Plasma RNA Research Articles

Congenital Co-infections Among HIV-Exposed Infants Born to Mothers on Antiretroviral Treatment in the United States.

BackgroundMany women living with HIV (WLHIV) are co-infected with cytomegalovirus (CMV), Toxoplasma gondii (T gondii), and/or hepatitis C virus (HCV). The rates of congenital or perinatal transmission of these co-infections are not well defined in the current era, when most WLHIV receive antiretroviral therapy (ART) during pregnancy.MethodsRetrospective review of infants of WLHIV born between 2009–2019. Mothers were screened for antibodies to CMV, T. gondii, and HCV; chronic HCV infection was confirmed using plasma RNA PCR. Infants whose mothers had positive/unknown serostatus were screened for CMV using urine or saliva DNA PCR or culture at ≤3 weeks of life; T. gondii using serology at ≤1 month; and HCV using plasma RNA PCR at ≤6 months and serology at ≥12 months.ResultsThe study included 264 infants from 255 pregnancies in 191 mothers. At delivery, the median (IQR) CD4 count was 569 (406–748) cells/mm3 and plasma HIV load was 0 (0–24) RNA copies/mL. Among 243 infants born to CMV-seropositive (209) or CMV-missed serostatus (25) mothers, 163 (67.1%) were tested for CMV. Four infants had CMV detected, resulting in a rate of congenital infection of 2.5%. Among 65 infants from 54 (21.2%) pregnancies in T. gondii-seropositive women and 8 in women with unknown T. gondii-serostatus, one acquired congenital toxoplasmosis in the setting of acute maternal T. gondii infection. There were no episodes of vertical transmission from mothers with latent toxoplasmosis. Among 18 infants from 13 (5.1%) pregnancies in HCV RNA PCR-positive women and 4 in women with unknown HCV serostatus, there were no congenital or perinatal HCV transmissions.ConclusionsIn a US cohort of pregnant WLHIV on ART, we identified high maternal CMV seroprevalence and a high rate of congenital CMV infection. We did not identify any congenital or perinatal transmissions of T. gondii or HCV among mothers who had latent or chronic infections. Our data support screening pregnant WLHIV and their infants for CMV and suggest that the rates of congenital and perinatal T. gondii and HCV infections among infants born to WLHIV on ART may be lower in the era of effective ART.

Plasma and urinary extracellular vesicle microRNAs and their related pathways in diabetic kidney disease

To explore extracellular vesicle microRNAs (EV miRNAs) and their target mRNAs in relation to diabetic kidney disease (DKD), we performed paired plasma and urinary EV small RNA sequencing (n = 18) in patients with type 2 diabetes and DKD (n = 5) and healthy subjects (n = 4) and metabolic network analyses using our own miRNA and public mRNA datasets. We found 13 common differentially expressed EV miRNAs in both fluids and 17 target mRNAs, including RRM2, NT5E, and UGDH. Because succinate dehydrogenase B was suggested to interact with proteins encoded by these three genes, we measured urinary succinate and adenosine in a validation study (n = 194). These two urinary metabolite concentrations were associated with DKD progression. In addition, renal expressions of NT5E and UGDH proteins were increased in db/db mice with DKD compared to control mice. In conclusion, we profiled DKD-related EV miRNAs in plasma and urine samples and found their relevant target pathways.

Abstract 3492: Exosome-based plasma RNA biomarkers for high-risk prostate cancer

Abstract Introduction: There is an unmet need for liquid biopsy tests to support the management of patients with intermediate and high-risk prostate cancer (PCa). In treatment naïve patients, risk stratification by standard pathology and tissue based genomic testing can be influenced by sampling errors inherent to PCa biopsy. Exosome-based liquid biopsies are emerging as a clinically effective platform for minimally invasive and highly sensitive diagnostics for diverse types of cancer. In this project we undertake the development of a plasma exosome-based liquid biopsy to stratify patients with clinically significant PCa that may circumvent the sampling challenges associated with tissue-based genomic tests. Exosomes are small double-lipid membrane vesicles that cells actively shed into various biofluids and that provide stable packages for RNA, DNA and protein molecules. Here we take advantage of a well-developed exosomal biomarker analysis platform to profile plasma exosomal RNA markers that differentiate treatment-naïve men diagnosed with NCCN low risk vs high risk PCa as the first step in the development of a plasma exosomal liquid biopsy to support the management of patients with intermediate and high-risk disease. Methods: We compared plasma exosomal RNA profiles from 11 high-risk and 9 low-risk, treatment-naïve, biopsy-confirmed patients together with 4 healthy controls. Exosomes were isolated from 1 ml plasma samples and exosomal RNA prepared using the ExosomeDx ExoLution platform. A hybrid-capture RNA Next Generation Sequencing analysis was performed to enrich for transcripts of exons, 3’ and 5’ untranslated RNA and long non-coding RNA (lncRNA) to identify differentially expressed RNAs. Results: We detected over 10,000 protein coding genes and ~400 lncRNAs in each plasma sample using exosomal RNAseq. 273 genes were differentially expressed between high-risk vs control but not in low-risk vs control. We identified four potential plasma exosomal biomarkers of high-risk PCa. These include three protein coding mRNA transcripts that showed greater than 20-fold lower expression in plasma from high-risk in comparison to low-risk patients; TCGA data show that two of these mRNA markers are also downregulated in PCa tissue in patients with worse survival. In addition, we identified one lncRNA that showed greater than 20-fold higher expression in plasma from high-risk in comparison to low-risk patients. We also detected multiple alternate androgen receptor transcripts in plasma exosomal RNA from the high-risk patients. Together, these represent early data for potentially novel liquid biopsy RNA biomarkers for high-risk PCa. Conclusions: Here we report results of a liquid biopsy biomarker discovery study to develop an exosome-based long RNA test for to support the management of patients with intermediate and high-risk PCa. Plasma exosomal RNA provides a rich reservoir of currently untapped biomarkers for high-risk PCa. Citation Format: Sandra M. Gaston, Johan K. Skog, Benjamin Spieler, Alan Dal Pra, Radka Stoyanova, Christian Fischer, Yevgenia Khodor, Grannum Sant, Seth Yu, Vasisht Tadigotla, Sudipto K. Chakrabortty, Sanoj Punnen, Alan Pollack. Exosome-based plasma RNA biomarkers for high-risk prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3492.

Abstract 1424: Prospective validation of a mRNA signature in plasma for the diagnosis of early stage lung cancer

Abstract Background: Non-small cell lung cancer (NSCLC) is usually diagnosed at stages IIIB-IV, with a median overall survival that does not exceed two years. In contrast, patients diagnosed at early and locally advanced stages (I-IIIA) can undergo surgery and have a significantly better prognosis. Imaging technologies often detect lung nodules of unknown significance that pose a diagnostic challenge. In a proof-of-concept study, based on a 76-patient cohort, we developed a preliminary mRNA expression signature in plasma that discriminated healthy individuals from early-stage NSCLC patients with AUC=0.98. Here, we aimed to expand the training cohort, to refine the diagnostic signature and to prospectively validate the final signature in the clinical setting. Methods: Two hundred and thirty individuals with pulmonary nodules suspicious of lung cancer have been enrolled in the training cohort. All of them underwent bronchoscopy, fine needle aspiration, percutaneous or surgical biopsy to confirm the diagnosis. Circulating-free RNA (cfRNA) has been isolated from plasma using an automatic extraction method (Qiasymphony, Qiagen). Purified cfRNA has been quantified using Qubit®, retrotranscribed and pre-amplified with 14 cycles using the Low RNA Input Amplification kit (NanoString Technologies). Gene expression analysis has been performed on the nCounter platform using the PanCancer IO360࣪ panel (NanoString Technologies), which can detect 770 transcripts related to tumor biology, micro-environment and the immune system. Results: One hundred twenty-six patients have been analyzed so far; plasma samples have been successfully analyzed by nCounter in all cases. Ongoing analysis reveal differential patterns of gene expression in early-stage NSCLC patients versus non-cancer individuals. Using a bioinformatics recursive feature elimination algorithm, we have selected a diagnostic signature with an area under the ROC curve of 0.89. The signature scores derived from the algorithm are significantly different between the non-cancer and NSCLC cases. Final results of the training and validation cohort will be presented at the meeting Conclusions: Plasma RNA expression signatures can be a useful tool to guide clinical decision in patients with pulmonary nodules suspicious of malignancy, orienting towards surgery or observation. Citation Format: Ana María Giménez Capitán, Pablo Rubisntein, Andrés Aguilar-Hernández, María González-cao, Irene Moya, Santiago Viteri, Carlos Cabrera, Santiago Ramón y Cajal, Karina Loor, Mario Culebras, Irene Sansano, Federico Rubisntein, Joselyn Valarezo, Clara Mayo-de las-Casas, Carlos Pedraz, Joseph Beechem, Sarah Warren, Rafael Rosell, Miguel Ángel Molina-Vila. Prospective validation of a mRNA signature in plasma for the diagnosis of early stage lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1424.

Detection of Embryonic Trisomy 21 in the First Trimester Using Maternal Plasma Cell-Free RNA.

Prenatal trisomy 21 (T21) screening commonly involves testing a maternal blood sample for fetal DNA aneuploidy. It is reliable but poses a cost barrier to universal screening. We hypothesized maternal plasma RNA screening might provide similar reliability but at a lower cost. Discovery experiments used plasma cell-free RNA from 20 women 11–13 weeks tested by RNA and miRNA microarrays followed by qRT-PCR. Thirty-six mRNAs and 18 small RNAs of the discovery cDNA were identified by qPCR as potential markers of embryonic T21. The second objective was validation of the RNA predictors in 998 independent pregnancies at 11–13 weeks including 50 T21. Initial analyses identified 9–15 differentially expressed RNA with modest predictive power (AUC < 0.70). The 54 RNAs were then subjected to machine learning. Eleven algorithms were trained on one partition and tested on an independent partition. The three best algorithms were identified by Kappa score and the effects of training/testing partition size and dataset class imbalance on prediction were evaluated. Six to ten RNAs predicted T21 with AUCs up to 1.00. The findings suggest that maternal plasma collected at 11–13 weeks, tested by qRT-PCR, and classified by machine learning, may accurately predict T21 for a lower cost than plasma DNA, thus opening the door to universal screening.

The Level of LINE-1 mRNA Is Increased in Extracellular Circulating Plasma RNA in Patients with Colorectal Cancer.

We performed a comparative quantitative analysis of LINE-1 mRNA levels in extracellular total plasma RNA in patients with colon cancer and practically healthy donors. Quantitative multiplex PCR with reverse transcription was used to assess the level of LINE-1 and 18S rRNA mRNA in extracellular total plasma RNA. The median of LINE-1 mRNA values in colon cancer patients (4.95) was significantly higher than in healthy donors (2.3) (p=0.037). It was shown for the first time that the level of LINE-1 mRNA in total RNA from blood plasma can be determined in the format of a liquid biopsy and serve as a new potential non-invasive marker of colon cancer.

Evaluation of a Maternal Plasma RNA Panel Predicting Spontaneous Preterm Birth and Its Expansion to the Prediction of Preeclampsia.

Preterm birth is the principal contributor to neonatal death and morbidity worldwide. We previously described a plasma cell-free RNA panel that between 16 and 20 weeks of pregnancy had potential to predict spontaneous preterm birth (sPTB) ≤ 32 weeks caused by preterm labor (PTL) or preterm premature rupture of membranes (PPROM). The present study had three objectives: (1) estimate the RNA panel prognostic accuracy for PTL/PPROM ≤ 32 weeks in a larger series; (2) improve accuracy by adding clinical characteristics to the predictive model; and (3) examine the association of the RNA panel with preeclampsia. We studied 289 women from Memphis TN prospectively sampled 16.0–20.7 weeks and found: (1) PSME2 and Hsa-Let 7g were differentially expressed in cases of PTL/PPROM ≤ 32 weeks and together provided fair predictive accuracy with AUC of 0.76; (2) combining the two RNAs with clinical characteristics improved good predictive accuracy for PTL/PPROM ≤ 32 weeks (AUC 0.83); (3) NAMPT and APOA1 were differentially expressed in women with ‘early-onset preeclampsia’ (EOP) and together provided good predictive accuracy with AUC of 0.89; and (4) combining the two RNAs with clinical characteristics provided excellent predictive accuracy (AUC 0.96). Our findings suggest an underlying common pathophysiological relationship between PTL/PPROM ≤ 32 weeks and EOP and open inroads for the prognostication of high-risk pregnancies.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA–treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.

W266 Detection of hepatitis G virus (GBV-C) RNA in blood plasma in patients with lymphoproliferative diseases

W266 Detection of hepatitis G virus (GBV-C) RNA in blood plasma in patients with lymphoproliferative diseases

Plasma cell-free RNA profiling distinguishes cancers from pre-malignant conditions in solid and hematologic malignancies

Cell-free RNA (cfRNA) in plasma reflects phenotypic alterations of both localized sites of cancer and the systemic host response. Here we report that cfRNA sequencing enables the discovery of messenger RNA (mRNA) biomarkers in plasma with the tissue of origin-specific to cancer types and precancerous conditions in both solid and hematologic malignancies. To explore the diagnostic potential of total cfRNA from blood, we sequenced plasma samples of eight hepatocellular carcinoma (HCC) and ten multiple myeloma (MM) patients, 12 patients of their respective precancerous conditions, and 20 non-cancer (NC) donors. We identified distinct gene sets and built classification models using Random Forest and linear discriminant analysis algorithms that could distinguish cancer patients from premalignant conditions and NC individuals with high accuracy. Plasma cfRNA biomarkers of HCC are liver-specific genes and biomarkers of MM are highly expressed in the bone marrow compared to other tissues and are related to cell cycle processes. The cfRNA level of these biomarkers displayed a gradual transition from noncancerous states through precancerous conditions and cancer. Sequencing data were cross-validated by quantitative reverse transcription PCR and cfRNA biomarkers were validated in an independent sample set (20 HCC, 9 MM, and 10 NC) with AUC greater than 0.86. cfRNA results observed in precancerous conditions require further validation. This work demonstrates a proof of principle for using mRNA transcripts in plasma with a small panel of genes to distinguish between cancers, noncancerous states, and precancerous conditions.

Coordination of inflammatory responses in children with perinatally acquired HIV infection.

We investigated dynamics of inflammatory biomarkers in children with perinatally acquired HIV (PHIV) who started antiretrovirals at age less than 3 years and achieved sustained virologic control (HIV plasma RNA <400 copies/ml). This was a retrospective analysis of inflammatory biomarkers in children enrolled in a randomized trial of early (<3 years of age) PI-based versus NNRTI-based regimens (P1060), who achieved sustained virologic control and participated in a neurodevelopmental follow-up study (P1104s) between ages 5 and 11 years. We measured 20 inflammatory biomarkers using ELISA or chemiluminescence at onset of sustained virologic control (Tc) and at P1104s entry (Te). The 213 participants had median ages of 1.2, 1.9, and 7 years at antiretroviral initiation, Tc, and Te, respectively, with 138 on protease inhibitor-based and 74 on NNRTI-based regimens at Tc. Eighteen markers decreased and two increased from Tc to Te (Te-Tc). Biomarker subsets, particularly cytokines, the chemokine IP-10, and adhesion molecules sICAM-1 and sVCAM-1, correlated at Tc, Te, and Te-Tc. At Tc, higher biomarker levels were associated with younger age, female sex, HIV plasma RNA at least 750 000 copies/ml, lower nadir CD4 + %, lower nadir weight z scores, and NNRTI-based treatment. Greater Te-Tc biomarker declines were associated with younger age, male sex, higher Tc biomarker levels, lower nadir CD4 + %, and NNRTI-based treatment. Duration of controlled viremia and nadir height z scores showed mixed associations. Biomarker expression showed substantial coordination. Most markers decreased after virologic control. Demographic and clinical variables associated with biomarker patterns were identified. Mechanistic studies of these biomarker patterns are needed to inform interventions to control inflammation.

Analysis of Macular Microvasculature in Human Immunodeficiency Virus Infection

ABSTRACT Purpose To evaluate the retinal and choroidal microvascular blood flow in patients with human immunodeficiency virus (HIV) infection using optical coherence tomography angiography (OCTA). Methods Macular OCTA parameters including vessel density (VD) of parafoveal superficial capillary plexus (SCP-VD) and deep capillary plexus (DCP-VD), foveal vessel density (FD), foveal avascular zone area, and flow area of choriocapillaris were analyzed in 42 HIV-positive patients, and compared with 42 healthy controls. Results The mean SCP-VD, DCP-VD and FD were significantly lower in HIV-positive group compared with controls (p < .001, p = .014, p = .026; respectively). Reduced SCP-VD was associated with higher HIV RNA plasma level (r = −0.400, p = .021) and lower CD4 + T cell count (r = 0.314, p = .046) in HIV-positive patients. Conclusions Macular microvascular blood flow is affected by HIV infection. OCTA can detect microvascular flow abnormalities in retinal capillary plexus in HIV-positive patients.

Transcript levels in plasma contribute substantial predictive value as potential Alzheimer's disease biomarkers in African Americans.

SummaryBackgroundAfrican Americans (AA) remain underrepresented in Alzheimer's disease (AD) research, despite the prevalence of AD being double in AA compared to non-Hispanic whites. To address this disparity, our group has established the Florida Consortium for African American Alzheimer's Disease Studies (FCA3DS), focusing on the identification of genetic risk factors and novel plasma biomarkers.MethodUtilizing FCA3DS whole exome sequence (WES) and plasma RNA samples from AD cases (n=151) and cognitively unimpaired (CU) elderly controls (n=269), we have performed differential gene expression (DGE) and expression quantitative trait locus (eQTL) analyses on 50 transcripts measured with a custom nanoString® panel. We designed this panel to measure, in plasma, cell-free mRNA (cf-mRNA) levels of AD-relevant genes.FindingsAssociation with higher plasma CLU in CU vs. AD remained significant after Bonferroni correction. Study-wide significant eQTL associations were observed with 105 WES variants in cis with 22 genes, including variants in genes previously associated with AD risk in AA such as ABCA7 and AKAP9. Results from this plasma eQTL analysis identified AD-risk variants in ABCA7 and AKAP9 that are significantly associated with lower and higher plasma mRNA levels of these genes, respectively. Receiver operating characteristic analysis of age, sex APOE-ε4 dosage, CLU, APP, CD14, ABCA7, AKAP9 and APOE mRNA levels, and ABCA7 and AKAP9 eQTLs, achieved 77% area under the curve to discriminate AD vs. CU, an 8% improvement over a model that only included age, sex and APOE-ε4 dosage.InterpretationIncorporating plasma mRNA levels could contribute to improved predictive value of AD biomarker panels.FundingThis work was supported by the National Institute on Aging [RF AG051504, U01 AG046139, R01 AG061796 to NET; P30 AG062677 to JAL and NGR]; Florida Health Ed and Ethel Moore Alzheimer's Disease grants [5AZ03 and 7AZ17 to NET; 7AZ07 to MMC; 8AZ08 to JAL].

Achieving virological control in pan-resistant HIV-1 infection: A case series.

SummaryBackgroundHIV-1 pan-resistance refers to a reduced susceptibility to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and integrase strand tranfer inhibitors. Although still anecdotal, its management remains a concern both for affected people living with HIV (PLWH) and for public health.MethodsWe described genotypic resistance testing (GRT) of three PLWH with a documented poor virological response to previous antiretroviral therapies, who started ibalizumab, an anti-CD4 monoclonal antibody, combined with an optimized background therapy. Both historical and most recent GRT on plasma RNA and peripheral blood mononuclear cell DNA were interpreted according to the Stanford HIVDb version 9.0 (last updated on 22 February, 2021). After the switch to a regimen including the monoclonal antibody, HIV-1 RNA has been quantified biweekly (PCR Cobas® HIV-1 test 6800 Systems, Roche Diagnostics). Follow-up was censored at data freezing (16 January, 2021).FindingsWe report findings from heavily treatment-experienced PLWH with a pan-resistant HIV-1 infection, who achieved virological control once introduced injections of ibalizumab, that is free from cross-resistance with all the antiretroviral drugs available and ensures patient adherence due to a close monitoring attributable to the route of administration, combined with recycled enfuvirtide and an optimized background regimen, selected on the basis of an accurate evaluation of resistance mutations.InterpretationIn these cases, this new approach has revealed to be a turning point in achieving virological control.FundingNone, this research was supported by internal funding.

Construction of a circRNA-miRNA-mRNA network based on differentially co-expressed circular RNA in gastric cancer tissue and plasma by bioinformatics analysis

BackgroundIncreasing evidence implicates circular RNAs (circRNAs) have been involved in human cancer progression. However, the mechanism remains unclear. In this study, we identified novel circRNAs related to gastric cancer and constructed a circRNA-miRNA-mRNA network.MethodsMicroarray datasets GSE83521 and GSE93541 were obtained from the Gene Expression Omnibus (GEO). Then, we used computational biology to identify circRNAs that were differentially expressed in both GC tissue and plasma compared to normal controls; then, we detected the expression of the selected circRNAs in gastric cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). We also identified circRNA-related candidate miRNAs and their target genes with online tools. Combining the predicted miRNAs and target mRNAs, a competing endogenous RNA regulatory network was established. Functional and pathway enrichment analyses were performed, and interactions between proteins were predicted by using String and Cytoscape. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to elucidate the possible functions of these differentially expressed circRNAs. The regulatory network constructed using the microarray datasets (GSE83521 and GSE93541) contained three differentially co-expressed circRNAs (DECs). A circRNA-miRNA-mRNA network was constructed based on 3 circRNAs, 43 miRNAs and 119 mRNAs.ResultsGO and KEGG analysis showed that the regulation of apoptotic signaling pathway and PI3K−Akt signaling pathway were highest degrees of enrichment respectively. We established a protein-protein interaction (PPI) network consisting of 165 nodes and 170 edges and identified hub genes by using MCODE plugin in Cytoscape. Furthermore, a core circRNA-miRNA-mRNA network was constructed based on hub genes. Hsa_circ_0001013 was finally determined to play an important role in the pathogenesis of GC according to the core circRNA-miRNA-mRNA network.ConclusionsWe propose a new circRNA-miRNA-mRNA network that is associated with the pathogenesis of GC. The network may become a new molecular biomarker and could be used to develop potential therapeutic strategies for gastric cancer.

Neurocognitive impact of Zika virus infection in adult rhesus macaques

BackgroundZika virus (ZIKV) is a mosquito-transmitted flavivirus that affects many regions of the world. Infection, in utero, causes microcephaly and later developmental and neurologic impairments. The impact of ZIKV infection on neurocognition in adults has not been well described. The objective of the study was to assess the neurocognitive impact of ZIKV infection in adult rhesus macaques.MethodsNeurocognitive assessments were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) via a touch screen and modified Brinkman Board before and after subcutaneous ZIKV inoculation. Immune activation markers were measured in the blood and cerebral spinal fluid (CSF) by multiplex assay and flow cytometry.ResultsAll animals (N = 8) had detectable ZIKV RNA in plasma at day 1 post-inoculation (PI) that peaked at day 2 PI (median 5.9, IQR 5.6–6.2 log10 genome equivalents/mL). In all eight animals, ZIKV RNA became undetectable in plasma by day 14 PI, but persisted in lymphoid tissues. ZIKV RNA was not detected in the CSF supernatant at days 4, 8, 14 and 28 PI but was detected in the brain of 2 animals at days 8 and 28 PI. Elevations in markers of immune activation in the blood and CSF were accompanied by a reduction in accuracy and reaction speed on the CANTAB in the majority of animals.ConclusionsThe co-occurrence of systemic and CSF immune perturbations and neurocognitive impairment establishes this model as useful for studying the impact of neuroinflammation on neurobehavior in rhesus macaques, as it pertains to ZIKV infection and potentially other pathogens.

Cell-free plasma RNA signatures as a surrogate biomarker of pregnancy health

Molecular diagnostics based on sequencing of plasma cell-free DNA has transformed obstetric care by accurately diagnosing fetal aneuploidy in early pregnancy. Rasmussen etal. now report plasmacell-free RNA profiling that could transform obstetrics by providing comprehensive molecular monitoring of pregnancy progression forboth healthy and complicated pregnancies such as preeclampsia.1.

Whole Blood Holding Time Prior to Plasma Processing Alters microRNA Expression Profile.

MicroRNAs (miRNAs) can exhibit aberrant expression under different physiological and pathological conditions. Therefore, differentially expressed circulating miRNAs have been a focus of biomarker discovery research. However, the use of circulating miRNAs comes with challenges which may hinder the reliability for their clinical application. These include varied sample collection protocols, storage times/conditions, sample processing and analysis methods. This study focused on examining the effect of whole blood holding time on the stability of plasma miRNA expression profiles. Whole blood samples were collected from healthy pregnant women and were held at 4°C for 30 min, 2 h, 6 h or 24 h prior to processing for plasma isolation. Plasma RNA was extracted and the expression of 179 miRNAs were analyzed. Unsupervised principal component analysis demonstrated that whole blood holding time was a major source of variation in miRNA expression profiles with 53 of 179 miRNAs showing significant changes in expression. Levels of specific miRNAs previously reported to be associated with pregnancy-associated complications such as hsa-miR-150-5p, hsa-miR-191-5p, and hsa-miR-29a-3p, as well as commonly used endogenous miRNA controls, hsa-miR-16-5p, hsa-miR-25-3p, and hsa-miR-223-3p were significantly altered with increase in blood holding time. Current protocols for plasma-based miRNA profiling for diagnostics describe major differences in whole blood holding periods ranging from immediately after collection to 26 h after. Our results demonstrate holding time can have dramatic effects on analytical reliability and reproducibility. This highlights the importance of standardization of blood holding time prior to processing for plasma in order to minimize introduction of non-biological variance in miRNA profiles.

Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed. We developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. The extracted RNA is then submitted to RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV virologic failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). In contrived samples, the in-house RNA extraction workflow achieved a detection limit of 200-copies per mL for HIV RNA in plasma and 100-copies per mL for SARS-CoV-2 RNA in NS. Similar detection limits were observed for HIV and SARS-CoV-2 in pooled plasma/NS contrived samples. When comparing in-house with standard extraction methods, we found high agreement (>0.91) between input and measured RNA copies for HIV LTR in contrived plasma; SARS-CoV-2 N1/N2 in contrived NS; and LTR, N1, and N2 in pooled plasma/NS samples. We further evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-positive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 combined plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house RNA extraction to those using a commercial kit (standard extraction method). The in-house extraction and standard extraction of clinical specimens were positively correlated: plasma HIV VL (R2 of 0.81) and NS SARS-CoV-2 VL (R2 of 0.95 and 0.99 for N1 and N2 genes, respectively); and pooled plasma/NS HIV VL (R2 of 0.71) and SARS-CoV-2 VL (R2 of 1 both for N1 and N2 genes). Our low-cost molecular test workflow ($1.85 per pooled sample extraction) for HIV RNA and SARS-CoV-2 RNA could serve as an alternative to current standard assays ($12 per pooled sample extraction) for laboratories in low-resource settings.

Quantitative Real-Time RT-PCR Systems to Detect SARS-CoV-2.

Since the outbreak of coronavirus disease 2019 (COVID-19) on the Diamond Princess cruise ship docked at Yokohama Port on February 3, 2020, real-time reverse transcription-polymerase chain reaction (RT-PCR) testing using nasopharyngeal swab samples from symptomatic and asymptomatic COVID-19 individuals has been the main way to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in almost all clinical laboratories in Japan. With the diffusion of sets containing the primers and probe, the gold standard real-time RT-PCR test has permeated throughout the country. To prevent the spread of infection, real-time RT-PCR testing is important to confirm whether people are positive, asymptomatic, or negative for COVID-19. Now, in addition to pharyngeal swab, saliva and blood samples can be used to detect SARS-CoV-2 RNA. Here, we introduce a clinical laboratory test performed using the High Pure viral nucleic acid kit and subsequent real-time RT-PCR system to detect SARS-CoV-2 RNA in serum, plasma, or whole blood in a hospital in Yokohama, Japan.