Studying temporal titre evolution of commercial SARS-CoV-2 assays reveals significant shortcomings of using BAU standardization for comparison.

BACKGROUND: Measuring specific anti-SARS-CoV-2 antibodies has become one of the main epidemiological tools to survey the ongoing SARS-CoV-2 pandemic, but also vaccination response. The WHO made available a set of well-characterized samples derived from recovered individuals to allow normalization between different quantitative anti-Spike assays to defined Binding Antibody Units (BAU). METHODS: To assess sero-responses longitudinally, a cohort of ninety-nine SARS-CoV-2 RT-PCR positive subjects was followed up together with forty-five vaccinees without previous infection but with two vaccinations. Sero-responses were evaluated using a total of six different assays: four measuring anti-Spike proteins (converted to BAU), one measuring anti-Nucleocapsid proteins and one SARS-CoV-2 surrogate virus neutralization. Both cohorts were evaluated using the Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and the Roche Elecsys Anti-SARS-CoV-2 anti-S1 assay. RESULTS: In SARS-CoV-2-convalesce subjects, the BAU-sero-responses of Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and Roche Elecsys Anti-SARS-CoV-2 anti-S1 peaked both at 47 (43-51) days, the first assay followed by a slow decay thereafter (> 208 days), while the second assay not presenting any decay within one year. Both assay values in BAUs are only equivalent a few months after infection, elsewhere correction factors up to 10 are necessary. In contrast, in infection-naive vaccinees the assays perform similarly. CONCLUSION: The results of our study suggest that the establishment of a protective correlate or vaccination booster recommendation based on different assays, although BAU-standardised, is still challenging. At the moment the characteristics of the available assays used are not related, and the BAU-standardisation is unable to correct for that.

Stage-Dependent Increase of Systemic Immune Activation and CCR5+CD4+ T Cells in Filarial Driven Lymphedema in Ghana and Tanzania.

Chronic lymphedema caused by infection of Wuchereria bancrofti is a disfiguring disease that leads to physical disability, stigmatization, and reduced quality of life. The edematous changes occur mainly on the lower extremities and can progress over time due to secondary bacterial infections. In this study, we characterized participants with filarial lymphedema from Ghana and Tanzania as having low (stage 1-2), intermediate (stage 3-4), or advanced (stage 5-7) lymphedema to determine CD4+ T cell activation patterns and markers associated with immune cell exhaustion. A flow cytometry-based analysis of peripheral whole blood revealed different T cell phenotypes within participants with different stages of filarial lymphedema. In detail, increased frequencies of CD4+HLA-DR+CD38+ T cells were associated with higher stages of filarial lymphedema in patients from Ghana and Tanzania. In addition, significantly increased frequencies of CCR5+CD4+ T cells were seen in Ghanaian participants with advanced LE stages, which was not observed in the Tanzanian cohort. The frequencies of CD8+PD-1+ T cells were augmented in individuals with higher stage lymphedema in both countries. These findings show distinct activation and exhaustion patterns in lymphedema patients but reveal that immunological findings differ between West and East African countries.

Assessment of tuberculosis disease activity in people infected with Mycobacterium tuberculosis and living with HIV: A longitudinal cohort study.

Background: Early detection of asymptomatic incipient tuberculosis (TB) could improve clinical outcomes and reduce the spread of Mycobacterium tuberculosis (MTB) infection, particularly in HIV endemic settings. This study assessed TB disease activity over 5 years in people living with HIV co-infected with MTB using a surrogate biomarker. Methods: Between Jan 1, 2013 and Aug 31, 2018, 2014 people living with HIV were screened annually for active TB using the Xpert MTB/RIF diagnostic assay in 11 clinics in Kenya, Tanzania, Uganda, and Nigeria. Longitudinal blood mononuclear cell samples from 46 selected patients with active and recurrent tuberculosis, latent infection, or incipient TB were further analysed for MTB-specific T-cell activation (defined by CD38 expression) as a well-defined surrogate marker for TB disease covering a total of 1758 person-months. Findings: MTB-specific CD4 T-cell activation differentiated active, Xpert MTB/RIF positive TB from latent TB with a sensitivity and specificity of 86% and was reduced upon TB treatment initiation. Activated MTB-specific T cells were present in 63% and 23% of incipient TB cases 6 and 12 months before diagnosis of active disease, respectively. Transient increases of MTB-specific T cell activation were also observed in individuals with latent infection, while persistent activation was a hallmark of recurrent TB after the end of treatment. Interpretation: In most cases, progression to active TB disease started 6-12 months before diagnosis by clinical symptoms and sputum occurrence of bacilli. Blood biomarkers could facilitate early detection of incipient TB, improve clinical outcomes, and reduce the transmission of MTB. Funding: This work was supported by the President's Emergency Plan for AIDS Relief via a cooperative agreement between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense [W81XWH-11-2-0174, W81XWH-18-2-0040] and by the Bundesministerium für Bildung und Forschung (BmBF) through funding of the Deutsches Zentrum für Infektionsforschung (DZIF, TTU-TB personalized medicine TTU 02_813).

The viability of utilising phone-based text messages in data capture and reporting morbidities due to lymphatic Filariasis by community health workers: a qualitative study in Kilwa district, Tanzania.

BACKGROUND: Globally, there is recognition of the value of using mobile phones among health providers in improving health systems performance. However, in many Low- and Middle-income countries where there is shortage of health providers, Community Health Workers have assumed some responsibilities especially relating to identifying and reporting on health problems within their communities. Despite the known benefits of using mobile phone technology to deliver health services, there is limited information on the extent to which Community Health Workers are able to effectively use the technology in data collection and reporting. The aim of this study was to determine the feasibility of utilizing phone-based text messages on Lymphatic Filariasis morbidity surveillance by Community Health Workers. METHODS: This was a cross sectional study whose data was collected through key informant interviews and focused group discussions among community health workers, health providers and staff of neglected tropical diseases control program. Collected transcripts were analysed through Thematic content analysis as it allowed for the identification of data codes through inductive reasoning. RESULTS: The use of mobile phone-based text messages in data collection and reporting lymphatic filariasis morbidity cases by Community Health Workers was perceived by study participants to be a relevant tool and feasible due to the ease of use of the technology. The tool was reported by end-users to significantly increase their performance and efficiency was gained in terms of reduced paper work, increased the number of patients accessing health care services and the ability to report in real-time. All respondents were confident that Community Health Workers were the right persons to interact with communities in tracking and reporting morbidity cases using mobile technology. CONCLUSION: Mobile phone-based text messages have proven to be effective in routine workflows such as, data collection and reporting on Lymphatic Filariasis morbidity cases, patient to provider communication, decision making and supportive supervision. Mobile phones have also improved efficiency and general performance of end users in terms of increased number of cases identified and efficiency gained in terms of reduced paper work and the ability to collect and report in real-time.

Step towards elimination of Wuchereria bancrofti in Southwest Tanzania 10 years after mass drug administration with Albendazole and Ivermectin.

BACKGROUND: Lymphatic filariasis is a mosquito transmitted parasitic infection in tropical regions. Annual mass treatment with ivermectin and albendazole is used for transmission control of Wuchereria bancrofti, the infective agent of lymphatic filariasis in many African countries, including Tanzania. METHODOLOGY: In a general population study in Southwest Tanzania, individuals were tested for circulating filarial antigen, an indicator of W. bancrofti adult worm burden in 2009 before mass drug administration commenced in that area. Seven annual rounds with ivermectin and albendazole were given between 2009 and 2015 with a population coverage of over 70%. Participants of the previous study took part in a follow-up activity in 2019 to measure the effect of this governmental activity. FINDINGS: One thousand two hundred and ninety nine inhabitants of Kyela district in Southwest Tanzania aged 14 to 65 years who had participated in the study activities in 2009 were revisited in 2010/11 and 2019. Among this group, the prevalence of lymphatic filariasis of the 14-65 years olds in 2009 was 35.1%. A follow-up evaluation in 2010/11 had shown a reduction to 27.7%. In 2019, after 7 years of annual treatment and an additional three years of surveillance, the prevalence had dropped to 1.7%, demonstrating successful treatment by the national control programme. Risk factors for W. bancrofti-infection were the occupation as farmer, male sex, and older age. Most infected individuals in the 2019 follow-up study already had a positive test for filarial antigen in 2009 and/or 2010/11. CONCLUSIONS: This data supports the findings of the Tanzanian Neglected Tropical Disease Control Programme (NTDCP), who conducted Transmission Assessment Surveys and found an impressive reduction in the prevalence of LF in children. Our results complement this data by showing a similar decrease in prevalence of LF in the adult population in the same area. The elimination of LF seems achievable in the near future.

Distinct Immune Profiles of Exhausted Effector and Memory CD8+ T Cells in Individuals With Filarial Lymphedema.

CD8+ T cells are crucial for the clearance of viral infections, and current research begins to highlight their importance in parasitic diseases too. In-depth research about characteristics of CD8+ T-cell subsets and exhaustion remains uncertain, especially during filariasis, a chronic helminth infection. Lymphatic filariasis, elicited by Wuchereria bancrofti, remains a serious health problem in endemic areas in Ghana, especially in those suffering from morbidity due to lymphedema (LE). In this observational study, the characteristics and profiles of CD8+ T cells were compared between asymptomatic Wuchereria bancrofti-infected individuals, uninfected endemic normals, and those with LE (grades 2-6). Focusing on exhausted memory (CD8+exmem: CD8+ T-betdimEomeshi) and effector (CD8+exeff: CD8+T-bethiEomesdim) CD8+ T-cell subsets, advanced flow cytometry revealed that LE individuals presented reduced frequencies of IFN-γ+CD8+exmem T cells expressing Tim-3 or LAG-3 which negatively correlated to the presence of LE. Moreover, the LE cohort further showed significantly higher frequencies of IL-10+CD8+exeff T cells expressing either Tim-3, LAG-3, CD39, KLRG-1, or PD-1, all associated markers of exhaustion, and that these frequencies positively correlated with the presence of LE. In summary, this study shows that distinct exhausted CD8+ T-cell subsets are prominent in individuals suffering from LE, suggesting that enhanced inflammation and constant immune activation might drive exhaustion of CD8+ T cells. Since T-cell exhaustion is known to be associated with insufficient control of persisting antigen, the data presented here reveals that these CD8+ T-cell exhaustion patterns in filarial LE should be taken into consideration for prevention and control management of LE.

Flow cytometric analysis of cell lineage and immune activation markers using minimal amounts of human whole blood-Field method for remote settings.

Remote laboratory settings - such as those where studies on neglected tropical diseases are performed - often lack specialized equipment required for flow cytometric analysis of immune cell subsets, which complicates evaluations on a single cell level using peripheral blood. Our aim was to establish a method to use whole blood for phenotypic characterization of T-cells for specific markers including CD3, CD4, HLA-DR, CD38, CCR5, CD27, CD45RA, CD25, and FoxP3. This method uses 100 µL whole blood which is stained for extracellular markers, lysed, and cryopreserved at -20 °C at a field laboratory before transferring to liquid nitrogen for long-term storage and transportation. Cells can then be transported to a central laboratory for flow cytometry analysis. The method was initially established using samples from healthy donors; expression levels after cryopreservation were comparable to fresh whole blood samples from the same individuals. Moreover, data sets were also comparable to those which were stored in liquid nitrogen for up to one year. The method was then transferred to field studies in a remote area of Ghana which was used to observe its practicality and robustness in limited resource settings. Collectively, the low amount of whole blood (such as that taken from a finger prick), lack of any specialized equipment, and ease of use make this method suitable for utilization in remote field locations.

DNA methylation and lipid metabolism: an EWAS of 226 metabolic measures.

BACKGROUND: The discovery of robust and trans-ethnically replicated DNA methylation markers of metabolic phenotypes, has hinted at a potential role of epigenetic mechanisms in lipid metabolism. However, DNA methylation and the lipid compositions and lipid concentrations of lipoprotein sizes have been scarcely studied. Here, we present an epigenome-wide association study (EWAS) (N = 5414 total) of mostly lipid-related metabolic measures, including a fine profiling of lipoproteins. As lipoproteins are the main players in the different stages of lipid metabolism, examination of epigenetic markers of detailed lipoprotein features might improve the diagnosis, prognosis, and treatment of metabolic disturbances. RESULTS: We conducted an EWAS of leukocyte DNA methylation and 226 metabolic measurements determined by nuclear magnetic resonance spectroscopy in the population-based KORA F4 study (N = 1662) and replicated the results in the LOLIPOP, NFBC1966, and YFS cohorts (N = 3752). Follow-up analyses in the discovery cohort included investigations into gene transcripts, metabolic-measure ratios for pathway analysis, and disease endpoints. We identified 161 associations (p value < 4.7 × 10-10), covering 16 CpG sites at 11 loci and 57 metabolic measures. Identified metabolic measures were primarily medium and small lipoproteins, and fatty acids. For apolipoprotein B-containing lipoproteins, the associations mainly involved triglyceride composition and concentrations of cholesterol esters, triglycerides, free cholesterol, and phospholipids. All associations for HDL lipoproteins involved triglyceride measures only. Associated metabolic measure ratios, proxies of enzymatic activity, highlight amino acid, glucose, and lipid pathways as being potentially epigenetically implicated. Five CpG sites in four genes were associated with differential expression of transcripts in blood or adipose tissue. CpG sites in ABCG1 and PHGDH showed associations with metabolic measures, gene transcription, and metabolic measure ratios and were additionally linked to obesity or previous myocardial infarction, extending previously reported observations. CONCLUSION: Our study provides evidence of a link between DNA methylation and the lipid compositions and lipid concentrations of different lipoprotein size subclasses, thus offering in-depth insights into well-known associations of DNA methylation with total serum lipids. The results support detailed profiling of lipid metabolism to improve the molecular understanding of dyslipidemia and related disease mechanisms.

Filarial Lymphedema Patients Are Characterized by Exhausted CD4+ T Cells.

Worldwide, more than 200 million people are infected with filariae which can cause severe symptoms leading to reduced quality of life and contribute to disability-adjusted life years (DALYs). In particular, lymphatic filariasis (LF) caused by Wuchereria bancrofti can lead to lymphedema (LE) and consequently presents a serious health problem. To understand why only a fraction of the infected individuals develop pathology, it is essential to understand how filariae regulate host immunity. The central role of T cells for immunity against filariae has been shown in several studies. However, there is little knowledge about T cell exhaustion, which causes T cell dysfunction and impaired immune responses, in this group of individuals. Recently, we showed that LE patients from Ghana harbor distinct patterns of exhausted effector and memory CD8+ T cell subsets. Based on these findings, we now characterized CD4+ T cell subsets from the same Ghanaian patient cohort by analyzing distinct markers within a 13-colour flow cytometry panel. We revealed that LE patients had increased frequencies of CD4+ T cells expressing exhaustion-associated receptors such as KLRG-1, TIM-3 and PD-1 compared to healthy endemic normal and W. bancrofti-infected individuals. Moreover, CD4+ T cells in LE patients were characterized by distinct co-expression patterns of inhibitory receptors. Collectively with the previous findings on CD8+ T cell exhaustion patterns, the data shown here demonstrates that filarial LE patients harbor distinct subsets of exhausted T cells. Thus, T cell exhaustion patterns in LE patients need attention especially in regards to susceptibility of concomitant infections and should be taken into consideration for LE management measures.

Versatile exclusion-based sample preparation platform for integrated rare cell isolation and analyte extraction.

Rare cell populations provide a patient-centric tool to monitor disease treatment, response, and resistance. However, understanding rare cells is a complex problem, which requires cell isolation/purification and downstream molecular interrogation - processes challenged by non-target populations, which vary patient-to-patient and change with disease. As such, cell isolation platforms must be amenable to a range of sample types while maintaining high efficiency and purity. The multiplexed technology for automated extraction (mTAE) is a versatile magnetic bead-based isolation platform that facilitates positive, negative, and combinatorial selection with integrated protein staining and nucleic acid isolation. mTAE is validated by isolating circulating tumor cells (CTCs) - a model rare cell population - from breast and prostate cancer patient samples. Negative selection yielded high efficiency capture of CTCs while positive selection yielded higher purity with an average of only 95 contaminant cells captured per milliliter of processed whole blood. With combinatorial selection, an overall increase in capture efficiency was observed, highlighting the potential significance of integrating multiple capture approaches on a single platform. Following capture (and staining), on platform nucleic acid extraction enabled the detection of androgen receptor-related transcripts from CTCs isolated from prostate cancer patients. The flexibility (e.g. negative, positive, combinatorial selection) and capabilities (e.g. isolation, protein staining, and nucleic acid extraction) of mTAE enable users to freely interrogate specific cell populations, a capability required to understand the potential of emerging rare cell populations and readily adapt to the heterogeneity presented across clinical samples.

Integrated Analysis of Multiple Biomarkers from Circulating Tumor Cells Enabled by Exclusion-Based Analyte Isolation.

PURPOSE: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. EXPERIMENTAL DESIGN: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. RESULTS: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. CONCLUSIONS: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 1-11. ©2016 AACR.