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    Serological assays to measure dimeric IgA antibodies in SARS-CoV-2 infections
    Wei, Z ; Angrisano, F ; Eriksson, EM ; Mazhari, R ; Van, H ; Zheng, S ; Center, RJ ; McMahon, J ; Lau, J ; Kiernan-Walker, N ; Ruybal-Pesantez, S ; Mueller, I ; Robinson, LJ ; Anderson, DA ; Drummer, HE (WILEY, 2023-10)
    Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.
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    Correction: Does Malaria Affect Placental Development? Evidence from In Vitro Models
    Umbers, AJ ; Stanisic, DI ; Ome, M ; Wangnapi, R ; Hanieh, S ; Unger, HW ; Robinson, LJ ; Lufele, E ; Baiwog, F ; Siba, PM ; King, CL ; Beeson, JG ; Mueller, I ; Aplin, JD ; Glazier, JD ; Rogerson, SJ ; Hviid, L (Public Library of Science (PLoS), 2013)
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    Co-infection of the four major Plasmodium species: Effects on densities and gametocyte carriage
    Holzschuh, A ; Gruenberg, M ; Hofmann, NE ; Wampfler, R ; Kiniboro, B ; Robinson, LJ ; Mueller, I ; Felger, I ; White, MT ; Fuehrer, H-P (PUBLIC LIBRARY SCIENCE, 2022-09)
    BACKGROUND: Co-infection of the four major species of human malaria parasite Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale sp. (Po) is regularly observed, but there is limited understanding of between-species interactions. In particular, little is known about the effects of multiple Plasmodium species co-infections on gametocyte production. METHODS: We developed molecular assays for detecting asexual and gametocyte stages of Pf, Pv, Pm, and Po. This is the first description of molecular diagnostics for Pm and Po gametocytes. These assays were implemented in a unique epidemiological setting in Papua New Guinea with sympatric transmission of all four Plasmodium species permitting a comprehensive investigation of species interactions. FINDINGS: The observed frequency of Pf-Pv co-infection for asexual parasites (14.7%) was higher than expected from individual prevalence rates (23.8%Pf x 47.4%Pv = 11.3%). The observed frequency of co-infection with Pf and Pv gametocytes (4.6%) was higher than expected from individual prevalence rates (13.1%Pf x 28.2%Pv = 3.7%). The excess risk of co-infection was 1.38 (95% confidence interval (CI): 1.09, 1.67) for all parasites and 1.37 (95% CI: 0.95, 1.79) for gametocytes. This excess co-infection risk was partially attributable to malaria infections clustering in some villages. Pf-Pv-Pm triple infections were four times more frequent than expected by chance alone, which could not be fully explained by infections clustering in highly exposed individuals. The effect of co-infection on parasite density was analyzed by systematic comparison of all pairwise interactions. This revealed a significant 6.57-fold increase of Pm density when co-infected with Pf. Pm gametocytemia also increased with Pf co-infection. CONCLUSIONS: Heterogeneity in exposure to mosquitoes is a key epidemiological driver of Plasmodium co-infection. Among the four co-circulating parasites, Pm benefitted most from co-infection with other species. Beyond this, no general prevailing pattern of suppression or facilitation was identified in pairwise analysis of gametocytemia and parasitemia of the four species. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov, Trial ID: NCT02143934.
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    Asia-Pacific International Center of Excellence in Malaria Research: Maximizing Impact on Malaria Control Policy and Public Health in Cambodia and Papua New Guinea
    Robinson, LJ ; Laman, M ; Makita, L ; Lek, D ; Dori, A ; Farquhar, R ; Vantaux, A ; Witkowski, B ; Karl, S ; Mueller, I (AMER SOC TROP MED & HYGIENE, 2022-10)
    The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) was funded in 2016 to conduct a coordinated set of field and in-depth biological studies in Cambodia and Papua New Guinea (PNG), in sites that span the range of transmission intensities currently found in the Asia-Pacific regions. The overall objective is to gain an understanding of key parasite, human host, and vector factors involved in maintaining transmission in the face of intensified control and elimination programs, and to develop novel approaches to identify and target residual transmission foci. In this article, we will describe how the ICEMR program was designed to address key knowledge gaps and priority areas for the malaria control programs in each country. In PNG, partners have worked together on two consecutive ICEMR grants (2009-2016 and 2017-2024) and we present a case study of the partnership and engagement approach that has led to stronger coordination of research activities and integration with program, informing country-level strategic planning and prioritization of control activities. In both settings, the ICEMR program has generated insights into transmission foci, risk factors for ongoing transmission, highlighting the hidden burden of vivax malaria, and the need for additional complementary vector control tools. Finally, we will summarize the emerging research questions and priority areas-namely surveillance, vivax malaria, new vector control tools, and community/health systems-oriented approaches-where further tool development and implementation research have been identified as being needed to guide policy.
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    Asia-Pacific ICEMR: Understanding Malaria Transmission to Accelerate Malaria Elimination in the Asia Pacific Region
    Mueller, I ; Vantaux, A ; Karl, S ; Laman, M ; Witkowski, B ; Pepey, A ; Vinit, R ; White, M ; Barry, A ; Beeson, JG ; Robinson, LJ (AMER SOC TROP MED & HYGIENE, 2022-10)
    Gaining an in-depth understanding of malaria transmission requires integrated, multifaceted research approaches. The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) is applying specifically developed molecular and immunological assays, in-depth entomological assessments, and advanced statistical and mathematical modeling approaches to a rich series of longitudinal cohort and cross-sectional studies in Papua New Guinea and Cambodia. This is revealing both the essential contribution of forest-based transmission and the particular challenges posed by Plasmodium vivax to malaria elimination in Cambodia. In Papua New Guinea, these studies document the complex host-vector-parasite interactions that are underlying both the stunning reductions in malaria burden from 2006 to 2014 and the significant resurgence in transmission in 2016 to 2018. Here we describe the novel analytical, surveillance, molecular, and immunological tools that are being applied in our ongoing Asia-Pacific ICEMR research program.
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    Population heterogeneity in Plasmodium vivax relapse risk
    Stadler, E ; Cromer, D ; Mehra, S ; Adekunle, AI ; Flegg, JA ; Anstey, NM ; Watson, JA ; Chu, CS ; Mueller, I ; Robinson, LJ ; Schlub, TE ; Davenport, MP ; Khouryid, DS ; Reithinger, R (PUBLIC LIBRARY SCIENCE, 2022-12)
    A key characteristic of Plasmodium vivax parasites is their ability to adopt a latent liver-stage form called hypnozoites, able to cause relapse of infection months or years after a primary infection. Relapses of infection through hypnozoite activation are a major contributor to blood-stage infections in P vivax endemic regions and are thought to be influenced by factors such as febrile infections which may cause temporary changes in hypnozoite activation leading to 'temporal heterogeneity' in reactivation risk. In addition, immunity and variation in exposure to infection may be longer-term characteristics of individuals that lead to 'population heterogeneity' in hypnozoite activation. We analyze data on risk of P vivax in two previously published data sets from Papua New Guinea and the Thailand-Myanmar border region. Modeling different mechanisms of reactivation risk, we find strong evidence for population heterogeneity, with 30% of patients having almost 70% of all P vivax infections. Model fitting and data analysis indicates that individual variation in relapse risk is a primary source of heterogeneity of P vivax risk of recurrences. Trial Registration: ClinicalTrials.gov NCT01640574, NCT01074905, NCT02143934.
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    Assessment of IgG3 as a serological exposure marker for Plasmodium vivax in areas with moderate-high malaria transmission intensity
    Tayipto, Y ; Rosado, J ; Gamboa, D ; White, MTT ; Kiniboro, B ; Healer, J ; Opi, DH ; Beeson, JGG ; Takashima, E ; Tsuboi, T ; Harbers, M ; Robinson, L ; Mueller, I ; Longley, RJJ (FRONTIERS MEDIA SA, 2022-08-09)
    A more sensitive surveillance tool is needed to identify Plasmodium vivax infections for treatment and to accelerate malaria elimination efforts. To address this challenge, our laboratory has developed an eight-antigen panel that detects total IgG as serological markers of P. vivax exposure within the prior 9 months. The value of these markers has been established for use in areas with low transmission. In moderate-high transmission areas, there is evidence that total IgG is more long-lived than in areas with low transmission, resulting in poorer performance of these markers in these settings. Antibodies that are shorter-lived may be better markers of recent infection for use in moderate-high transmission areas. Using a multiplex assay, the antibody temporal kinetics of total IgG, IgG1, IgG3, and IgM against 29 P. vivax antigens were measured over 36 weeks following asymptomatic P. vivax infection in Papua New Guinean children (n = 31), from an area with moderate-high transmission intensity. IgG3 declined faster to background than total IgG, IgG1, and IgM. Based on these kinetics, IgG3 performance was then assessed for classifying recent exposure in a cohort of Peruvian individuals (n = 590; age 3-85 years) from an area of moderate transmission intensity. Using antibody responses against individual antigens, the highest performance of IgG3 in classifying recent P. vivax infections in the prior 9 months was to one of the Pv-fam-a proteins assessed (PVX_125728) (AUC = 0.764). Surprisingly, total IgG was overall a better marker of recent P. vivax infection, with the highest individual classification performance to RBP2b1986-2653 (PVX_094255) (AUC = 0.838). To understand the acquisition of IgG3 in this Peruvian cohort, relevant epidemiological factors were explored using a regression model. IgG3 levels were positively associated with increasing age, living in an area with (relatively) higher transmission intensity, and having three or more PCR-detected blood-stage P. vivax infections within the prior 13 months. Overall, we found that IgG3 did not have high accuracy for detecting recent exposure to P. vivax in the Peruvian cohort, with our data suggesting that this is due to the high levels of prior exposure required to acquire high IgG3 antibody levels.
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    Developing sero-diagnostic tests to facilitate Plasmodium vivax Serological Test-and-Treat approaches: modeling the balance between public health impact and overtreatment
    Obadia, T ; Nekkab, N ; Robinson, LJ ; Drakeley, C ; Mueller, I ; White, MT (BMC, 2022-03-18)
    BACKGROUND: Eliminating Plasmodium vivax will require targeting the hidden liver-stage reservoir of hypnozoites. This necessitates new interventions balancing the benefit of reducing vivax transmission against the risk of over-treating some individuals with drugs which may induce haemolysis. By measuring antibodies to a panel of vivax antigens, a strategy of serological-testing-and-treatment (PvSeroTAT) can identify individuals with recent blood-stage infections who are likely to carry hypnozoites and target them for radical cure. This provides a potential solution to selectively treat the vivax reservoir with 8-aminoquinolines. METHODS: PvSeroTAT can identify likely hypnozoite carriers with ~80% sensitivity and specificity. Diagnostic test sensitivities and specificities ranging 50-100% were incorporated into a mathematical model of vivax transmission to explore how they affect the risks and benefits of different PvSeroTAT strategies involving hypnozoiticidal regimens. Risk was measured as the rate of overtreatment and benefit as reduction of community-level vivax transmission. RESULTS: Across a wide range of combinations of diagnostic sensitivity and specificity, PvSeroTAT was substantially more effective than bloodstage mass screen and treat strategies and only marginally less effective than mass drug administration. The key test characteristic determining of the benefit of PvSeroTAT strategies is diagnostic sensitivity, with higher values leading to more hypnozoite carriers effectively treated and greater reductions in vivax transmission. The key determinant of risk is diagnostic specificity: higher specificity ensures that a lower proportion of uninfected individuals are unnecessarily treated with primaquine. These relationships are maintained in both moderate and low transmission settings (qPCR prevalence 10% and 2%). Increased treatment efficacy and adherence can partially compensate for lower test performance. Multiple rounds of PvSeroTAT with a lower performing test may lead to similar or higher reductions in vivax transmission than fewer rounds with a higher performing test, albeit with higher rate of overtreatment. CONCLUSIONS: At current performance, PvSeroTAT is predicted to be a safe and efficacious option for targeting the hypnozoite reservoir towards vivax elimination. P. vivax sero-diagnostic tests should aim for both high performance and ease of use in the field. The target product profiles informing such development should thus reflect the trade-offs between impact, overtreatment, and ease of programmatic implementation.
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    Infectivity of Symptomatic Malaria Patients to Anopheles farauti Colony Mosquitoes in Papua New Guinea
    Timinao, L ; Vinit, R ; Katusele, M ; Koleala, T ; Nate, E ; Czeher, C ; Burkot, TR ; Schofield, L ; Felger, I ; Mueller, I ; Laman, M ; Robinson, LJ ; Karl, S (FRONTIERS MEDIA SA, 2021-12-22)
    Plasmodium transmission from humans to mosquitoes is an understudied bottleneck in the transmission of malaria. Direct membrane feeding assays (DMFA) allow detailed malaria transmission studies from humans to mosquitoes. Especially for Plasmodium vivax, which cannot be cultured long-term under laboratory conditions, implementation of DMFAs requires proximity to P. vivax endemic areas. In this study, we investigated the infectivity of symptomatic Plasmodium infections to Anopheles farauti colony mosquitoes in Papua New Guinea (PNG). A total of 182 DMFAs were performed with venous blood collected from rapid diagnostic test (RDT) positive symptomatic malaria patients and subsequently analysed by light microscopy and quantitative real time polymerase chain reaction (qPCR). DMFAs resulted in mosquito infections in 20.9% (38/182) of cases. By light microscopy and qPCR, 10 - 11% of P. falciparum and 32 - 44% of P. vivax positive individuals infected An. farauti. Fifty-eight percent of P. vivax and 15% of P. falciparum gametocytaemic infections infected An farauti.
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    SARS-CoV-2 Multi-Antigen Serology Assay
    Mazhari, R ; Ruybal-Pesantez, S ; Angrisano, F ; Kiernan-Walker, N ; Hyslop, S ; Longley, RJ ; Bourke, C ; Chen, C ; Williamson, DA ; Robinson, LJ ; Mueller, I ; Eriksson, EM (MDPI, 2021-12)
    Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens.