Molecular surveillance of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in Mozambique, 2023 

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However, deletions of the pfhrp2 and pfhrp3 genes can lead to false negative test results and compromise appropriate case management. Due to the high burden of malaria in Mozambique, it is crucial to monitor the potential emergence of parasites with pfhrp2/3 gene deletions in the country. Methods The presence of pfhrp2/3 deletions was assessed during the 2023 high transmission season in 34 health facilities from 9 districts across 6 provinces in Mozambique. Children between 2 and 10 years of age attending the health facility with fever were tested by both the routine HRP2-RDT and a P. falciparum lactate dehydrogenase (PfLDH)-RDT, and dried blood spots (DBS) were collected from those testing positive by one or both RDTs. DBS from children with a negative HRP2-RDT but positive PfLDH-RDT were tested for the presence of pfhrp2/3 deletions by multiplex real time quantitative polymerase chain reaction (qPCR). Results 3208 children attended the health facilities during the study. 81.6% (2612/3208) participants were positive for at least one malaria RDT and, among them, 0.8% (210/2612) had discrepant RDT results (22 HRP2- but LDH + and 188 HRP2 + but LDH-). The overall prevalence of suspected false-negative HRP2-RDT results in Mozambique was 0.91% (95% CI 0.58–1.39; 22/2424). pfhrp2/3 gene deletions were confirmed in 4 cases (1 in Nampula and 3 in Inhambane). Therefore, the prevalence of P. falciparum confirmed cases with pfhrp2/3 gene deletions in the six provinces sampled was 0.16% (95% CI 0.15–2.57; 4/2424), being 0.27% (95% CI 0.01–1.75; 1/367) in Nampula and 0.59% (95% CI 0.15–1.88; 3/503) in Inhambane. Conclusion pfhrp2/3 gene deletions were detected in 2 out of 6 provinces surveyed in Mozambique, but at a prevalence far below the 5% threshold recommended for a change in HRP2 based-RDT. Molecular surveillance diagnostics RDT malaria pfhrp2/3 gene deletions Mozambique Figures Figure 1 Figure 2 BACKGROUND Despite large efforts to control and eradicate malaria, the disease continues to be responsible for the loss of human life worldwide [ 1 ]. Plasmodium resulted in around 608 000 deaths worldwide in 2022, and the global burden of malaria cases reached 249 million. In 2022, Mozambique accounted for 4.2% of global malaria cases, the large majority caused by Plasmodium (P.) falciparum [ 2 , 3 ]. Effective and easy access diagnostic tools are critical for adequate case management and treatment, reducing morbidity and mortality from malaria [ 4 ]. The World Health Organization (WHO) currently recommends the use of microscopy or rapid diagnostic tests (RDTs) to confirm suspected cases of malaria. RDTs are most commonly used in malaria-endemic areas, where results are needed fast, and both availability of microscopes and training of health facility staff is limited [ 5 ][ 6 ]. The most commonly used RDTs are based on the detection of histidine-rich protein-2 (HRP2) [ 6 – 8 ]. These RDTs generally have high sensitivity, good heat-stability profiles and their targeted antigens are expressed exclusively in P. falciparum asexual and gametocytes stages of infection [ 6 , 9 , 10 ]. Despite the advantages of this diagnostic tool, false negative results can compromise adequate diagnosis. Common causes of false-negative RDT results are operator errors, inadequate storage conditions or low parasitemia. However, false-negatives can also occur in infections with parasites deleted in pfhrp2 and/or pfhrp3 genes [ 11 – 13 ]. Although these two genes are distant from each other, they are structurally identical and HRP3 can potentially still be detected by HRP2-RDT in parasites lacking HRP2 due to antibody cross-reactivity [ 9 , 14 ]. The first record of deletions in the pfhrp2/3 genes in clinical samples was reported in Peru in 2010 [ 11 , 15 ], followed by multiple reports on this phenomenon in other parts of the world, including Central America [ 16 , 17 ], Asia [ 18 , 19 ] and Africa [ 20 – 23 ]. Of note, rapid emergence of parasites with pfhrp2 gene deletions has been recently reported in some African countries like Eritrea [ 24 ], Djibouti [ 25 ] or Ethiopia [ 26 ]. WHO recommends countries with any report of pfhrp2/3 deletions as well as neighbouring countries to conduct surveillance for pfhrp2/3 deletion, and has published guidelines on surveillance with this purpose [ 27 ]. Countries where the prevalence of parasites with pfhrp2/3 gene deletions exceeds 5%, should replace HRP2-based RDTs by a non-HRP2 based diagnostic approach. This is the prevalence at which false negatives due to lack of HRP2 antigen expression are likely to exceed the false negative rate observed using alternative RDTs based on lactate dehydrogenase (LDH) parasite protein [ 27 , 28 ]. Due to Mozambique’s contribution to global annual malaria cases, it is crucial to monitor the potential rise of pfhrp2/3 in this country. To date, two studies have been carried out in Mozambique. One analysed samples from Cabo Delgado, Zambézia and Maputo collected in 2018, using a bead-based multiplex immunoassay technique to detect presence of HRP2 antigen in P. falciparum [ 23 ]. The second study, carried out in the districts of Manhiça and Magude (Maputo province) between 2010 and 2016 [ 21 ]. Both showed a very low prevalence of parasites carrying pfhrp2/3 deletions (< 1.5%). However, no study has systematically addressed the prevalence of gene deletions and its impact on malaria diagnostic as a primary objective, and up to date data is lacking. This study aimed to determine the prevalence of deletions in the pfhrp2/3 genes causing false negative RDT results among symptomatic patients recruited in six Mozambican provinces during the rainy season in 2023, in order to inform the National Malaria Control Programme (NMCP) on RDT policy recommendations. METHODS Study design A convenience health facility survey (HFS) was conducted between January and March 2023 in 6 Mozambican provinces (Inhambane, Manica, Nampula, Niassa, Sofala and Zambézia). The number of health facilities (HF) included in each province was 8 in Nampula, 6 in Niassa, 6 in Manica, 6 in Sofala, 4 in Zambézia and 4 in Inhambane, respectively (Fig. 1 ). Under the hypothesis that the prevalence of pfhrp2/3 gene deletion causing false negative RDTs within P. falciparum symptomatic patients is below 5%, a sample size of 370 per sampling domain ( i.e. province) was targeted based on WHO guidelines for surveillance of pfhrp2/3 deletions published in 2020 [ 27 ]. Assuming a 10% of loss of samples or uninterpretable analysis, target sample size per province was 407 individuals with a confirmed P. falciparum infection. Participant recruitment and sample collection Children aged between 2 and 10 years of age presenting at outpatients’ clinic with fever (axillary temperature > 37.5ºC) or self-reported history of fever in the previous 24 h were invited to take part in the study. Once the child's legal representative agreed to participate by signing a written informed consent, each child was tested for malaria using both the routine HRP2-based RDTs (SD BIOLINE Malaria Antigen P. f (Abbott, 05FK50) in Inhambane, Manica and Sofala, or First Response® Malaria Antigen P.falciparum HRP2 (Premier Medical, PI13FRC25) in Zambézia, Nampula and Niassa) and the confirmatory PfLDH-based RDT (BIOCREDIT Malaria Ag Pf pLDH , Rapigen Inc, C14RHG25). Dried blood spots (DBS) were collected from children who tested positive for at least one of the two RDTs. Samples with discrepancies in RDT results (HRP2-/PfLDH + and HRP2+/PfLDH-), were selected for molecular characterization. Molecular analyses DNA was extracted from a 5mm DBS punch using a Tween-Chelex based method as previously described [ 29 ]. Briefly, the method consisted in washing punched DBS disks with Phosphate-buffered saline (to balance the salt concentration around the cells, preventing osmosis), and finally adding Chelex (Merck) at a concentration of 20%, followed by an incubation at 95ºc for 10 minutes. The supernatant containing DNA was then transferred to 1.5ml eppendorf to be stored at -20°C. The parasite density of discrepant samples was estimated using the pf18S ribosomal qPCR reaction as previously described [ 30 ]. Parasite densities were quantified from the Ct values by extrapolation to an standard curve composed of six (1:10) dilutions of P . falciparum laboratory strain 3D7 from 100000 to 1 parasite/µL. Deletions in pfhrp2 and pfhrp3 were analyzed by adapting the qPCR method by Grignard et al for the multiplex detection of pfhrp2 , pfhrp3 , pfldh (parasite DNA control) and human tubulin (humtubb; DNA extraction control ) [ 31 ]. Briefly, reactions were set up using TaqMan Multiplex Master Mix (Thermo Fisher), previously published primers and probes at custom concentrations ( Supplementary Table 1 ), and run in a 7500 Real- Time PCR Instrument (Applied Biosystems™) under the following cycling conditions: 20s at 95°C, 45cyles of 3s at 95°C and 33s at 60°C. Results were visualised and analysed using the 7500Real-Time SDS software version 2.3 (Applied Biosystems). Laboratory strains with known different deletion profiles were used to validate the assay: 3D7 (no deletions), Dd2 ( pfhrp2 -deleted), and HB3 ( pfhrp3 -deleted; see Supplementary Fig. 1) . Sample results were considered valid if amplification in humtubb ( Ct < 30) and pfldh (Ct 30). Samples without amplification (or Ct40) in pfhrp2 or pfhrp3 were considered fully deleted for one or both genes. To estimate presence of infections with mixed pfhrp2/3 deleted and non-deleted clones we used ΔΔCt relative method quantification as described in [ 31 ], normalizing by total DNA quantity and using 3D7 as calibrator. Definitions and statistical analyses The number of PfLDH-RDT + patients was used as denominator to calculate prevalence of suspected false-negative HRP2-RDT results and pfhrp2/3 gene deletions [ 32 ]. All calculations were done at the provincial level with 95% confidence intervals (CI) estimated for all point estimates. Areas of residence had been pre-categorized as rural or urban based on definitions that consider distance from large towns or cities, access to infrastructure and connectivity. Ethical considerations The study protocol obtaining ethical clearance by the National Bioethics Committee for Health of Mozambique (Ref: 131/CNBS/2021; October 2021). RESULTS Malaria RDT positivity and patient characteristics Overall, 3208 children aged 2–10 years were tested with both the HRP2-based RDT and the confirmatory PfLDH-based RDT across six provinces (Fig. 2 ). A total of 2612 of the participants (81.4%) were positive for at least one malaria RDT (i.e. HRP2 + or PfLDH+), and DBS were collected. By RDT type, positivity rate among febrile individuals was 80.7% (2590/3208) using routine HRP2-RDT testing, and 75.6% (2424/3208) using PfLDH-based RDT. The children had a median age of 5.2 years and 51% (1329/2612) were female. Most of the study participants (84%) were recruited in rural areas (Table 1 ). Overall, 8% (210/2612) of study participants who were positive by any RDT had discrepant RDT results, most of them (188/210, 89,5%) due to a positive HRP2 but negative PfLDH result. qPCR analysis of these samples confirmed 87/188 (46.27%) positives for P. falciparum DNA, with a median parasite density of 406.1 parasites/µL (interquartile range [IQR] 24.4, 11890.4). Table 1 Characteristics of study participants with a positive malaria RDT result in six provinces of Mozambique, 2023. Overall Inhambane Manica Sofala Zambézia Nampula Niassa N = 2612 N = 537 N = 417 N = 344 N = 449 N = 431 N = 434 Sex, n (%) Female 1329 (51%) 285 (53%) 211 (51%) 184 (53%) 235 (52%) 218 (51%) 196 (45%) Male 1283 (49%) 252 (47%) 206 (49%) 160 (47%) 214 (48%) 213 (49%) 238 (55%) Age (years), median (IQR) 5.2 (3.5, 7.4) 6.1 (4.0, 8.3) 5.4 (3.7, 7.4) 5.6 (3.9, 7.1) 5.1 (3.5, 7.3) 4.2 (2.8, 6.3) 5.1 (3.3, 7.0) Residence, n (%) Rural 2184 (84%) 367 (68%) 326 (78%) 344 (100%) 350 (78%) 431 (100%) 366 (84%) Urban 428 (16%) 170 (32%) 91 (22%) 0 (0%) 99 (22%) 0 (0%) 68 (16%) IQR, inter-quartile range Prevalence of false-negative HRP2-based RDT and pfhrp2/3 gene deletions For subsequent analysis on pfhrp2/3 deletion surveillance, patients positive with PfLDH-RDT positive result (n = 2424) were considered as confirmed malaria cases. Twenty-two of these participants (0.91%;95% CI 0.15–1.38) had negative HRP2 RDT result (Fig. 2 ). The prevalence of suspected false-negative HRP2-RDT per province ranged from 0.47% (95% CI 0.08–1.89; 2/422) in Zambézia to 1.68%, 95% CI 0.67–3.70; 6/367) in Nampula (Table 2 ). Table 2 False-negative HRP2-RDT results in six provinces of Mozambique, 2023. Provinces displayed form North to South. Province Children screened PfLDH RDT+ Prevalence of suspected false negative Total HRP2 RDT+ HRP2 RDT- Niassa 527 404 401 3 0.74% CI (0.19–2.34) Nampula 527 367 361 6 0.16% CI (0.67–3.70) Zambézia 511 422 420 2 0.47% CI (0.08–1.89) Sofala 389 325 322 3 0.92% CI (0.24–2.90) Manica 520 403 401 2 0.48% CI (0.09–1.98) Inhambane 734 503 497 6 1.19% CI (0.49–2.71) Total 3208 2424 2402 22 0.91% CI (0.15–2.57) CI, 95% Confidence interval Out of the 22 HRP2-/PfLDH + samples analyzed by qPCR, eight had no detectable parasite DNA (despite human DNA presence confirming adequate DNA extraction) and 3 samples had an invalid Ct result in the multiplex qPCR (Table 3 ). Among 11 valid samples, pfhrp2 and pfhrp3 genes were not amplified in 4 cases from Nampula and Inhambane (1 pfhrp2 deletion, 2 pfhrp3 deletions and 1 pfhrp2/3 double deletion). Taking into account confirmed RDT tests, the prevalence of confirmed uncomplicated P. falciparum infections with pfhrp2/3 gene deletions was 0.27% (95% CI 0.01–1.75; 1/367) in Nampula, 0.59% (95% CI 0.15–1.88; 3/503) in Inhambane and not detected in the other four other provinces. The overall prevalence for the six provinces sampled was 0.16% (95% CI 0.15–2.57; 4/2424). Median parasite densities in infections with pfhrp2/3 deletions were 1419.6 parasites/µl (IQR 61.9, 41690.2). Another six samples presented evidence of mixed pfhrp2/3 deleted and non-deleted parasites, with 4 of them showing a reduction in pfhrp2 as compared to calibrator wild type sample, and 2 a reduction in both pfhrp2 and pfhrp3 . Table 3 Molecular analysis of suspected false-negative HRP2 RDT cases in six provinces of Mozambique, 2023. Province Samples tested Pf DNA positive pfhrp2/3 qPCR Valid No deletion pfhrp2 deletion pfhrp3 deletion double deletion partial deletions* Niassa 3 0 - - - - - - Nampula 7 3 1 0 0 1 0 0 Zambézia 2 2 2 0 0 0 0 2 Sofala 2 2 2 1 0 0 0 1 Manica 1 1 1 0 0 0 0 1 Inhambane 7 6 5 0 1 1 1 2 Total 22 14 11 1 1 2 1 6 *P. falciparum infection with evidence of mixed clones with and without pfhrp2/3 deletions DISCUSSION This study provides an up-to-date prevalence of pfhrp2/3 gene deletions in Mozambique, covering 6 provinces and all regions of the country, namely the south, centre and north. This survey found that P. falcipurum infections with these gene deletions are rare in Mozambique (4 deletions detected in 2 provinces, with an estimated prevalence of 0.16%,95% CI 0.15–2.57%), and prevalence is still way below the 5% threshold established by the WHO for RDT type replacement [ 27 ], suggesting that HRP2-based RDTs are currently effective for P. falciparum diagnosis in Mozambique. The low prevalence of pfhrp2 /3 deletions is similar to results reported in neighbouring countries like Zambia (2009–2017 [ 33 ]), Eswatini, (2012 to 2014 [ 34 ] and Tanzania (2021; [ 35 ]) which also suggest that HRP2-based RDTs remain effective tools for malaria diagnosis. This contrasts with trends in other African countries, like Ethiopia [ 26 ] and Eritrea [ 24 ], which have reported substantial increase in pfhrp2/3 deletions in recent years, sometimes leading to RDT policy changes. In Mozambique, similar low very low levels of pfhrp2/3 deletions have been reported in previous studies [ 21 , 23 ], although those studies were limited in the number of areas covered and did not follow a standardized study design for the purpose of gene deletion surveillance. The present survey was based on WHO guidelines available on 2020 for both sample size and RDT selection. The PfLDH-based RDT confirmatory test selected (BIOCREDIT Malaria Ag Pf pLDH) has a confirmed acceptable diagnostic performance against both HRP2 expressing and HRP2 non-expressing infections at 200 parasites/µL ( pfhrp2/3 single or double deletions) based on the most recent WHO laboratory assessment [ 27 ]. As for the molecular characterization, we applied a multiplex qPCR protocol which is being used as alternative to conventional PCR also in other pfhrp2/3 deletion surveys [ 31 , 32 ][ 31 ][ 30 ][ 29 ][ 28 ]. This method is faster and more cost-effective, and includes a human gene target to inform success of the DNA purification process. Moreover, the method can provide a relative quantification of deleted pfhrp2/3 parasites in mixed infections (deleted and non-deleted parasites in the same infection). Although these mixed infections are not taken into account in the calculation of deletion prevalence according to WHO guidelines, it provides additional information on the presence of mutated parasites in the population to inform future studies. Importantly, even if all samples with partial deletions and with non-valid pfhrp2/3 qPCR results had been computed as deletions, prevalence of deletions would still be below < 1.1% in all provinces. This study also showed that HRP2+/PfLDH- RDT results discrepancies can occur frequently (7.2% of HRP2 positive test results, 188/2590). These discrepancies could be related to the lower sensitivity of tests based on the PfLDH antigen (46% of the discrepancies were positive by confirmed positive by qPCR), or to false-positives due to detection of HRP2 antigens that are still in the bloodstream despite recent effective parasite clearance [ 36 ]. Considering that 54% (101/188) of discrepancies tested negative for P. falciparum DNA, up to 3.9% (101/2590) of HRP2 routine RDT testing could be false-positive, with fever possibly attributable to non-malaria causes. Study limitations First, the study was conducted using convenience selection of health facilities and districts rather than a cluster randomised based approach, that would consider all health facilities in the province as eligible [ 27 ]. This factor could miss heterogeneity at the province level. Second, sample size targeted was 370 samples per domain (province) [ 27 ]; however, the WHO protocol was amended in 2023 with a recommendation to increase sample size from 370 samples to 600 [ 37 ]. Third, this study does not provide estimates of pfhrp2/3 deletion prevalence among the overall circulating parasite population, as there is a high incidence of multi-clonal infections that can potentially contain both deleted and non-deleted parasites and still give a positive PfHRP2-RDT result. Finally, we conducted the study among children, to optimize resources by focussing on paediatric admissions that do not cover adults’ outpatients’ visits and limiting the use of secondary RDT tests among the population of highest malaria risk infection. CONCLUSIONS pfhrp2/3 gene deletions were detected in 2 out of 6 provinces surveyed in 2023 in Mozambique, with a prevalence of 0.27% (Nampula) and 0.59% (Inhambane), way below the 5% threshold recommended by the WHO for an RDT change [ 27 ]. PfHRP2-based diagnostics are still effective for P. falciparum diagnosis in Mozambique. These results were communicated with the National Malaria Control Programme and incorporated into the Integrated Malaria Information Storage System (SIIM). However, given the emergence of pfhrp2/3 deletions in some African countries and the growing concern, surveillance efforts have to be maintained to prevent the use of ineffective diagnostics. Declarations ACKNOWLEDGMENTS We are grateful to the children and their families for accepting to participate in the study, the clinical teams at the health facilities and personnel at CISM, Malaria Consortium and ISGlobal who made this study possible. Author contributions AM, CdS, ERV, FS and BC: Designed the study protocol; CdS, PdC, NN, AT, HM, AH, GM, SE, NC and BR: Conducted the field activities (sample collection); CdS, DT, SB and PC: Conducted laboratory activities; CdS, DT, PC, ERV, AP, AAD and AM: Analyzed the data; CdS, DT, ERV and AM: Wrote the first draft of the manuscript. All authors read, reviewed and approved the final manuscript. Funding This work was supported by the Bill & Melinda Gates Foundation (INV-019032, A.M.). CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID). This research is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces. We acknowledge support from the grant CEX2023-0001290-S funded by MCIN/AEI/ 10.13039/501100011033, from the Generalitat de Catalunya through the CERCA Program and from the Departament d’Universitats i Recerca de la Generalitat de Catalunya (AGAUR; grant 2017 SGR 664). This work received the support of a fellowship from the ”laCaixa” Foundation (ID 100010434). The fellowship code is LCF/BQ/PR24/12050009. 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A novel multiplex qPCR assay for detection of Plasmodium falciparum with histidine-rich protein 2 and 3 (pfhrp2 and pfhrp3) deletions in polyclonal infections. EBioMedicine. 2020;55. Agaba BB, Smith D, Travis J, Pasay C, Nabatanzi M, Arinaitwe E. Limited threat of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletion to the utility of HRP2 – based malaria RDTs in Northern. Malar J [Internet]. BioMed Central; 2024;1–9. https://doi.org/10.1186/s12936-023-04830-w Kobayashi T, Sikalima J, Parr JB, Chaponda M, Stevenson JC, Thuma PE, et al. The search for plasmodium falciparum histidine-rich protein 2/3 deletions in Zambia and implications for plasmodium falciparum histidine-rich protein 2-based rapid diagnostic tests. Am J Trop Med Hyg. 2019;100:842–5. Ranadive N, Kunene S, Darteh S, Ntshalintshali N, Nhlabathi N, Dlamini N, et al. Limitations of rapid diagnostic testing in patients with suspected malaria: A diagnostic accuracy evaluation from Swaziland, a low-endemicity country aiming for malaria elimination. Clin Infect Dis. 2017;64:1221–7. Rogier E, Battle N, Bakari C, Seth MD, Nace D, Herman C et al. Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021. Sci Rep [Internet]. Nature Publishing Group UK; 2024;14:1–10. https://doi.org/10.1038/s41598-024-58455-3 Das S, Jang IK, Barney B, Peck R, Rek JC, Arinaitwe E, et al. Performance of a high-sensitivity rapid diagnostic test for Plasmodium falciparum malaria in asymptomatic individuals from Uganda and Myanmar and naive human challenge infections. Am J Trop Med Hyg. 2017;97:1540–50. WHO. Master protocol for surveillance of pfhrp2/pfhrp3 deletions and biobanking to support future research CORRIGENDA (19 January 2023). 2023;6:1–7. Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTARYMATERIAL.docx Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Malaria Journal → Version 1 posted Editorial decision: Revision requested 18 Oct, 2024 Reviews received at journal 18 Oct, 2024 Reviewers agreed at journal 18 Oct, 2024 Reviews received at journal 06 Oct, 2024 Reviews received at journal 05 Oct, 2024 Reviewers agreed at journal 02 Oct, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviewers invited by journal 27 Sep, 2024 Editor assigned by journal 21 Sep, 2024 Submission checks completed at journal 21 Sep, 2024 First submitted to journal 20 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5121569","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":367865477,"identity":"6ba8968c-bff3-454f-8516-14c1454bd745","order_by":0,"name":"Clemente da 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Francisco","correspondingAuthor":false,"prefix":"","firstName":"Andrés","middleName":"","lastName":"Aranda-Díaz3","suffix":""},{"id":367865485,"identity":"f1695217-cd9f-4a33-b486-140cd957fc3a","order_by":8,"name":"Henriques Mbeve","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Henriques","middleName":"","lastName":"Mbeve","suffix":""},{"id":367865486,"identity":"79729747-7475-4c65-ba52-be84c1973ba2","order_by":9,"name":"Nelo Ndimande","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Nelo","middleName":"","lastName":"Ndimande","suffix":""},{"id":367865487,"identity":"fbd892c0-8a0e-4107-a3b0-dbaa6053024a","order_by":10,"name":"Alcido Timana","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Alcido","middleName":"","lastName":"Timana","suffix":""},{"id":367865488,"identity":"55588fd9-becd-4776-8989-883e867fb1a2","order_by":11,"name":"Aura Hunguana","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Aura","middleName":"","lastName":"Hunguana","suffix":""},{"id":367865490,"identity":"90852c8e-6afb-4c0a-9a99-70382abcbf76","order_by":12,"name":"Simone Boene","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Simone","middleName":"","lastName":"Boene","suffix":""},{"id":367865491,"identity":"11a90266-f763-418d-b4cf-ef115752eb43","order_by":13,"name":"ARNAU PUJOL","email":"","orcid":"","institution":"Barcelona Institute for Global Health","correspondingAuthor":false,"prefix":"","firstName":"ARNAU","middleName":"","lastName":"PUJOL","suffix":""},{"id":367865492,"identity":"3c152fda-82c8-408b-8f77-613c09d8d388","order_by":14,"name":"Bernardete Rafael","email":"","orcid":"","institution":"National Malaria Control Program","correspondingAuthor":false,"prefix":"","firstName":"Bernardete","middleName":"","lastName":"Rafael","suffix":""},{"id":367865493,"identity":"299775d7-f68b-41d9-945f-c507a297e7ca","order_by":15,"name":"Sonia Maria Enosse","email":"","orcid":"","institution":"Malaria Consortium","correspondingAuthor":false,"prefix":"","firstName":"Sonia","middleName":"Maria","lastName":"Enosse","suffix":""},{"id":367865494,"identity":"b1901594-ef69-4b67-9c12-3e760047fa40","order_by":16,"name":"Baltazar Candrinho","email":"","orcid":"","institution":"National Malaria Control Program","correspondingAuthor":false,"prefix":"","firstName":"Baltazar","middleName":"","lastName":"Candrinho","suffix":""},{"id":367865495,"identity":"62de8590-7f49-47ae-a798-27b74a26224f","order_by":17,"name":"Francisco Saute","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Saute","suffix":""},{"id":367865496,"identity":"fea6c894-fa4d-4fc8-b04c-06b24758c28f","order_by":18,"name":"Alfredo Mayor","email":"","orcid":"","institution":"Manhiça Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Alfredo","middleName":"","lastName":"Mayor","suffix":""}],"badges":[],"createdAt":"2024-09-20 07:32:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5121569/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5121569/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-024-05230-4","type":"published","date":"2024-12-26T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69850794,"identity":"d0a0485f-f1d6-48a3-90d4-e08c633506f0","added_by":"auto","created_at":"2024-11-25 23:31:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMap of Mozambique with the study sites. \u003c/strong\u003eYellow dots represent health facilities where \u003cem\u003eP. falciparum\u003c/em\u003e samples were collected. The map was created using R software version 4.2.2.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5121569/v1/fe958b067534775141314aad.png"},{"id":69850263,"identity":"8992de0b-e5ff-45c7-accd-f4aad30328e5","added_by":"auto","created_at":"2024-11-25 23:23:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvey flow chart and sample selection for molecular characterization.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5121569/v1/667de0aad66f0f4dad59a5e2.png"},{"id":72640252,"identity":"bcad5c2d-cf0f-4833-94b0-fc4f4fa8b6df","added_by":"auto","created_at":"2024-12-30 16:03:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1077910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5121569/v1/4c4e51fd-81d9-42f1-a553-9b553c8c22b4.pdf"},{"id":69850265,"identity":"71b913eb-88d6-47d5-b2c4-1b1759705a65","added_by":"auto","created_at":"2024-11-25 23:23:47","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":475876,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-5121569/v1/f21ff42c587f73d1d308b510.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular surveillance of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in Mozambique, 2023 ","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eDespite large efforts to control and eradicate malaria, the disease continues to be responsible for the loss of human life worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003ePlasmodium\u003c/em\u003e resulted in around 608 000 deaths worldwide in 2022, and the global burden of malaria cases reached 249\u0026nbsp;million. In 2022, Mozambique accounted for 4.2% of global malaria cases, the large majority caused by \u003cem\u003ePlasmodium (P.) falciparum\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEffective and easy access diagnostic tools are critical for adequate case management and treatment, reducing morbidity and mortality from malaria [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The World Health Organization (WHO) currently recommends the use of microscopy or rapid diagnostic tests (RDTs) to confirm suspected cases of malaria. RDTs are most commonly used in malaria-endemic areas, where results are needed fast, and both availability of microscopes and training of health facility staff is limited [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The most commonly used RDTs are based on the detection of histidine-rich protein-2 (HRP2) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These RDTs generally have high sensitivity, good heat-stability profiles and their targeted antigens are expressed exclusively in \u003cem\u003eP. falciparum\u003c/em\u003e asexual and gametocytes stages of infection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite the advantages of this diagnostic tool, false negative results can compromise adequate diagnosis. Common causes of false-negative RDT results are operator errors, inadequate storage conditions or low parasitemia. However, false-negatives can also occur in infections with parasites deleted in \u003cem\u003epfhrp2\u003c/em\u003e and/or \u003cem\u003epfhrp3\u003c/em\u003e genes [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although these two genes are distant from each other, they are structurally identical and HRP3 can potentially still be detected by HRP2-RDT in parasites lacking HRP2 due to antibody cross-reactivity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe first record of deletions in the \u003cem\u003epfhrp2/3\u003c/em\u003e genes in clinical samples was reported in Peru in 2010 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], followed by multiple reports on this phenomenon in other parts of the world, including Central America [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], Asia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and Africa [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Of note, rapid emergence of parasites with \u003cem\u003epfhrp2\u003c/em\u003e gene deletions has been recently reported in some African countries like Eritrea [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Djibouti [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] or Ethiopia [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. WHO recommends countries with any report of \u003cem\u003epfhrp2/3\u003c/em\u003e deletions as well as neighbouring countries to conduct surveillance for \u003cem\u003epfhrp2/3\u003c/em\u003e deletion, and has published guidelines on surveillance with this purpose [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Countries where the prevalence of parasites with \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions exceeds 5%, should replace HRP2-based RDTs by a non-HRP2 based diagnostic approach. This is the prevalence at which false negatives due to lack of HRP2 antigen expression are likely to exceed the false negative rate observed using alternative RDTs based on lactate dehydrogenase (LDH) parasite protein [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to Mozambique\u0026rsquo;s contribution to global annual malaria cases, it is crucial to monitor the potential rise of \u003cem\u003epfhrp2/3\u003c/em\u003e in this country. To date, two studies have been carried out in Mozambique. One analysed samples from Cabo Delgado, Zamb\u0026eacute;zia and Maputo collected in 2018, using a bead-based multiplex immunoassay technique to detect presence of HRP2 antigen in \u003cem\u003eP. falciparum\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The second study, carried out in the districts of Manhi\u0026ccedil;a and Magude (Maputo province) between 2010 and 2016 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Both showed a very low prevalence of parasites carrying \u003cem\u003epfhrp2/3\u003c/em\u003e deletions (\u0026lt;\u0026thinsp;1.5%). However, no study has systematically addressed the prevalence of gene deletions and its impact on malaria diagnostic as a primary objective, and up to date data is lacking. This study aimed to determine the prevalence of deletions in the \u003cem\u003epfhrp2/3\u003c/em\u003e genes causing false negative RDT results among symptomatic patients recruited in six Mozambican provinces during the rainy season in 2023, in order to inform the National Malaria Control Programme (NMCP) on RDT policy recommendations.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eA convenience health facility survey (HFS) was conducted between January and March 2023 in 6 Mozambican provinces (Inhambane, Manica, Nampula, Niassa, Sofala and Zamb\u0026eacute;zia). The number of health facilities (HF) included in each province was 8 in Nampula, 6 in Niassa, 6 in Manica, 6 in Sofala, 4 in Zamb\u0026eacute;zia and 4 in Inhambane, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder the hypothesis that the prevalence of \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletion causing false negative RDTs within \u003cem\u003eP. falciparum\u003c/em\u003e symptomatic patients is below 5%, a sample size of 370 per sampling domain (\u003cem\u003ei.e.\u003c/em\u003e province) was targeted based on WHO guidelines for surveillance of \u003cem\u003epfhrp2/3\u003c/em\u003e deletions published in 2020 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Assuming a 10% of loss of samples or uninterpretable analysis, target sample size per province was 407 individuals with a confirmed \u003cem\u003eP. falciparum\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParticipant recruitment and sample collection\u003c/h3\u003e\n\u003cp\u003eChildren aged between 2 and 10 years of age presenting at outpatients\u0026rsquo; clinic with fever (axillary temperature\u0026thinsp;\u0026gt;\u0026thinsp;37.5\u0026ordm;C) or self-reported history of fever in the previous 24 h were invited to take part in the study. Once the child's legal representative agreed to participate by signing a written informed consent, each child was tested for malaria using both the routine HRP2-based RDTs (SD BIOLINE Malaria Antigen \u003cem\u003eP. f\u003c/em\u003e (Abbott, 05FK50) in Inhambane, Manica and Sofala, or First Response\u0026reg; Malaria Antigen \u003cem\u003eP.falciparum\u003c/em\u003e HRP2 (Premier Medical, PI13FRC25) in Zamb\u0026eacute;zia, Nampula and Niassa) and the confirmatory PfLDH-based RDT (BIOCREDIT Malaria Ag \u003cem\u003ePf pLDH\u003c/em\u003e, Rapigen Inc, C14RHG25). Dried blood spots (DBS) were collected from children who tested positive for at least one of the two RDTs. Samples with discrepancies in RDT results (HRP2-/PfLDH\u0026thinsp;+\u0026thinsp;and HRP2+/PfLDH-), were selected for molecular characterization.\u003c/p\u003e\n\u003ch3\u003eMolecular analyses\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from a 5mm DBS punch using a Tween-Chelex based method as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, the method consisted in washing punched DBS disks with Phosphate-buffered saline (to balance the salt concentration around the cells, preventing osmosis), and finally adding Chelex (Merck) at a concentration of 20%, followed by an incubation at 95\u0026ordm;c for 10 minutes. The supernatant containing DNA was then transferred to 1.5ml eppendorf to be stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe parasite density of discrepant samples was estimated using the \u003cem\u003epf18S\u003c/em\u003e ribosomal qPCR reaction as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Parasite densities were quantified from the Ct values by extrapolation to an standard curve composed of six (1:10) dilutions of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e laboratory strain 3D7 from 100000 to 1 parasite/\u0026micro;L. Deletions in \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e were analyzed by adapting the qPCR method by Grignard et al for the multiplex detection of \u003cem\u003epfhrp2\u003c/em\u003e, \u003cem\u003epfhrp3\u003c/em\u003e, \u003cem\u003epfldh\u003c/em\u003e (parasite DNA control) and human \u003cem\u003etubulin (humtubb;\u003c/em\u003e DNA extraction control\u003cem\u003e)\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, reactions were set up using TaqMan Multiplex Master Mix (Thermo Fisher), previously published primers and probes at custom concentrations (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e), and run in a 7500 Real- Time PCR Instrument (Applied Biosystems\u0026trade;) under the following cycling conditions: 20s at 95\u0026deg;C, 45cyles of 3s at 95\u0026deg;C and 33s at 60\u0026deg;C. Results were visualised and analysed using the 7500Real-Time SDS software version 2.3 (Applied Biosystems). Laboratory strains with known different deletion profiles were used to validate the assay: 3D7 (no deletions), Dd2 (\u003cem\u003epfhrp2\u003c/em\u003e-deleted), and HB3 (\u003cem\u003epfhrp3\u003c/em\u003e-deleted; see \u003cb\u003eSupplementary Fig.\u0026nbsp;1)\u003c/b\u003e. Sample results were considered valid if amplification in \u003cem\u003ehumtubb (\u003c/em\u003eCt\u0026thinsp;\u0026lt;\u0026thinsp;30) and \u003cem\u003epfldh\u003c/em\u003e (Ct\u0026thinsp;\u0026lt;\u0026thinsp;35) targets was detected. Sample results were considered invalid if amplification in \u003cem\u003ehumtubb (\u003c/em\u003eCt\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;30). Samples without amplification (or Ct40) in \u003cem\u003epfhrp2\u003c/em\u003e or \u003cem\u003epfhrp3\u003c/em\u003e were considered fully deleted for one or both genes. To estimate presence of infections with mixed \u003cem\u003epfhrp2/3\u003c/em\u003e deleted and non-deleted clones we used ΔΔCt relative method quantification as described in [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], normalizing by total DNA quantity and using 3D7 as calibrator.\u003c/p\u003e\n\u003ch3\u003eDefinitions and statistical analyses\u003c/h3\u003e\n\u003cp\u003eThe number of PfLDH-RDT\u0026thinsp;+\u0026thinsp;patients was used as denominator to calculate prevalence of suspected false-negative HRP2-RDT results and \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. All calculations were done at the provincial level with 95% confidence intervals (CI) estimated for all point estimates. Areas of residence had been pre-categorized as rural or urban based on definitions that consider distance from large towns or cities, access to infrastructure and connectivity.\u003c/p\u003e\n\u003ch3\u003eEthical considerations\u003c/h3\u003e\n\u003cp\u003e The study protocol obtaining ethical clearance by the National Bioethics Committee for Health of Mozambique (Ref: 131/CNBS/2021; October 2021).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMalaria RDT positivity and patient characteristics\u003c/h2\u003e \u003cp\u003eOverall, 3208 children aged 2\u0026ndash;10 years were tested with both the HRP2-based RDT and the confirmatory PfLDH-based RDT across six provinces (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A total of 2612 of the participants (81.4%) were positive for at least one malaria RDT (i.e. HRP2\u0026thinsp;+\u0026thinsp;or PfLDH+), and DBS were collected. By RDT type, positivity rate among febrile individuals was 80.7% (2590/3208) using routine HRP2-RDT testing, and 75.6% (2424/3208) using PfLDH-based RDT. The children had a median age of 5.2 years and 51% (1329/2612) were female. Most of the study participants (84%) were recruited in rural areas (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, 8% (210/2612) of study participants who were positive by any RDT had discrepant RDT results, most of them (188/210, 89,5%) due to a positive HRP2 but negative PfLDH result. qPCR analysis of these samples confirmed 87/188 (46.27%) positives for \u003cem\u003eP. falciparum\u003c/em\u003e DNA, with a median parasite density of 406.1 parasites/\u0026micro;L (interquartile range [IQR] 24.4, 11890.4).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of study participants with a positive malaria RDT result in six provinces of Mozambique, 2023.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverall\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInhambane\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eManica\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSofala\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eZamb\u0026eacute;zia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNampula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNiassa\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;2612\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;537\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;417\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;344\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;449\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;431\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;434\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1329 (51%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e285 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e211 (51%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e184 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e235 (52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e218 (51%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e196 (45%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1283 (49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e206 (49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e160 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e214 (48%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e213 (49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e238 (55%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years), \u003c/p\u003e \u003cp\u003emedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.2 (3.5, 7.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.1 (4.0, 8.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.4 (3.7, 7.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.6 (3.9, 7.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.1 (3.5, 7.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.2 (2.8, 6.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.1 (3.3, 7.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidence, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2184 (84%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e367 (68%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e326 (78%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e344 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e350 (78%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e431 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e366 (84%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e428 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e170 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e68 (16%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eIQR, inter-quartile range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePrevalence of false-negative HRP2-based RDT and\u003c/b\u003e \u003cb\u003epfhrp2/3\u003c/b\u003e \u003cb\u003egene deletions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor subsequent analysis on \u003cem\u003epfhrp2/3\u003c/em\u003e deletion surveillance, patients positive with PfLDH-RDT positive result (n\u0026thinsp;=\u0026thinsp;2424) were considered as confirmed malaria cases. Twenty-two of these participants (0.91%;95% CI 0.15\u0026ndash;1.38) had negative HRP2 RDT result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The prevalence of suspected false-negative HRP2-RDT per province ranged from 0.47% (95% CI 0.08\u0026ndash;1.89; 2/422) in Zamb\u0026eacute;zia to 1.68%, 95% CI 0.67\u0026ndash;3.70; 6/367) in Nampula (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eFalse-negative HRP2-RDT results in six provinces of Mozambique, 2023.\u003c/b\u003e Provinces displayed form North to South.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProvince\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChildren screened\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ePfLDH RDT+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePrevalence of suspected false negative\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHRP2 RDT+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHRP2 RDT-\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNiassa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.74% CI (0.19\u0026ndash;2.34)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNampula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16% CI (0.67\u0026ndash;3.70)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZamb\u0026eacute;zia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.47% CI (0.08\u0026ndash;1.89)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSofala\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.92% CI (0.24\u0026ndash;2.90)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eManica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48% CI (0.09\u0026ndash;1.98)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInhambane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e734\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.19% CI (0.49\u0026ndash;2.71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2424\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.91% CI (0.15\u0026ndash;2.57)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI, 95% Confidence interval\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOut of the 22 HRP2-/PfLDH\u0026thinsp;+\u0026thinsp;samples analyzed by qPCR, eight had no detectable parasite DNA (despite human DNA presence confirming adequate DNA extraction) and 3 samples had an invalid Ct result in the multiplex qPCR (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among 11 valid samples, \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e genes were not amplified in 4 cases from Nampula and Inhambane (1 \u003cem\u003epfhrp2\u003c/em\u003e deletion, 2 \u003cem\u003epfhrp3\u003c/em\u003e deletions and 1 \u003cem\u003epfhrp2/3\u003c/em\u003e double deletion). Taking into account confirmed RDT tests, the prevalence of confirmed uncomplicated \u003cem\u003eP. falciparum\u003c/em\u003e infections with \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions was 0.27% (95% CI 0.01\u0026ndash;1.75; 1/367) in Nampula, 0.59% (95% CI 0.15\u0026ndash;1.88; 3/503) in Inhambane and not detected in the other four other provinces. The overall prevalence for the six provinces sampled was 0.16% (95% CI 0.15\u0026ndash;2.57; 4/2424). Median parasite densities in infections with \u003cem\u003epfhrp2/3\u003c/em\u003e deletions were 1419.6 parasites/\u0026micro;l (IQR 61.9, 41690.2). Another six samples presented evidence of mixed \u003cem\u003epfhrp2/3\u003c/em\u003e deleted and non-deleted parasites, with 4 of them showing a reduction in \u003cem\u003epfhrp2\u003c/em\u003e as compared to calibrator wild type sample, and 2 a reduction in both \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular analysis of suspected false-negative HRP2 RDT cases in six provinces of Mozambique, 2023.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProvince\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSamples tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePf DNA positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003epfhrp2/3\u003c/em\u003e qPCR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eValid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo deletion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003epfhrp2\u003c/em\u003e deletion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003epfhrp3\u003c/em\u003e deletion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003edouble deletion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003epartial deletions*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNiassa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNampula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZamb\u0026eacute;zia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSofala\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eManica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInhambane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003e*P. falciparum infection with evidence of mixed clones with and without pfhrp2/3 deletions\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study provides an up-to-date prevalence of \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions in Mozambique, covering 6 provinces and all regions of the country, namely the south, centre and north. This survey found that \u003cem\u003eP. falcipurum\u003c/em\u003e infections with these gene deletions are rare in Mozambique (4 deletions detected in 2 provinces, with an estimated prevalence of 0.16%,95% CI 0.15\u0026ndash;2.57%), and prevalence is still way below the 5% threshold established by the WHO for RDT type replacement [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], suggesting that HRP2-based RDTs are currently effective for \u003cem\u003eP. falciparum\u003c/em\u003e diagnosis in Mozambique.\u003c/p\u003e \u003cp\u003eThe low prevalence of \u003cem\u003epfhrp2\u003c/em\u003e/3 deletions is similar to results reported in neighbouring countries like Zambia (2009\u0026ndash;2017 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]), Eswatini, (2012 to 2014 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and Tanzania (2021; [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]) which also suggest that HRP2-based RDTs remain effective tools for malaria diagnosis. This contrasts with trends in other African countries, like Ethiopia [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and Eritrea [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which have reported substantial increase in \u003cem\u003epfhrp2/3\u003c/em\u003e deletions in recent years, sometimes leading to RDT policy changes. In Mozambique, similar low very low levels of \u003cem\u003epfhrp2/3\u003c/em\u003e deletions have been reported in previous studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], although those studies were limited in the number of areas covered and did not follow a standardized study design for the purpose of gene deletion surveillance. The present survey was based on WHO guidelines available on 2020 for both sample size and RDT selection. The PfLDH-based RDT confirmatory test selected (BIOCREDIT Malaria Ag Pf pLDH) has a confirmed acceptable diagnostic performance against both HRP2 expressing and HRP2 non-expressing infections at 200 parasites/\u0026micro;L (\u003cem\u003epfhrp2/3\u003c/em\u003e single or double deletions) based on the most recent WHO laboratory assessment [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs for the molecular characterization, we applied a multiplex qPCR protocol which is being used as alternative to conventional PCR also in other \u003cem\u003epfhrp2/3\u003c/em\u003e deletion surveys [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This method is faster and more cost-effective, and includes a human gene target to inform success of the DNA purification process. Moreover, the method can provide a relative quantification of deleted \u003cem\u003epfhrp2/3\u003c/em\u003e parasites in mixed infections (deleted and non-deleted parasites in the same infection). Although these mixed infections are not taken into account in the calculation of deletion prevalence according to WHO guidelines, it provides additional information on the presence of mutated parasites in the population to inform future studies. Importantly, even if all samples with partial deletions and with non-valid \u003cem\u003epfhrp2/3\u003c/em\u003e qPCR results had been computed as deletions, prevalence of deletions would still be below \u0026lt;\u0026thinsp;1.1% in all provinces.\u003c/p\u003e \u003cp\u003eThis study also showed that HRP2+/PfLDH- RDT results discrepancies can occur frequently (7.2% of HRP2 positive test results, 188/2590). These discrepancies could be related to the lower sensitivity of tests based on the PfLDH antigen (46% of the discrepancies were positive by confirmed positive by qPCR), or to false-positives due to detection of HRP2 antigens that are still in the bloodstream despite recent effective parasite clearance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Considering that 54% (101/188) of discrepancies tested negative for \u003cem\u003eP. falciparum\u003c/em\u003e DNA, up to 3.9% (101/2590) of HRP2 routine RDT testing could be false-positive, with fever possibly attributable to non-malaria causes.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy limitations\u003c/h2\u003e \u003cp\u003eFirst, the study was conducted using convenience selection of health facilities and districts rather than a cluster randomised based approach, that would consider all health facilities in the province as eligible [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This factor could miss heterogeneity at the province level. Second, sample size targeted was 370 samples per domain (province) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; however, the WHO protocol was amended in 2023 with a recommendation to increase sample size from 370 samples to 600 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Third, this study does not provide estimates of \u003cem\u003epfhrp2/3\u003c/em\u003e deletion prevalence among the overall circulating parasite population, as there is a high incidence of multi-clonal infections that can potentially contain both deleted and non-deleted parasites and still give a positive PfHRP2-RDT result. Finally, we conducted the study among children, to optimize resources by focussing on paediatric admissions that do not cover adults\u0026rsquo; outpatients\u0026rsquo; visits and limiting the use of secondary RDT tests among the population of highest malaria risk infection.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003e \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions were detected in 2 out of 6 provinces surveyed in 2023 in Mozambique, with a prevalence of 0.27% (Nampula) and 0.59% (Inhambane), way below the 5% threshold recommended by the WHO for an RDT change [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. PfHRP2-based diagnostics are still effective for \u003cem\u003eP. falciparum\u003c/em\u003e diagnosis in Mozambique. These results were communicated with the National Malaria Control Programme and incorporated into the Integrated Malaria Information Storage System (SIIM). However, given the emergence of \u003cem\u003epfhrp2/3\u003c/em\u003e deletions in some African countries and the growing concern, surveillance efforts have to be maintained to prevent the use of ineffective diagnostics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the children and their families for accepting to participate in the study, the clinical teams at the health facilities and personnel at CISM, Malaria Consortium and ISGlobal who made this study possible.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAM, CdS, ERV, FS and BC: Designed the study protocol; CdS, PdC, NN, AT, HM, AH, GM, SE, NC and BR:\u0026nbsp;Conducted the field activities (sample collection); CdS, DT,\u0026nbsp;SB\u0026nbsp;and PC: Conducted laboratory activities; CdS, DT, PC, ERV, AP, AAD and AM: Analyzed the data; CdS, DT, ERV and AM: Wrote the first draft of the manuscript. All authors read, reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Bill \u0026amp; Melinda Gates Foundation (INV-019032, A.M.). CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID).\u0026nbsp;This research is part of the ISGlobal\u0026rsquo;s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundaci\u0026oacute;n Ram\u0026oacute;n Areces. We acknowledge support from the grant CEX2023-0001290-S funded by MCIN/AEI/ 10.13039/501100011033, from the Generalitat de Catalunya through the CERCA Program and from the Departament d\u0026rsquo;Universitats i Recerca de la Generalitat de Catalunya (AGAUR; grant 2017 SGR 664).\u0026nbsp;This work received the support of a fellowship from the \u0026rdquo;laCaixa\u0026rdquo; Foundation (ID 100010434). The fellowship code is LCF/BQ/PR24/12050009.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data related to this study are included in the manuscript or its supplementary materials, or is available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e: The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSato S. Correction to: Plasmodium\u0026mdash;a brief introduction to the parasites causing human malaria and their basic biology (Journal of Physiological Anthropology, (2021), 40, 1, (1), 10.1186/s40101-020-00251-9). J Physiol Anthropol. Journal of Physiological Anthropology; 2021;40:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. 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Genomic malaria surveillance of antenatal care users detects reduced transmission following elimination interventions in Mozambique. Nat Commun Engl. 2024;15:2402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMayor A, Bardaj\u0026iacute; A, Macete E, Nhampossa T, Fonseca AM, Gonz\u0026aacute;lez R, et al. Changing Trends in P. falciparum Burden, Immunity, and Disease in Pregnancy. N Engl J Med. 2015;373:1607\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrignard L, Nolder D, Sep\u0026uacute;lveda N, Berhane A, Mihreteab S, Kaaya R et al. A novel multiplex qPCR assay for detection of Plasmodium falciparum with histidine-rich protein 2 and 3 (pfhrp2 and pfhrp3) deletions in polyclonal infections. EBioMedicine. 2020;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgaba BB, Smith D, Travis J, Pasay C, Nabatanzi M, Arinaitwe E. 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Master protocol for surveillance of pfhrp2/pfhrp3 deletions and biobanking to support future research CORRIGENDA (19 January 2023). 2023;6:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Molecular surveillance, diagnostics, RDT, malaria, pfhrp2/3 gene deletions, Mozambique","lastPublishedDoi":"10.21203/rs.3.rs-5121569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5121569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRapid diagnostic tests (RDTs) based on the detection of \u003cem\u003eP. falciparum\u003c/em\u003e histidine rich protein 2 (\u003cem\u003ePf\u003c/em\u003eHRP2) are widely used for the diagnostic of \u003cem\u003eP. falciparum\u003c/em\u003e in Africa. However, deletions of the \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e genes can lead to false negative test results and compromise appropriate case management. Due to the high burden of malaria in Mozambique, it is crucial to monitor the potential emergence of parasites with \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions in the country.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe presence of \u003cem\u003epfhrp2/3\u003c/em\u003e deletions was assessed during the 2023 high transmission season in 34 health facilities from 9 districts across 6 provinces in Mozambique. Children between 2 and 10 years of age attending the health facility with fever were tested by both the routine HRP2-RDT and a \u003cem\u003eP. falciparum\u003c/em\u003e lactate dehydrogenase (PfLDH)-RDT, and dried blood spots (DBS) were collected from those testing positive by one or both RDTs. DBS from children with a negative HRP2-RDT but positive PfLDH-RDT were tested for the presence of \u003cem\u003epfhrp2/3\u003c/em\u003e deletions by multiplex real time quantitative polymerase chain reaction (qPCR).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e3208 children attended the health facilities during the study. 81.6% (2612/3208) participants were positive for at least one malaria RDT and, among them, 0.8% (210/2612) had discrepant RDT results (22 HRP2- but LDH\u0026thinsp;+\u0026thinsp;and 188 HRP2\u0026thinsp;+\u0026thinsp;but LDH-). The overall prevalence of suspected false-negative HRP2-RDT results in Mozambique was 0.91% (95% CI 0.58\u0026ndash;1.39; 22/2424). \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions were confirmed in 4 cases (1 in Nampula and 3 in Inhambane). Therefore, the prevalence of \u003cem\u003eP. falciparum\u003c/em\u003e confirmed cases with \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions in the six provinces sampled was 0.16% (95% CI 0.15\u0026ndash;2.57; 4/2424), being 0.27% (95% CI 0.01\u0026ndash;1.75; 1/367) in Nampula and 0.59% (95% CI 0.15\u0026ndash;1.88; 3/503) in Inhambane.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions were detected in 2 out of 6 provinces surveyed in Mozambique, but at a prevalence far below the 5% threshold recommended for a change in HRP2 based-RDT.\u003c/p\u003e","manuscriptTitle":"Molecular surveillance of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in Mozambique, 2023 ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 23:23:42","doi":"10.21203/rs.3.rs-5121569/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-19T03:48:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-19T03:46:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289980151429071842808344179882472631707","date":"2024-10-19T01:46:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-06T22:59:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-05T13:07:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231583115214630006406850555380467498420","date":"2024-10-02T13:42:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255255122430031564106373341083289858700","date":"2024-09-27T15:26:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-27T04:02:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-21T07:45:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-21T07:43:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2024-09-20T07:30:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a10b33f4-879e-4b95-8f79-3e6c6d188c6b","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T15:58:12+00:00","versionOfRecord":{"articleIdentity":"rs-5121569","link":"https://doi.org/10.1186/s12936-024-05230-4","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2024-12-26 15:56:51","publishedOnDateReadable":"December 26th, 2024"},"versionCreatedAt":"2024-11-25 23:23:42","video":"","vorDoi":"10.1186/s12936-024-05230-4","vorDoiUrl":"https://doi.org/10.1186/s12936-024-05230-4","workflowStages":[]},"version":"v1","identity":"rs-5121569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5121569","identity":"rs-5121569","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}