Plasmodium vivax and SARS-CoV-2 co-infection in Venezuelan pregnant women: a case series.

BACKGROUND: Malaria-endemic areas are not spared from the impact of coronavirus disease 2019 (COVID-19), leading to co-infection scenarios where overlapping symptoms impose serious diagnostic challenges. Current knowledge on Plasmodium spp. and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infection in pregnant women remains limited, especially in Latin America, where Plasmodium vivax infection is highly prevalent. METHODS: This is a case series of five pregnant women with P. vivax and SARS-CoV-2 co-infection hospitalized in two main malaria referral centers of the Capital District and Bolivar state, Venezuela between March 13, 2020 and December 31, 2021. RESULTS: Clinical and laboratory data from five pregnant women with a mean age of 22 years were analyzed; three of them were in the third trimester of pregnancy. Comorbidities included obesity in two cases, hypertension in one, and asthma in one. Three out of five patients had severe to critical COVID-19 disease. Dry cough, fever, chills, and headache were the most frequent symptoms reported. Laboratory analyses showed elevated aspartate/alanine aminotransferase and creatinine levels, thrombocytopenia, and severe anemia as the most relevant abnormalities. The mean period between symptom onset and a positive molecular test for SARS-CoV-2 infection or positive microscopy for Plasmodium spp. was 4.8 ± 2.5 days and 2.8 ± 1.6 days, respectively. The mean hospital stay was 5.4 ± 7 days. Three women recovered and were discharged from the hospital. Two women died, one from cerebral malaria and one from respiratory failure. Three adverse fetal outcomes were registered, two miscarriages and one stillbirth. CONCLUSION: This study documented a predominance of severe/critical COVID-19 disease and a high proportion of adverse maternal-fetal outcomes among pregnant women with malaria and COVID-19 co-infection. More comprehensive prospective cohort studies are warranted to explore the risk factors, management challenges, and clinical outcomes of pregnant women with this co-infection.

Increased circulating myeloid-derived suppressor cells in vivax malaria and severe falciparum malaria.

BACKGROUND: Circulating myeloid-derived-suppressor-cells (MDSC) with immunosuppressive function are increased in human experimental Plasmodium falciparum infection, but have not been studied in clinical malaria. METHODS: Using flow-cytometry, circulating polymorphonuclear-MDSC were evaluated in cryopreserved samples from patients with uncomplicated Plasmodium vivax (n = 8) and uncomplicated (n = 4) and severe (n = 16) falciparum malaria from Papua, Indonesia. RESULTS: The absolute number of circulating polymorphonuclear-MDSC were significantly elevated in severe falciparum malaria patients compared to controls (n = 10). Polymorphonuclear-MDSC levels in uncomplicated vivax malaria were also elevated to levels comparable to that seen in severe falciparum malaria. CONCLUSION: Control of expansion of immunosuppressive MDSC may be important for development of effective immune responses in falciparum and vivax malaria.

The spectrum of clinical biomarkers in severe malaria and new avenues for exploration.

Globally, malaria is a public health concern, with severe malaria (SM) contributing a major share of the disease burden in malaria endemic countries. In this context, identification and validation of SM biomarkers are essential in clinical practice. Some biomarkers (C-reactive protein, angiopoietin 2, angiopoietin-2/1 ratio, platelet count, histidine-rich protein 2) have yielded interesting results in the prognosis of Plasmodium falciparum severe malaria, but for severe P. vivax and P. knowlesi malaria, similar evidence is missing. The validation of these biomarkers is hindered by several factors such as low sample size, paucity of evidence-evaluating studies, suboptimal values of sensitivity/specificity, poor clinical practicality of measurement methods, mixed Plasmodium infections, and good clinical value of the biomarkers for concurrent infections (pneumonia and current COVID-19 pandemic). Most of these biomarkers are non-specific to pathogens as they are related to host response and hence should be regarded as prognostic/predictive biomarkers that complement but do not replace pathogen biomarkers for clinical evaluation of SM patients. This review highlights the importance of research on diagnostic/predictive/therapeutic biomarkers, neglected malaria species, and clinical practicality of measurement methods in future studies. Finally, the importance of omics technologies for faster identification/validation of SM biomarkers is also included.

An algorithm based on molecular protocols to improve the detection of Plasmodium in autochthonous malarial areas in the Atlantic Forest biome.

Malaria is the most important vector-borne disease in the world and a challenge for control programs. In Brazil, 99% of cases occur in the Amazon region. In the extra-Amazonian region, a non-endemic area, epidemiological surveillance focuses on imported malaria and on autochthonous outbreaks, including cases with mild symptoms and low parasitemia acquired in the Atlantic Forest biome. In this scenario, cases are likely to be underreported, since submicroscopic parasitemias are not detected by thick blood smear, considered the reference test. Molecular tests are more sensitive, detecting asymptomatic individuals and mixed infections. The aim of this study was to propose a more efficient alternative to detect asymptomatic individuals living in areas of low malaria endemicity, as they are reservoirs of Plasmodium that maintain transmission locally. In total, 955 blood samples from residents of 16 municipalities with autochthonous malaria outbreaks in the Sao Paulo State were analyzed; 371 samples were collected in EDTA tubes and 584 in filter paper. All samples were initially screened by a genus-specific qPCR targeting ssrRNA genes (limit of detection of 1 parasite/µL). Then, positive samples were subjected to a nested PCR targeting ssrRNA and dihydrofolate reductase-thymidylate synthase genes (limit of detection of 10 parasites/µL) to determine Plasmodium species. The results showed a statistically significant difference (K = 0.049; p < 0.0001) between microscopy positivity (6.9%) and qPCR (22.9%) for EDTA-blood samples. Conversely, for samples collected in filter paper, no statistical difference was observed, with 2.6% positivity by thick blood smear and 3.1% for qPCR (K = 0.036; p = 0.7). Samples positive by qPCR were assayed by a species-specific nested PCR that was in turn positive in 26% of samples (16 P. vivax and 4 P. malariae ). The results showed that molecular protocols applied to blood samples from residents in areas with autochthonous transmission of malaria were useful to detect asymptomatic patients who act as a source of transmission. The results showed that the genus-specific qPCR was useful for screening positives, with the subsequent identification of species by nested PCR. Additional improvements, such as standardization of blood plotting on filter paper and a more sensitive protocol for species determination, are essential. The qPCR-based algorithm for screening positives followed by nested PCR will contribute to more efficient control of malaria transmission, offering faster and more sensitive tools to detect asymptomatic Plasmodium reservoirs.

Asymptomatic malaria infection at the China-Vietnam border: Knowledge and implications for the cross-border migrant population during the COVID-19 pandemic.

BACKGROUND: Eliminating malaria along the China-Vietnam border remains one of the greatest challenges in China, especially during the coronavirus disease 2019 (COVID-19) pandemic, which has disrupted the continuity of malaria control and elimination programs. Understanding the factors associated with asymptomatic malaria infection will inform control interventions aimed at elimination of the disease among migrants from Vietnam working in China, who constitute an at-risk population. METHODS: From March 2018 to September 2019, 108 migrants from Vietnam working in Ningming County, Guangxi, were enrolled in this study. Each person was interviewed using a structured questionnaire. Blood samples were collected and sent for PCR detection and sequencing. The obtained sequences were analyzed using the BLAST program and DNAMAN software. RESULTS: The proportion of participants with malaria knowledge was low, with 19.4% (21/108) reporting knowledge about transmission, 23.2% (25/108) reporting knowledge about clinical symptoms, 7.4% (8/108) reporting awareness of the risk of death and 14.8% (16/108) reporting awareness of prevention methods. No significant difference in the malaria knowledge rate was found among occupational groups, except in the migrant worker group, whose knowledge rate was higher than those in the other occupational groups (χ2 = 32.452, p < 0.001). Although most of the participants (80.6%, 87/108) owned mosquito nets, only approximately half of the participants (49.1%, 53/108) reported using bed nets. The parasitological analysis revealed that 5.6% (6/108) of all the participants were positive for malaria, including 5 participants with Plasmodium falciparum and 1 participant with Plasmodium vivax malaria. There were no statistically significant differences in the positivity rates among the different age, sex, family-size, nationality, occupational, and behavior groups. The positivity rates in individuals who did not use mosquito nets, did not use mosquito coils, and did not install mosquito nets were 4.8% (1/21), 6.8% (3/44), and 3.6% (2/55), respectively. CONCLUSION: Health education focused on high-risk populations, such as migrant workers and forest goers, should be strengthened. Verbal communication and information transmission via the internet, radio, and mobile phone platforms may be required during the COVID-19 pandemic. Further risk assessments and proactive case detection should also be performed in Ningming County and other border counties in Guangxi to detect active and asymptomatic infections in a timely manner and prevent re-establishment of the disease in these communities.

Haematological response in experimental human Plasmodium falciparum and Plasmodium vivax malaria.

BACKGROUND: Malaria-associated anaemia, arising from symptomatic, asymptomatic and submicroscopic infections, is a significant cause of morbidity worldwide. Induced blood stage malaria volunteer infection studies (IBSM-VIS) provide a unique opportunity to evaluate the haematological response to early Plasmodium falciparum and Plasmodium vivax infection. METHODS: This study was an analysis of the haemoglobin, red cell counts, and parasitaemia data from 315 participants enrolled in IBSM-VIS between 2012 and 2019, including 269 participants inoculated with the 3D7 strain of P. falciparum (Pf3D7), 15 with an artemisinin-resistant P. falciparum strain (PfK13) and 46 with P. vivax. Factors associated with the fractional fall in haemoglobin (Hb-FF) were evaluated, and the malaria-attributable erythrocyte loss after accounting for phlebotomy-related losses was estimated. The relative contribution of parasitized erythrocytes to the malaria-attributable erythrocyte loss was also estimated. RESULTS: The median peak parasitaemia prior to treatment was 10,277 parasites/ml (IQR 3566-27,815), 71,427 parasites/ml [IQR 33,236-180,213], and 34,840 parasites/ml (IQR 13,302-77,064) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. The median Hb-FF was 10.3% (IQR 7.8-13.3), 14.8% (IQR 11.8-15.9) and 11.7% (IQR 8.9-14.5) in those inoculated with Pf3D7, PfK13 and P. vivax, respectively, with the haemoglobin nadir occurring a median 12 (IQR 5-21), 15 (IQR 7-22), and 8 (IQR 7-15) days following inoculation. In participants inoculated with P. falciparum, recrudescence was associated with a greater Hb-FF, while in those with P. vivax, the Hb-FF was associated with a higher pre-treatment parasitaemia and later day of anti-malarial treatment. After accounting for phlebotomy-related blood losses, the estimated Hb-FF was 4.1% (IQR 3.1-5.3), 7.2% (IQR 5.8-7.8), and 4.9% (IQR 3.7-6.1) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. Parasitized erythrocytes were estimated to account for 0.015% (IQR 0.006-0.06), 0.128% (IQR 0.068-0.616) and 0.022% (IQR 0.008-0.082) of the malaria-attributable erythrocyte loss in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. CONCLUSION: Early experimental P. falciparum and P. vivax infection resulted in a small but significant fall in haemoglobin despite parasitaemia only just at the level of microscopic detection. Loss of parasitized erythrocytes accounted for < 0.2% of the total malaria-attributable haemoglobin loss.

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex technology (Bio-Plex 200 or MAGPIX).

Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.