Impact of ACE I gene insertion/deletion, A-240T polymorphisms and the renin-angiotensin-aldosterone system on COVID-19 disease.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which has led to an enormous burden on patient morbidity and mortality. The renin-angiotensin-aldosterone system (RAAS) plays a significant role in various pulmonary diseases. Since SARS-CoV-2 utilizes the angiotensin-converting enzyme (ACE)2 receptor to exert its virulence and pathogenicity, the RAAS is of particular importance in COVID 19. METHODS: Our preliminary study investigates retrospectively the influence of selected ACE-polymorphisms (I/D location at intron 16 in the B-coding sequence (rs4646994) and A-240T (rs 4291) at the A-promoter) as well as ACE1 and ACE2 serum levels on disease severity and the inflammatory response in inpatients and outpatients with COVID-19. RESULTS: Our study included 96 outpatients and 88 inpatients (65.9% male, mean age 60 years) with COVID-19 from April to December 2020 in four locations in Germany. Of the hospitalized patients, 88.6% participants were moderately ill (n = 78, 64% male, median age 60 years), and 11.4% participants were severely ill or deceased (n = 10, 90% male, median age 71 years). We found no polymorphism-related difference in disease, in age distribution, time to hospitalization and time of hospitalization for the inpatient group. ACE1 serum levels were significantly increased in the DD compared to the II polymorphism and in the TT compared to the AA polymorphism. There was no significant difference in ACE 1 serum levels l between moderately ill and severely ill patients. However, participants requiring oxygen supplementation had significantly elevated ACE1 levels compared to participants not requiring oxygen, with no difference in ACE2 levels whereas females had significantly higher ACE2 levels. CONCLUSIONS: Although there were no differences in the distribution of ACE polymorphisms in disease severity, we found increased proinflammatory regulation of the RAAS in patients with oxygen demand and increased serum ACE2 levels in women, indicating a possible enhanced anti-inflammatory immune response. CLINICAL TRIAL REGISTRATION: PreBiSeCov: German Clinical Trials Register, DRKS-ID: DRKS00021591, Registered on 27th April 2020.

Follow-up SARS-CoV-2 serological study of a health care worker cohort following COVID-19 booster vaccination.

BACKGROUND: Studies have shown that Omicron breakthrough infections can occur at higher SARS-CoV-2 antibody levels compared to previous variants. Estimating the magnitude of immunological protection induced from COVID-19 vaccination and previous infection remains important due to varying local pandemic dynamics and types of vaccination programmes, particularly among at-risk populations such as health care workers (HCWs). We analysed a follow-up SARS-CoV-2 serological survey of HCWs at a tertiary COVID-19 referral hospital in Germany following the onset of the Omicron variant. METHODS: The serological survey was conducted in January 2022, one year after previous surveys in 2020 and the availability of COVID-19 boosters including BNT162b2, ChAdOx1-S, and mRNA-1273. HCWs voluntarily provided blood for serology and completed a comprehensive questionnaire. SARS-CoV-2 serological analyses were performed using an Immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). Antibody levels were reported according to HCW demographic and occupational characteristics, COVID-19 vaccination and SARS-CoV-2 infection history, and multivariate linear regression was used to evaluate these associations. RESULTS: In January 2022 (following the fourth COVID-19 wave in Germany including the onset of the Omicron variant), 1482/1517 (97.7%) HCWs tested SARS-CoV-2 seropositive, compared to 4.6% in December 2020 (second COVID-19 wave). Approximately 80% had received three COVID-19 vaccine doses and 15% reported a previous laboratory-confirmed SARS-CoV-2 infection. SARS-CoV-2 IgG geometric mean titres ranged from 335 (95% Confidence Intervals [CI]: 258-434) among those vaccinated twice and without previous infection to 2204 (95% CI: 1919-2531) among those vaccinated three times and with previous infection. Heterologous COVID-19 vaccination combinations including a mRNA-1273 booster were significantly associated with the highest IgG antibody levels compared to other schemes. There was an 8-to 10-fold increase in IgG antibody levels among 31 HCWs who reported a SARS-CoV-2 infection in May 2020 to January 2022 after COVID-19 booster vaccination. CONCLUSIONS: Our findings demonstrate the importance of ongoing COVID-19 booster vaccination strategies in the context of variants such as Omicron and despite hybrid immunity from previous SARS-CoV-2 infections, particularly for at-risk populations such as HCWs. Where feasible, effective types of booster vaccination, such as mRNA vaccines, and the appropriate timing of administration should be carefully considered.

Serological methods for the detection of antibodies against monkeypox virus applicable for laboratories with different biosafety levels.

The monkeypox virus (MPXV) outbreak in 2022 has renewed interest in the detection of antibodies against orthopox viruses (OPXV) and MPXV, as serological methods can aid diagnostics and are key to epidemiological studies. Here three complementary serological methods are described with different strengths to aid the development and evaluation of in-house assays: An immunofluorescence assay (IFA) for specific detection of IgG and IgM, an enzyme-linked immunosorbent assay for higher sample throughput to aid epidemiological studies and a neutralization test to detect virus neutralizing antibodies. As implementation of MPXV-specific diagnostics is often hampered by the requirement for a dedicated biosafety level 3 laboratory (BSL-3), the focus of this study is on biosafety aspects to facilitate safe testing also under BSL-2 conditions. To this aim, it was analyzed whether OPXV, which can be handled under BSL-2 conditions, could be used as less virulent alternatives to MPXV. Furthermore, an inactivation method was established to remove up to five log-steps of infectious virus particles from viraemic sera without compromising antibody detection. The results show that immunological cross-reactivity between OPXV provides an opportunity for the interchangeable usage of different OPXV species in serological assays, enabling MPXV serology outside of BSL-3 facilities.

Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic.

BACKGROUND: Over the course of the COVID-19 pandemic, laboratories worldwide have been facing an unprecedented increase in demand for PCR testing because of the high importance of diagnostics for prevention and control of virus spread. Moreover, testing demand has been varying considerably over time, depending on the epidemiological situation, rendering efficient resource allocation difficult. Here, we present a scalable workflow which we implemented in our laboratory to increase PCR testing capacity while maintaining high flexibility regarding the number of samples to be processed. METHODS: We compared the performance of five automated extraction instruments, using dilutions of SARS-CoV-2 cell culture supernatant as well as clinical samples. To increase PCR throughput, we combined the two duplex PCR reactions of our previously published SARS-CoV-2 PCR assay into one quadruplex reaction and compared their limit of detection as well as their performance on the detection of low viral loads in clinical samples. Furthermore, we developed a sample pooling protocol with either two or four samples per pool, combined with a specifically adapted SARS-CoV-2 quadruplex PCR assay, and compared the diagnostic sensitivity of pooled testing and individual testing. RESULTS: All tested automated extraction instruments yielded comparable results regarding the subsequent sensitivity of SARS-CoV-2 detection by PCR. While the limit of detection of the quadruplex SARS-CoV-2 PCR assay (E-Gene assay: 28.7 genome equivalents (ge)/reaction, orf1ab assay: 32.0 ge/reaction) was slightly higher than that of our previously published duplex PCR assays (E-Gene assay: 9.8 ge/reaction, orf1ab assay: 6.6 ge/reaction), the rate of correctly identified positive patient samples was comparable for both assays. Sample pooling with optimized downstream quadruplex PCR showed no loss in diagnostic sensitivity compared to individual testing. CONCLUSION: Specific adaptation of PCR assays can help overcome the potential loss of sensitivity due to higher levels of PCR multiplexing or sample dilution in pooled testing. Combining these adapted PCR assays with different sample processing strategies provides a simple and highly adjustable workflow for resource-efficient SARS-CoV-2 diagnostics. The presented principles can easily be adopted in a variety of laboratory settings as well as be adapted to pathogens other than SARS-CoV-2, making it feasible for any laboratory that conducts PCR diagnostics.

Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

Longitudinal SARS-CoV-2 seroepidemiological investigation among healthcare workers at a tertiary care hospital in Germany.

BACKGROUND: SARS-CoV-2 cases in Germany increased in early March 2020. By April 2020, cases among health care workers (HCW) were detected across departments at a tertiary care hospital in Berlin, prompting a longitudinal investigation to assess HCW SARS-CoV-2 serostatus with an improved testing strategy and associated risk factors. METHODS: In May/June and December 2020, HCWs voluntarily provided blood for serology and nasopharyngeal/oropharyngeal (NP/OP) samples for real-time polymerase chain reaction (PCR) and completed a questionnaire. A four-tiered SARS-CoV-2 serological testing strategy including two different enzyme-linked immunosorbent assays (ELISA) and biological neutralization test (NT) was used. ELISA-NT correlation was assessed using Pearson's correlation coefficient. Sociodemographic and occupational factors associated with seropositivity were assessed with multivariate logistic regression. RESULTS: In May/June, 18/1477 (1.2%) HCWs were SARS-CoV-2 seropositive, followed by 56/1223 (4.6%) in December. Among those tested in both, all seropositive in May/June remained seropositive by ELISA and positive by NT after 6 months. ELISA ratios correlated well with NT titres in May/June (R = 0.79) but less so in December (R = 0.41). Those seropositive reporting a past SARS-CoV-2 positive PCR result increased from 44.4% in May/June to 85.7% in December. HCWs with higher occupational risk (based on profession and working site), nurses, males, and those self-reporting COVID-19-like symptoms had significantly higher odds of seropositivity. CONCLUSIONS: This investigation provides insight into the burden of HCW infection in this local outbreak context and the antibody dynamics over time with an improved robust testing strategy. It also highlights the continued need for effective infection control measures particularly among HCWs with higher occupational risk.

Intense and Mild First Epidemic Wave of Coronavirus Disease, The Gambia.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is evolving differently in Africa than in other regions. Africa has lower SARS-CoV-2 transmission rates and milder clinical manifestations. Detailed SARS-CoV-2 epidemiologic data are needed in Africa. We used publicly available data to calculate SARS-CoV-2 infections per 1,000 persons in The Gambia. We evaluated transmission rates among 1,366 employees of the Medical Research Council Unit The Gambia (MRCG), where systematic surveillance of symptomatic cases and contact tracing were implemented. By September 30, 2020, The Gambia had identified 3,579 SARS-CoV-2 cases, including 115 deaths; 67% of cases were identified in August. Among infections, MRCG staff accounted for 191 cases; all were asymptomatic or mild. The cumulative incidence rate among nonclinical MRCG staff was 124 infections/1,000 persons, which is >80-fold higher than estimates of diagnosed cases among the population. Systematic surveillance and seroepidemiologic surveys are needed to clarify the extent of SARS-CoV-2 transmission in Africa.

qRT-PCR versus IFA-based Quantification of Male and Female Gametocytes in Low-Density Plasmodium falciparum Infections and Their Relevance for Transmission.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Clinical relevance of low-density Plasmodium falciparum parasitemia in untreated febrile children: A cohort study.

BACKGROUND: Low-density (LD) Plasmodium infections are missed by standard malaria rapid diagnostic tests (standard mRDT) when the blood antigen concentration is below the detection threshold. The clinical impact of these LD infections is unknown. This study investigates the clinical presentation and outcome of untreated febrile children with LD infections attending primary care facilities in a moderately endemic area of Tanzania. METHODS/FINDINGS: This cohort study includes 2,801 febrile pediatric outpatients (median age 13.5 months [range 2-59], female:male ratio 0.8:1.0) recruited in Dar es Salaam, Tanzania between 01 December 2014 and 28 February 2016. Treatment decisions were guided by a clinical decision support algorithm run on a mobile app, which also collected clinical data. Only standard mRDT+ cases received antimalarials. Outcomes (clinical failure, secondary hospitalization, and death) were collected in follow-up visits or interviews on days 3, 7, and 28. After patient recruitment had ended, frozen blood from all 2,801 patients was tested for Plasmodium falciparum (Pf) by ultrasensitive-quantitative polymerase chain reaction (qPCR), standard mRDT, and "ultrasensitive" mRDT. As the latter did not improve sensitivity beyond standard mRDT, it is hereafter excluded. Clinical features and outcomes in LD patients (standard mRDT-/ultrasensitive-qPCR+, not given antimalarials) were compared with those with no detectable (ND) parasitemia (standard mRDT-/ultrasensitive-qPCR-) or high-density (HD) infections (standard mRDT+/ultrasensitive-qPCR+, antimalarial-treated). Pf positivity rate was 7.1% (n = 199/2,801) and 9.8% (n = 274/2,801) by standard mRDT and ultrasensitive qPCR, respectively. Thus, 28.0% (n = 76/274) of ultrasensitive qPCR+ cases were not detected by standard mRDT and labeled "LD". LD patients were, on average, 10.6 months younger than those with HD infections (95% CI 7.0-14.3 months, p < 0.001). Compared with ND, LD patients more frequently had the diagnosis of undifferentiated fever of presumed viral origin (risk ratio [RR] = 2.0, 95% CI 1.3-3.1, p = 0.003) and were more often suffering from severe malnutrition (RR = 3.2, 95% CI 1.1-7.5, p = 0.03). Despite not receiving antimalarials, outcomes for the LD group did not differ from ND regarding clinical failures (2.6% [n = 2/76] versus 4.0% [n = 101/2,527], RR = 0.7, 95% CI 0.2-3.5, p = 0.7) or secondary hospitalizations (2.6% [n = 2/76] versus 2.8% [n = 72/2,527], RR = 0.7,95% CI 0.2-3.2, p = 0.9), and no deaths were reported in any Pf-positive groups. HD patients experienced more secondary hospitalizations (10.1% [n = 20/198], RR = 0.3, 95% CI 0.1-1.0, p = 0.005) than LD patients. All the patients in this cohort were febrile children; thus, the association between parasitemia and fever cannot be investigated, nor can the conclusions be extrapolated to neonates and adults. CONCLUSIONS: During a 28-day follow-up period, we did not find evidence of a difference in negative outcomes between febrile children with untreated LD Pf parasitemia and those without Pf parasitemia. These findings suggest LD parasitemia may either be a self-resolving fever or an incidental finding in children with other infections, including those of viral origin. These findings do not support a clinical benefit nor additional risk (e.g. because of missed bacterial infections) to using ultrasensitive malaria diagnostics at a primary care level.

Molecular methods for tracking residual Plasmodium falciparum transmission in a close-to-elimination setting in Zanzibar.

BACKGROUND: Molecular detection of low-density Plasmodium falciparum infections is essential for surveillance studies conducted to inform malaria control strategies in close-to-elimination settings. Molecular monitoring of residual malaria infections usually requires a large study size, therefore sampling and diagnostic processes need to be economical and optimized for high-throughput. A method comparison was undertaken to identify the most efficient diagnostic procedure for processing large collections of community samples with optimal test sensitivity, simplicity, and minimal costs. METHODS: In a reactive case detection study conducted on Zanzibar, parasitaemia of 4590 individuals of all ages was investigated by a highly sensitive quantitative (q) PCR that targets multiple var gene copies per parasite genome. To reduce cost, a first round of positivity screening was performed on pools of dried blood spots from five individuals. Ten cycles of a pre-PCR were performed directly on the filter paper punches, followed by qPCR. In a second round, samples of positive pools were individually analysed by pre-PCR and qPCR. RESULTS: Prevalence in household members and neighbors of index cases was 1.7% (78/4590) with a geometric mean parasite density of 58 parasites/µl blood. Using qPCR as gold standard, diagnostic sensitivity of rapid diagnostic tests (RDTs) was 37% (29/78). Infections positive by qPCR but negative by RDT had mean densities of 15 parasites/µl blood. CONCLUSION: The approach of pre-screening reactive case detection samples in pools of five was ideal for a low prevalence setting such as in Zanzibar. Performing direct PCR on filter paper punches saves substantial time and justifies the higher cost for a polymerase suitable for amplifying DNA directly from whole blood. Molecular monitoring in community samples provided a more accurate picture of infection prevalence, as it identified a potential reservoir of infection that was largely missed by RDT. The developed qPCR-based methodology for screening large sample sets represents primarily a research tool that should inform the design of malaria elimination strategies. It may also prove beneficial for diagnostic tasks in surveillance-response activities.

Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities.

BACKGROUND: The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome. METHODS: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities. RESULTS: In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG. CONCLUSION: The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

Diagnostic Performance of Conventional and Ultrasensitive Rapid Diagnostic Tests for Malaria in Febrile Outpatients in Tanzania.

BACKGROUND: A novel ultrasensitive malaria rapid diagnostic test (us-RDT) has been developed for improved active Plasmodium falciparum infection detection. The usefulness of this us-RDT in clinical diagnosis and fever management has not been evaluated. METHODS: Diagnostic performance of us-RDT was compared retrospectively to that of conventional RDT (co-RDT) in 3000 children and 515 adults presenting with fever to Tanzanian outpatient clinics. The parasite density was measured by an ultrasensitive qPCR (us-qPCR), and the HRP2 concentration was measured by an enzyme-linked immunosorbent assay. RESULTS: us-RDT identified few additional P. falciparum-positive patients as compared to co-RDT (276 vs 265 parasite-positive patients detected), with only a marginally greater sensitivity (75% vs 73%), using us-qPCR as the gold standard (357 parasite-positive patients detected). The specificity of both RDTs was >99%. Five of 11 additional patients testing positive by us-RDT had negative results by us-qPCR. The HRP2 concentration was above the limit of detection for co-RDT (>3653 pg of HRP2 per mL of blood) in almost all infections (99% [236 of 239]) with a parasite density >100 parasites per µL of blood. At parasite densities <100 parasites/µL, the HRP2 concentration was above the limits of detection of us-RDT (>793 pg/mL) and co-RDT in 29 (25%) and 24 (20%) of 118 patients, respectively. CONCLUSION: There is neither an advantage nor a risk of using us-RDT, rather than co-RDT, for clinical malaria diagnosis. In febrile patients, only a small proportion of infections are characterized by a parasite density or an HRP2 concentration in the range where use of us-RDT would confer a meaningful advantage over co-RDT.

Plasmodium vivax and Plasmodium falciparum infection dynamics: re-infections, recrudescences and relapses.

BACKGROUND: In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation. RESULTS: For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses. CONCLUSION: The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics.

Plasmodium vivax molecular diagnostics in community surveys: pitfalls and solutions.

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers.

Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.

Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets.

BACKGROUND: Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. METHODS AND FINDINGS: Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/µl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum-positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. CONCLUSIONS: Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts.

Strategies for understanding and reducing the Plasmodium vivax and Plasmodium ovale hypnozoite reservoir in Papua New Guinean children: a randomised placebo-controlled trial and mathematical model.

BACKGROUND: The undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children. METHODS AND FINDINGS: From 17 August 2009 to 20 May 2010, 524 children aged 5-10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes. CONCLUSIONS: These results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission. TRIAL REGISTRATION: ClinicalTrials.gov NCT02143934.

Identification and deconvolution of cross-resistance signals from antimalarial compounds using multidrug-resistant Plasmodium falciparum strains.

Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

Dynein light chain 8a of Toxoplasma gondii, a unique conoid-localized ß-strand-swapped homodimer, is required for an efficient parasite growth.

Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins (TgDLC8a-d), of which only TgDLC8a clusters in the mainstream LC8 class. TgDLC8b-d proteins form a divergent and alveolate-specific clade. TgDLC8b-d proteins are largely cytosolic, whereas TgDLC8a resides in the conoid at the apical end of T. gondii. The apical location of TgDLC8a is also not shared by its nearly identical Eimeria (EtDLC8a), Plasmodium (PfDLC8), or human (HsDLC8) orthologs. Notwithstanding an exclusive conoid targeting, TgDLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the ß strands that interact with the antiparallel ß' strands of the opposing monomers. The TgDLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported PfDLC8 homodimer is shaped by binding of the ß strand with the parallel ß' strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the TgDLC8a-S88E mutant monomeric and cytosolic in T. gondii, and its overexpression inhibits the parasite growth in human fibroblasts.