Comparison of diagnostic methods for the detection and quantification of the four sympatric Plasmodium species in field samples from Papua New Guinea

Accurate diagnosis of Plasmodium infections is essential for malaria morbidity and mortality reduction in tropical areas. Despite great advantages of light microscopy (LM) for malaria diagnosis, its limited sensitivity is a critical shortfall for epidemiological studies. Robust molecular diagnostics tools are thus needed.; The present study describes the development of a duplex quantitative real time PCR (qPCR) assay, which specifically detects and quantifies the four human Plasmodium species. Performance of this method was compared to PCR-ligase detection reaction-fluorescent microsphere assay (PCR_LDR_FMA), nested PCR (nPCR) and LM, using field samples collected from 452 children one to five years of age from the Sepik area in Papua New Guinea. Agreement between diagnostic methods was calcualted using kappa statistics.; The agreement of qPCR with other molecular diagnostic methods was substantial for the detection of P. falciparum, but was moderate for the detection of P. vivax, P. malariae and P. ovale. P. falciparum and P. vivax prevalence by qPCR was 40.9% and 65.7% respectively. This compares to 43.8% and 73.2% by nPCR and 47.1% and 67.5% by PCR_LDR_FMA. P. malariae and P. ovale prevalence was 4.7% and 7.3% by qPCR, 3.3% and 3.8% by nPCR, and 7.7% and 4.4% by PCR_LDR_FMA. Prevalence by LM was lower for all four species, being 25.4% for P. falciparum, 54.9% for P. vivax, 2.4% for P. malariae and 0.0% for P. ovale. The quantification by qPCR closely correlated with microscopic quantification for P. falciparum and P. vivax samples (R2 = 0.825 and R2 = 0.505, respectively). The low prevalence of P. malariae and P. ovale did not permit a solid comparative analysis of quantification for these species.; The qPCR assay developed proved optimal for detection of all four Plasmodium species. Densities by LM were well reflected in quantification results by qPCR, whereby congruence was better for P. falciparum than for P. vivax. This likely is a consequence of the generally lower P. vivax densities. Easy performance of the qPCR assay, a less laborious workflow and reduced risk of contamination, together with reduced costs per sample through reduced reaction volume, opens the possibility to implement qPCR in endemic settings as a suitable diagnostic tool for large epidemiological studies

Implementing a secure instant messaging app in the COVID-19 pandemic: Usage experiences of primary care physicians and local health authorities

Background: At the onset of the COVID-19 pandemic general practitioners complained about feeling uninformed and lacking a sufficient flow of information from the local health authorities. Secure instant messaging describes a digital, chat-based form of communication enabling ambulatory care providers to connect in real-time and share information across medial sectors. KomPan, a proof-of-concept study, established a secure instant messaging structure in two model regions in Germany to improve commu-nication between general practitioners and local health authorities via an additional communication pathway. This paper presents results of a qualitative user survey.Methods: We recruited general practitioners (n = 43) and staff of local health authorities (n = 10) in two Hessian model regions for using the secure instant messaging (SIM) app of the Famedly GmbH, starting in December 2020 (2nd COVID-19 wave). We asked participants to share their usage experiences after a usage time of multiple months. In guided telephone interviews, we primarily asked how communication between the two user groups had changed while using secure instant messaging. The interviews were transcribed verbatim and analysed using thematic analysis.Results: We conducted ten interviews with general practitioners and two with staff of the participating local health authorities. Using our SIM-based platform promoted professional exchange between general practitioners. In contrast, the app had little impact on communication between local health authorities and general practitioners. Opposing expectations and usage patterns of the two user groups, among other reasons, probably led to a reduced direct trans-sectoral communication via secure instant messaging.Conclusion: Establishing local chat groups for general practitioners was welcomed, especially during the pandemic situation, to improve professional exchange while experiencing challenging working condi-tions. To use secure instant messaging effectively for trans-sectoral communication a more comprehen-sive approach seems to be needed, such as digitalisation of institutional communication structures and improved networks of local healthcare providers

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1

Co-localization of Tex1 with SBP1.

<p>P27-specific polyclonal rabbit sera was used to detect Tex1 (red). Co-localization was performed using SBP1 polyclonal mouse sera (green). Co-localization was performed in ring (A) trophozoite (B) and schizont stage (C) infected RBCs. Nuclear DNA was stained with DAPI (blue), Transmission image (DIC), Scale bar: 5 µm.</p

Brefeldin A sensitivity of Tex1 export.

<p>3D7 infected RBC were treated with BFA and fixed (+BFA). Tex1 was stained using P27 or P27A-specific mouse antibodies (in red, upper panel: early trophozoite, middle panel: trophozoite). Tex1 visible inside the parasite in close proximity to the nucleus. A control culture (+ETOH) was incubated with equivalent concentration of ethanol, the solvent of Brefeldin A. In the control culture Tex1 was correctly exported and associated to MC (in red). The nucleus was stained with DAPI (in blue). Transmission image (DIC). scale bar: 5 µm.</p

Immunofluorescence staining of erythrocytes infected by <i>P. falciparum</i> (ring, trophozoites and schizont stages) using P27-specific polyclonal rabbit sera.

<p>P27-specific polyclonal rabbit sera was used to detect Tex1 (green) A) in late ring stages B) in trophozoite stages C) in schizont stages. Nucleus stained with DAPI (blue), transmission picture of the infected red blood cell (DIC) and merged picture of the two signals or the signals merged with transmission picture (merge), Scale bar: 5 µm.</p

Equinatoxin II assay.

<p>A) 3D7 infected RBC lysed with equinatoxin II. Integrity of MCs is demonstrated by the absence of the SBP1 signal after using SBP1 N-terminus specific polyclonal mouse sera (note: N-terminus of SBP1 faces the lumen of MCs). Tex1 signal on the MC surface was obtained with P27-specific polyclonal rabbit sera (in green). B) 3D7 infected RBC lysed with equinatoxin followed by Triton lysis. MC lumen is now accessible for antibodies as shown by the SBP1 signal (in red). Nuclear DNA stained with DAPI (blue), Transmission image (DIC). Scale bar: 5 µm.</p

Co-localization of Tex1 with MAHRP1.

<p>P27-specific polyclonal rabbit sera was used to detect Tex1 (red). Co-localization was performed using MAHRP1 polyclonal mouse sera (green). Co-localization was performed in ring stage (A) trophozoite (B) and schizont stage (C) infected RBC. Nuclear DNA was stained with DAPI (blue), Transmission image (DIC), Scale bar: 5 µm.</p