Molecular epidemiology of asymptomatic Plasmodium species infections in Papua New Guinea

Asymptomatic malaria infections present a major challenge to malaria control and elimination in endemic settings, as they contribute to maintaining malaria transmission but are missed during clinical surveillance. Little is known on the true extent and basic epidemiological parameters of asymptomatic malaria infections, such as the infection rate or the duration of infection. In case of P. vivax, an additional challenge is its ability to form dormant liver stages that evade diagnostic tools. The aim of this thesis was to advance our basic understanding of asymptomatic P. falciparum and P. vivax infections and their relevance for malaria transmission. To this end, the extent of low-density malaria infections and the presence of gametocytes, i.e. the parasite stage responsible for human-to-mosquito transmission, is examined using highly sensitive molecular tools in Papua New Guinea and Tanzania. Longitudinal tracking of P. falciparum and P. vivax clones in a cohort study in Papua New Guinean children provides novel insights into the molecular epidemiology of malaria infections, allowing to explore small-scale geographical heterogeneity in malaria exposure, the relationship of exposure and clinical disease, the duration of untreated asymptomatic P. falciparum infections, and the contribution of P. vivax relapses from dormant liver stages to infection and disease

Evaluation of a commercial ELISA as alternative to plaque reduction neutralization test to detect neutralizing antibodies against SARS-CoV-2

High-throughput detection of neutralizing antibodies against SARS-CoV-2 presents a valuable tool for vaccine trials or investigations of population immunity. We evaluate the performance of the first commercial surrogate virus neutralization test (sVNT, GenScript Biotech) against SARS-CoV-2 plaque reduction neutralization test (PRNT) in convalescent and vaccinated individuals. We compare it to five other ELISAs, two of which are designed to detect neutralizing antibodies. In 491 pre-vaccination serum samples, sVNT missed 23.6% of PRNT-positive samples when using the manufacturer-recommended cutoff of 30% binding inhibition. Introducing an equivocal area between 15 and 35% maximized sensitivity and specificity against PRNT to 72.8–93.1% and 73.5–97.6%, respectively. The overall diagnostic performance of the other ELISAs for neutralizing antibodies was below that of sVNT. Vaccinated individuals exhibited higher antibody titers by PRNT (median 119.8, IQR 56.7–160) and binding inhibition by sVNT (median 95.7, IQR 88.1–96.8) than convalescent patients (median 49.1, IQR 20–62; median 52.9, IQR 31.2–76.2). GenScript sVNT is suitable to screen for SARS-CoV-2-neutralizing antibodies; however, to obtain accurate results, confirmatory testing by PRNT in a equivocal area is required. This equivocal area may require adaptation for use in vaccinated individuals, due to higher antibody titers.Peer Reviewe

Deciphering archeological contexts from the magnetic map: Determination of daub distribution and mass of Chalcolithic house remains

The unique size and development of prehistoric megasites of the north Pontic Cucuteni-Tripolye Chalcolithic groups (4100–3600 BCE) challenge modern archeology and paleoecology. The extremely large number of houses (approximately 3000, mostly burned) necessitates the development of multidisciplinary technologies to gain a holistic understanding of such sites. In this contribution, we introduce a novel geophysical methodology and a detailed analysis of magnetic data – including evolved modeling techniques – to provide critical information about the setup of findings, enabling a thorough understanding of the settlement dynamics, apart from invasive excavation techniques. The case study is based on data from magnetic field maps and distribution maps of the daub and pottery find categories. This information is used to infer magnetic models for each find category to numerically calculate their magnetic fields for comparison with the archeological data. The comparison quantifies the sensitivity of the magnetic measurements with respect to the distribution of the different find categories. Next, via inversion computation, the characteristic depth functions of soil magnetization are used to generate maps of magnetization from the measured magnetic field maps. To validate the inverted soil magnetization maps, the magnetic excavation models are used, providing an interpretational frame for the application to magnetic anomalies outside excavated areas. This joint magnetic and archeological methodology allows estimating the find density and testing hypotheses about the burning processes of the houses. In this paper, we show internal patterns of burned houses, comparable to archeological house models, and their calculated masses as examples of the methodology. An application of the new approach to complete megasites has the potential to enable a better understanding of the settlement structure and its evolution, improve the quality of population estimations, and thus calculate the human impact on the forest steppe environment and address questions of resilience and carrying capacity

On‐site non‐destructive determination of the remanent magnetization of archaeological finds using field magnetometers

The determination of the natural remanent magnetization (NRM) of archaeological features can be used for magnetic modelling, joining of shards, archaeomagnetic dating or the investigation of the firing–cooling–collapsing order of ancient buildings. The measurement of NRM is normally conducted on cylindrical or cubic samples in the laboratory. Nevertheless, archaeological finds should preferably not be destroyed, and laboratory instruments are high in costs. Therefore, we propose a lightweight and portable measurement set-up including already available field magnetometers (preferably caesium magnetometers) in which the archaeological sample of arbitrary shape, in our case a piece of daub, is mounted inside a gimbal to be rotated in all directions. The magnetic field of the sample is measured at a large number of rotational positions with the magnetometer kept at fixed position. In these measurements, the unknown direction of the NRM vector of the sample is rotated, whereas the average magnetic susceptibility of the sample and the ambient magnetic field are constant and known. Hence, the vector of NRM can be determined through least-squares inversion. For the inversion computation, the sample volume is discretized either as voxel model or approximated as an equivalent sphere. Under certain conditions depending on sample–sensor distance, dipole moment and radius of the sample, the approximation by a sphere is valid without effect on the accuracy of results.This is accurate enough, for example, to determine from daub pieces of burnt house remains whether the building was burnt and cooled before or after it collapsed

Prevalence and zoonotic transmission of colistin-resistant and carbapenemase-producing Enterobacterales on German pig farms

The treatment of infections due to colistin-resistant (Col-E) and carbapenemase-producing (CPE) Enterobacterales challenges clinicians both in human and veterinary medicine. Preventing zoonotic transmission of these multidrug-resistant bacteria is a Public Health priority. This study investigates the prevalence of Col-E and CPE on 81 pig farms in North-West Germany as well as among 138 directly exposed humans working on these farms. Between March 2018 and September 2020, 318 samples of porcine feces were taken using boot swabs. Farm workers provided a stool sample. Both a selective culture-based approach and a molecular detection of colistin (mcr-1 to mcr-5) and carbapenem resistance determinants (blaOXA-48/blaVIM/blaKPC/blaNDM) was used to screen all samples. Isolates from farm workers and farms were compared using core genome multilocus-sequence typing (cgMLST) and plasmid-typing. CPE were cultured neither from porcine feces nor from human stool samples. In one stool sample, blaOXA-48 was detected, but no respective CPE isolate was found. Col-E were found in 18/318 porcine (5.7%) samples from 10/81 (12.3%) farms and 2/138 (1.4%) farmers, respectively. All Col-E isolates were Escherichia coli harboring mcr-1. Both farm workers colonized with Col-E worked on farms where no Col-E were detected in porcine samples. In conclusion, CPE were absent on German pig farms. This supports findings of culture-based national monitoring systems and provides evidence that even when improving the diagnostic sensitivity by using molecular detection techniques in addition to culture, CPE are not prevalent. Col-E were prevalent in porcine feces despite a recent decrease in colistin usage among German livestock and absence of colistin treatments on the sampled farms. Farmers carried Col-E, but zoonotic transmission was not confirmed.Peer Reviewe

Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections

Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum.; Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis.; Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold.; To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones

Effects of liver-stage clearance by Primaquine on gametocyte carriage of Plasmodium vivax and P. falciparum

Primaquine (PQ) is the only currently licensed antimalarial that prevents Plasmodium vivax (Pv) relapses. It also clears mature P. falciparum (Pf) gametocytes, thereby reducing post-treatment transmission. Randomized PQ treatment in a treatment-to-reinfection cohort in Papua New Guinean children permitted the study of Pv and Pf gametocyte carriage after radical cure and to investigate the contribution of Pv relapses.; Children received radical cure with Chloroquine, Artemether-Lumefantrine plus either PQ or placebo. Blood samples were subsequently collected in 2-to 4-weekly intervals over 8 months. Gametocytes were detected by quantitative reverse transcription-PCR targeting pvs25 and pfs25.; PQ treatment reduced the incidence of Pv gametocytes by 73%, which was comparable to the effect of PQ on incidence of blood-stage infections. 92% of Pv and 79% of Pf gametocyte-positive infections were asymptomatic. Pv and to a lesser extent Pf gametocyte positivity and density were associated with high blood-stage parasite densities. Multivariate analysis revealed that the odds of gametocytes were significantly reduced in mixed-species infections compared to single-species infections for both species (ORPv = 0.39 [95% CI 0.25-0.62], ORPf = 0.33 [95% CI 0.18-0.60], p<0.001). No difference between the PQ and placebo treatment arms was observed in density of Pv gametocytes or in the proportion of Pv infections that carried gametocytes. First infections after blood-stage and placebo treatment, likely caused by a relapsing hypnozoite, were equally likely to carry gametocytes than first infections after PQ treatment, likely caused by an infective mosquito bite.; Pv relapses and new infections are associated with similar levels of gametocytaemia. Relapses thus contribute considerably to the Pv reservoir highlighting the importance of effective anti-hypnozoite treatment for efficient control of Pv.; ClinicalTrials.gov NCT02143934

qRT-PCR versus IFA-based quantification of male and female gametocytes in low-density Plasmodium falciparum infections and their relevance for transmission

Accurate quantification of female and male gametocytes and sex ratios in asymptomatic low-density malaria infections are important for assessing their transmission potential. Gametocytes often escape detection even by molecular methods, therefore ultralow gametocyte densities were quantified in large blood volumes.; Female and male gametocytes were quantified in 161 PCR-positive Plasmodium falciparum infections from a cross-sectional survey in Papua New Guinea. Ten-fold concentrated RNA from 800 µL blood was analyzed using female-specific pfs25 and male-specific pfmget or mssp qRT-PCR. Gametocyte sex ratios from qRT-PCR were compared with those from immunofluorescence assays (IFA).; Gametocytes were identified in 58% (93/161) P. falciparum-positive individuals. Mean gametocyte densities were frequently below 1 female and 1 male gametocyte/µL by qRT-PCR. The mean proportion of males was 0.39 (95% confidence interval, 0.33-0.44) by pfs25/pfmget qRT-PCR; this correlated well with IFA results (Pearsons r2 = 0.91; P < .001). A Poisson model fitted to our data predicted 16% P. falciparum-positive individuals that are likely to transmit, assuming at least 1 female and 1 male gametocyte per 2.5 µL mosquito bloodmeal.; Based on model estimates of female and male gametocytes per 2.5 µL blood, P. falciparum-positive individuals detected exclusively by ultrasensitive diagnostics are negligible for human-to-mosquito transmission.Estimating the transmission potential of ultralow-density malaria infections informs interventions. Almost all infections with ≥1 female and male gametocyte per 2.5 µL mosquito bloodmeal, and thus with highest likelihood of contributing to human-to-mosquito transmission, were detectable by standard molecular diagnostics

First results from stratigraphic investigation of Chiselet tell (Romania) using seismic full waveform inversion

In this paper we analyze the applicability of seismic full-waveform inversion (FWI) for investigation of tells, which are often characterized by heterogeneous and small-scale stratigraphy. Major aim is to identify location and shape of settlement layers and house remains. Furthermore, a paleochannel, which is important to understand the interaction between tell and the surrounding landscape, was found at the tell flank