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In a Plasmodium vivax infection, it was shown a proportionally increased on gametocyte distribution within the bone marrow aspirant, suggesting a role of this organ as a reservoir for this parasite stage. Here, we evaluated the ex vivo cytoadhesive capacity of P. vivax gametocytes to bone marrow endothelial cells (HBMEC) and investigated the involvement of some receptors in the cytoadhesion process by using transfected CHO cells (CHO-ICAM1, CHO-CD36 and CHO-VCAM), wild type (CHO-K1) or deficient in heparan and chondroitin sulfate (CHO-745). Ex-vivo cytoadhesion assays were performed using a total of 44  P. vivax isolates enriched in gametocyte stages by Percoll gradient in the different cell lines. The majority of isolates (88.9%) were able to adhere to HBMEC monolayer. ICAM1 seemed to be the sole receptor significantly involved. CD-36 was the receptor with higher adhesion rate, despite no significance was noticed when compared to CHO-745. We demonstrated that gametocyte P. vivax adheres ex vivo to bone marrow endothelial cells. Moreover, P. vivax gametocytes display the ability to adhere to all CHO cells investigated, especially to CHO-ICAM1. These findings bring insights to the comprehension of the role of the bone marrow as a P. vivax reservoir and the potential impact on parasite transmission to the vector., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Salazar Alvarez, Vera Lizcano, da Silva Barros, Baia-da-Silva, Monteiro, Pimenta, de Lacerda, Costa and Lopes.)

Background: Malaria and HIV are two important public health issues. However, evidence on HIV-Plasmodium vivax co-infection (HIV/PvCo) is scarce, with most of the available information related to Plasmodium falciparum on the African continent. It is unclear whether HIV can change the clinical course of vivax malaria and increase the risk of complications. In this study, a systematic review of HIV/PvCo studies was performed, and recent cases from the Brazilian Amazon were included., Methods: Medical records from a tertiary care centre in the Western Brazilian Amazon (2009-2018) were reviewed to identify HIV/PvCo hospitalized patients. Demographic, clinical and laboratory characteristics and outcomes are reported. Also, a systematic review of published studies on HIV/PvCo was conducted. Metadata, number of HIV/PvCo cases, demographic, clinical, and outcome data were extracted., Results: A total of 1,048 vivax malaria patients were hospitalized in the 10-year period; 21 (2.0%) were HIV/PvCo cases, of which 9 (42.9%) had AIDS-defining illnesses. This was the first malaria episode in 11 (52.4%) patients. Seven (33.3%) patients were unaware of their HIV status and were diagnosed on hospitalization. Severe malaria was diagnosed in 5 (23.8%) patients. One patient died. The systematic review search provided 17 articles (12 cross-sectional or longitudinal studies and 5 case report studies). A higher prevalence of studies involved cases in African and Asian countries (35.3 and 29.4%, respectively), and the prevalence of reported co-infections ranged from 0.1 to 60%., Conclusion: Reports of HIV/PvCo are scarce in the literature, with only a few studies describing clinical and laboratory outcomes. Systematic screening for both co-infections is not routinely performed, and therefore the real prevalence of HIV/PvCo is unknown. This study showed a low prevalence of HIV/PvCo despite the high prevalence of malaria and HIV locally. Even though relatively small, this is the largest case series to describe HIV/PvCo.

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.

Background: Plasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development., Results: Absence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity., Conclusions: This study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection.

Methylene blue (MB) is an alternative for combating drug-resistant malaria parasites. Its transmission-blocking potential has been demonstrated in vivo in murine models, in vitro, and in clinical trials. MB shows high efficacy against Plasmodium vivax asexual stages; however, its efficacy in sexual stages is unknown. In this study, we evaluated the potential of MB against asexual and sexual forms of P. vivax isolated from the blood of patients residing in the Brazilian Amazon. An ex vivo schizont maturation assay, zygote to ookinete transformation assay, direct membrane feed assay (DMFA), and standard membrane feed assay (SMFA) using P. vivax gametocytes with MB exposure were performed. A cytotoxicity assay was also performed on freshly collected peripheral blood mononuclear cells (PBMCs) and the hepatocyte carcinoma cell line HepG2. MB inhibited the P. vivax schizont maturation and demonstrated an IC50 lower than that of chloroquine (control drug). In the sexual forms, the MB demonstrated a high level of inhibition in the transformation of the zygotes into ookinetes. In the DMFA, MB did not considerably affect the infection rate and showed low inhibition, but it demonstrated a slight decrease in the infection intensity in all tested concentrations. In contrast, in the SMFA, MB was able to completely block the transmission at the highest concentration (20 μM). MB demonstrated low cytotoxicity to fresh PBMCs but demonstrated higher cytotoxicity to the hepatocyte carcinoma cell line HepG2. These results show that MB may be a potential drug for vivax malaria treatment. [ABSTRACT FROM AUTHOR]

Globally, malaria and human immunodeficiency virus (HIV) are both independently associated with a massive burden of disease and death. While their co-infection has been well studied for Plasmodium falciparum, scarce data exist regarding the association of P. vivax and HIV. In this cohort study, we assessed the effect of HIV on the risk of vivax malaria infection and recurrence during a 4-year follow-up period in an endemic area of the Brazilian Amazon. For the purpose of this study, we obtained clinical information from January 2012 to December 2016 from two databases. HIV screening data were acquired from the clinical information system at the tropical hospital Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD). The National Malaria Surveillance database (SIVEP malaria) was utilized to identify malaria infections during a 4-year follow-up period after diagnosis of HIV. Both datasets were combined via data linkage. Between 2012 and 2016, a total of 42,121 people were screened for HIV, with 1569 testing positive (3.7%). Out of all the patients diagnosed with HIV, 198 had at least one episode of P. vivax malaria in the follow-up. In the HIV-negative group, 711 participants had at least one P. vivax malaria episode. When comparing both groups, HIV patients had a 6.48 [(5.37–7.83); P < 0.0001] (adjusted relative risk) greater chance of acquiring P. vivax malaria. Moreover, being of the male gender [ARR = 1.41 (1.17–1.71); P < 0.0001], Amerindian ethnicity [ARR = 2.77 (1.46–5.28); P < 0.0001], and a resident in a municipality of the Metropolitan region of Manaus [ARR = 1.48 (1.02–2.15); P = 0.038] were independent risk factors associated with an increased risk of clinical malaria. Education ≥ 8 years [ARR = 0.41 (0.26–0.64); P < 0.0001] and living in the urban area [ARR = 0.44 (0.24–0.80); P = 0.007] were associated to a lower risk of P. vivax malaria. A total of 28 (14.1%) and 180 (25.3%) recurrences (at least a second clinical malaria episode) were reported in the HIV-positive and HIV-negative groups, respectively. After adjusting for sex and education, HIV-positive status was associated with a tendency towards protection from P. vivax malaria recurrences [ARR = 0.55 (0.27–1.10); P = 0.090]. HIV status was not associated with hospitalizations due to P. vivax malaria. CD4 + counts and viral load were not associated with recurrences of P. vivax malaria. No significant differences were found in the distribution of parasitemia between HIV-negative and HIV-positive P. vivax malaria patients. Our results suggest that HIV-positive status is a risk factor for vivax malaria infection, which represents an additional challenge that should be addressed during elimination efforts. [ABSTRACT FROM AUTHOR]

The control and elimination of malaria caused by Plasmodium vivax both represent a great challenge due to the biological aspects of the species. Gametocytes are the forms responsible for the transmission of the parasite to the vector and the search for new strategies for blocking transmission are essential in a scenario of control and elimination The challenges in this search in regard to P. vivax mainly stem from the lack of a long-term culture and the limitation of studies of gametocytes. This study evaluated the viability and infectivity of P. vivax gametocytes in short-term culture. The samples enriched in gametocytes using Percoll (i), using magnetic-activated cell sorting (MACS®) (ii), and using non-enriched samples (iii) were evaluated. After the procedures, gametocytes were cultured in IMDM medium for up to 48 h. Cultured P. vivax gametocytes were viable and infectious for up to 48 h, however differences in viability and infectivity were observed in the samples after 12 h of culture in relation to 0 h. Percoll-enriched samples were shown to be viable in culture for longer intervals than those purified using MACS®. Gametocyte viability after enrichment procedures and short-term culture may provide new avenues in the development of methods for evaluating P. vivax TB. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development. Results: Absence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity. Conclusions: This study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection. [ABSTRACT FROM AUTHOR]