Identification of Reticulocyte Binding Domain of Plasmodium ovale curtisi Duffy Binding Protein (PocDBP) Involved in Reticulocyte Invasion

The Plasmodium ovale curtisi (Poc) prevalence has increased substantially in sub-Saharan African countries as well as regions of Southeast Asia. Poc parasite biology has not been explored much to date; in particular, the invasion mechanism of this malaria parasite remains unclear. In this study, the binding domain of the Duffy binding protein of P. ovale curtisi (PocDBP) was characterized as an important ligand for reticulocyte invasion. The homologous region of the P. vivax Duffy binding protein in PocDBP, named PocDBP-RII herein, was selected, and the recombinant PocDBP-RII protein was expressed in an Escherichia coli system. This was used to analyze reticulocyte binding activity using fluorescence-activated cell sorting and immune serum production in rabbits. The binding specificity was proven by treating reticulocytes with trypsin, chymotrypsin and neuraminidase. The amino acid sequence homology in the N-terminal Cys-rich region was found to be ~ 44% between PvDBP and PocDBP. The reticulocyte binding activity of PocDBP-RII was significantly higher than the erythrocyte binding activity and was concentration dependent. Erythrocyte binding was reduced significantly by chymotrypsin treatment and inhibited by an anti-PocDBP-RII antibody. This finding suggests that PocDBP may be an important ligand in the reticulocyte invasion process of P. ovale curtisi.

Neglected malaria parasites in hard-to-reach areas of Odisha, India: implications in elimination programme

Background Information on the foci of Plasmodium species infections is essential for any country heading towards elimination. Odisha, one of the malaria-endemic states of India is targeting elimination of malaria by 2030. To support decision-making regarding targeted intervention, the distribution of Plasmodium species infections was investigated in hard-to-reach areas where a special malaria elimination drive, namely Durgama Anchalare Malaria Nirakaran (DAMaN) began in 2017. Methods A cross-sectional survey was conducted in 2228 households during July to November 2019 in six districts, to evaluate the occurrence of Plasmodium species. The species were identified by polymerase chain reaction (PCR) followed by sequencing, in case of Plasmodium ovale. Results Of the 3557 blood specimens tested, malaria infection was detected in 282 (7.8%) specimens by PCR. Of the total positive samples, 14.1% were P. ovale spp. and 10.3% were Plasmodium malariae infections. The majority of P. ovale spp. (75.8%) infections were mixed with either Plasmodium falciparum and/or Plasmodium vivax and found to be distributed in three geophysical regions (Northern-plateau, Central Tableland and Eastern Ghat) of the State, while P. malariae has been found in Northern-plateau and Eastern Ghat regions. Speciation revealed occurrence of both Plasmodium ovale curtisi (classic type) and Plasmodium ovale wallikeri (variant type). Conclusions In the present study a considerable number of P. ovale spp. and P. malariae were detected in a wide geographical areas of Odisha State, which contributes around 40% of the country’s total malaria burden. For successful elimination of malaria within the framework of national programme, P. ovale spp. along with P. malariae needs to be incorporated in surveillance system, especially when P. falciparum and P. vivax spp. are in rapid decline.

Genetic diversity and immunogenicity of the merozoite surface protein 1 C-terminal 19-kDa fragment of Plasmodium ovale imported from Africa into China

Background Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119. Methods A total of 37 clinical Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012–2016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp119 (pocmsp119) and P. ovale wallikeri (pow) msp119 (powmsp119) genes by polymerase chain reaction. The genetic diversity of pomsp119 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (GST) proteins were expressed in an Escherichia coli expression system and analyzed by western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays. Results Sequences of pomsp119 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as ~ 37-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively. Conclusions This study revealed highly conserved gene sequences of pomsp119. In addition, naturally acquired humoral immune responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design. Graphical abstract

Epidemiological, Physiological and Diagnostic Comparison of Plasmodium ovale curtisi and Plasmodium ovale wallikeri

Nowadays, Plasmodium ovale is divided into two non-recombinant sympatric species: Plasmodium ovale wallikeri and Plasmodium ovale curtisi. In this mini review, we summarize the available knowledge on the clinical/biological aspects of P. ovale spp. malaria and current techniques for the diagnosis/characterisation of P. ovale curtisi and P. ovale wallikeri. P. ovale wallikeri infections are characterized by a deeper thrombocytopenia and shorter latency compared to P. ovale curtisi infections, indicating that P. ovale wallikeri is more pathogenic than P. ovale curtisi. Rapid diagnosis for effective management is difficult for P. ovale spp., since specific rapid diagnostic tests are not available and microscopic diagnosis, which is recognized as the gold standard, requires expert microscopists to differentiate P. ovale spp. from other Plasmodium species. Neglect in addressing these issues in the prevalence of P. ovale spp. represents the existing gap in the fight against malaria.

Genetic diversity and immunogenicity of the merozoite surface protein 1 C-terminal 19-kDa fragment (MSP119) of Plasmodium ovale imported from Africa to China

Background Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa of MSP1 has long been considered as one of the major candidate antigens for malaria blood-stage vaccine in Plasmodium falciparum. However, there are limited information on the C-terminal 19-kDa region of Plasmodium ovale merozoite surface protein 1 (PoMSP119). This study, therefore, aims to analyse genetic diversity and immunogenicity of PoMSP119. Methods A total of 37 clinical P. ovale isolates including P. ovale curtisi (Poc) and P. ovale wallikeri (Pow) imported from Africa to China and collected between 2012 and 2016 were used. Genomic DNA were used to amplify pocmsp119 and powmsp119 genes by polymerase chain reaction (PCR). Genetic diversity of pomsp119 was analyzed using the MegAlign and GeneDoc v.6 programs. Recombinant PoMSP119-GST proteins were expressed in an Escherichia coli expression system and analyzed by Western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assays (ELISA). In addition, antigen-specific T-cell responses were performed by lymphocyte proliferation assays. A total of 49 serum samples from P. ovale infections and healthy people were used to evaluate natural immune responses through protein microarray assays. Results Sequences of pomsp119 were found thoroughly conserved in all clinical isolates. Recombinant PoMSP119 proteins were efficiently expressed and purified as ~ 37 kDa proteins. High antibody responses in immunized mice with rPoMSP119-GST were observed. The rPoMSP119-GST induced high avidity indexes with an average of 92.57% and 85.32% for Poc and Pow, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found during splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for natural immune responses detection in patients infected with P. ovale were 89.96% and 75%, respectively. Conclusions This study revealed high conservation in sequences of pomsp119 and high immunogenicity of rPoMSP119. The rPoMSP119 proteins detected humoral immune responses in patients with P. ovale infection. Such informative results advance our understanding of natural immunity to P. ovale infection and contribute to the knowledge base for the development of a PoMSP119-based vaccine.

COVID-19 and Plasmodium ovale Malaria: A Rare Case of Co-Infection

The COVID-19 pandemic continues to be a major health problem worldwide. Timely diagnosis of co-infections mimicking COVID-19, such as malaria, might be challenging particularly in non-endemic areas. We report the first case of COVID-19 and Plasmodium ovale malaria co-infection from our region aiming to highligt the importance of travel history and prophylaxis in malaria management in the context of pandemic. The galloping sound can sometimes be a harbinger of zebra besides the horse.

Plasmodium vivax and Plasmodium ovale in the Malaria Elimination Agenda in Africa

In recent times, several countries in sub-Saharan Africa have reported cases of Plasmodium vivax (Pv) with a considerable number being Duffy negative. Current efforts at malaria elimination are focused solely on Plasmodium falciparum (Pf) excluding non-falciparum malaria. Pv and Plasmodium ovale (Po) have hypnozoite forms that can serve as reservoirs of infection and sustain transmission. The burden of these parasites in Africa seems to be more than acknowledged, playing roles in migrant and autochthonous infections. Considering that elimination and eradication is a current aim for WHO and Roll Back Malaria (RBM), the inclusion of Pv and Po in the elimination agenda cannot be over-emphasized. The biology of Pv and Po are such that the same elimination strategies as are used for Pf cannot be applied so, going forward, new approaches will be required to attain elimination and eradication targets.

Evaluation of a novel real-time PCR assay for the detection, identification and quantification of Plasmodium species causing malaria in humans

Background The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value. Methods The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions. Results No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10–3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%. Conclusion Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.