawal of the use of CQ as a treatment in the country has been effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in SP resistance markers evidence its misuse alone or in combination with AS or AQ in some areas of the country, this allows selective pressure from SP to continue. SNPs 540E and 581G do not exceed the limit of 50% and 10% respectively, which means that SP as an IPT is still effective in the country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACTs. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.

Background: Deletion of pfhrp2 and/or pfhrp3 genes cause false negatives in malaria rapid diagnostic test (RDT) and threating malaria control strategies. This systematic review aims to assess the main methodological aspects in the study of pfhrp2 and pfhrp3 gene deletions and its global epidemiological status, with special focus on their distribution in Africa; and its possible impact in RDT. Methods: The systematic review was conducted by examining the principal issues of study design and methodological workflow of studies addressing pfhrp2 deletion. Meta-analysis was applied to represent reported prevalences of pfhrp2 and pfhrp3 single and double deletion in the World Health Organization (WHO) region. Pooled-prevalence of deletions was calculated using DerSimonnian-Laird random effect model. Then, in-deep analysis focused on Africa was performed to assess possible variables related with these deletions. Finally, the impact of these deletions in RDT results was analysed combining reported information about RDT sensitivity and deletion prevalences. Results: 49 articles were included for the systematic review and 37 for the meta-analysis, 13 of them placed in Africa. Study design differs significantly, especially in terms of population sample and information reported, resulting in high heterogeneity between studies that difficulties comparisons and merged conclusions. Reported prevalences vary widely in all the WHO regions, significantly higher deletion were reported in South-Central America, following by Africa and Asia. Pfhrp3 deletion is more prevalent (43% in South-Central America; 3% in Africa; and 1% in Asia) than pfhrp2 deletion (18% in South-Central America; 4% in Africa; and 3% in Asia) worldwide. In Africa, there were not found differences in deletion prevalence by geographical or population origin of samples. The prevalence of deletion among false negatives ranged from 0 to 100% in Africa, but in Asia and South-Central America was only up to 90% and 48%, respectively, showing substantial relation between deletions and false negatives. Conclusion: The concerning prevalence of pfhrp2, pfhrp3 and pfhrp2/3 gene deletions, as its possible implications in malaria control, highlights the importance of regular and systematic surveillance of these deletions. This review has also outlined that a standardized methodology could play a key role to ensure comparability between studies to get global conclusions.

This study describes the alcohol environment comparing residents’ perceptions and objective measures in two different income-level districts. Measures were gathered between 2017 and 2018 in two districts with different income levels in Madrid, Spain. We obtained perceived measures using Photovoice. We procured objective measures through social systematic observation. Data were integrated using triangulation. Perceived and objective measures of the alcohol environment were characterized and compared in terms of alcohol availability, alcohol promotion, and signs of alcohol consumption. The integration was classified as agreement, partial agreement, or dissonance. The results related to alcohol availability and signs of its consumption showed high agreement. Availability was high in both areas, which was recognized by residents. Residents of the high-income district (HID) discussed fewer signs of alcohol consumption, whilst those in the low-income district (LID) reported extensive signs of consumption. Such observations agreed with the objective measures. There were dissonances between the approaches for alcohol promotion. Although the alcohol promotion was higher in HID according to the objective measures, it was deeply discussed by LID residents. Both methodologies helped us deepen the understanding of the alcohol environment. These results may help design more effective interventions to prevent hazardous drinking.