Transcription-mediated amplification blood donation screening for Babesia.

BACKGROUND: Transfusion-transmitted Babesia microti is well recognized in the Northeast and upper Midwestern United States. Blood donation screening in Babesia-endemic states has occurred under investigational protocols prior to US Food and Drug Administration-licensed test availability. Here, we provide a prospective screening summary of nucleic acid testing (NAT) as part of a multicenter Babesia pivotal trial followed by extended investigational use. METHODS: From June 2017 to February 2018, 176,928 donation samples were tested with Procleix Babesia Assay (Grifols Diagnostic Solutions), a blood screening NAT for Babesia species ribosomal RNA detection using whole blood samples. During the pivotal trial, donations were collected in 11 endemic states plus Washington, DC, and Florida (nonendemic). Whole blood lysate samples were either tested in pools of 16 or individually. Reactive samples were confirmed by Babesia microti antibody and polymerase chain reaction (PCR) testing. If unconfirmed, further testing used a second PCR assay capable of detecting multiple Babesia species. Follow-up samples were also tested. Extended investigational testing followed pivotal trial completion. RESULTS: The pivotal trial identified 61 confirmed positives (176,608 donations): 35 (57%) PCR positive, 59 (97%) antibody positive, and two (3%) NAT positive/antibody negative, for a total yield of one positive per 2895 donations, including one Florida resident; others were from seven endemic states. During extended investigational testing of 496,270 donations in endemic states through January 2019, 211 (1:2351) repeat reactive donations were identified. CONCLUSIONS: Babesia was detected in donors from multiple US states, including one previously not associated with positive blood donors. This study supports the use of the Procleix Babesia Assay using individual testing or pools of up to 16.

Prevalence of Babesia in Canadian blood donors: June-October 2018.

BACKGROUND: The erythrocytic protozoan parasite Babesia microti, the cause of human babesiosis, is transmitted not only by tick bites but also via blood transfusion. B. microti is endemic in the northeastern/upper midwestern United States, where partial screening of blood donations has been implemented. In Canada, a 2013 study of approximately 14,000 donors found no B. microti antibody-positive samples, suggesting low risk at that time. METHODS: Between June and October 2018, 50,752 Canadian donations collected from sites near the US border were tested for Babesia nucleic acid by transcription-mediated amplification (TMA). Reactive donations were tested for B. microti by IgG immunofluorescence assay and polymerase chain reaction. A subset of 14,758 TMA nonreactive samples was also screened for B. microti antibody. Donors who tested reactive/positive were deferred, asked about risk factors, and were requested to provide a follow-up sample for supplemental testing. RESULTS: One sample from Winnipeg, Manitoba, was TMA and antibody reactive. Of the 14,758 TMA-nonreactive donations tested for antibody, four reactive donations were identified from southwestern Ontario near Lake Erie. None of the interviewed donors remembered any symptoms, likely tick exposure, or relevant travel within Canada or the United States. CONCLUSIONS: This is the largest B. microti prevalence study performed in Canada. The results indicate very low prevalence, with only one TMA-confirmed-positive donation of 50,752 tested. This donor was from the only region in Canada where autochthonous infection has been reported. Seropositive donations in southwestern Ontario suggest low prevalence; travel should not be ruled out given the proximity to the US border.

Minimal infectious dose and dynamics of Babesia microti parasitemia in a murine model.

BACKGROUND: Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. STUDY DESIGN AND METHODS: Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. RESULTS: In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. CONCLUSION: The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

SKIP interacts with c-Myc and Menin to promote HIV-1 Tat transactivation.

The Ski-interacting protein SKIP/SNW1 associates with the P-TEFb/CDK9 elongation factor and coactivates inducible genes, including HIV-1. We show here that SKIP also associates with c-Myc and Menin, a subunit of the MLL1 histone methyltransferase (H3K4me3) complex and that HIV-1 Tat transactivation requires c-Myc and Menin, but not MLL1 or H3K4me3. RNAi-ChIP experiments reveal that SKIP acts downstream of Tat:P-TEFb to recruit c-Myc and its partner TRRAP, a scaffold for histone acetyltransferases, to the HIV-1 promoter. By contrast, SKIP is recruited by the RNF20 H2B ubiquitin ligase to the basal HIV-1 promoter in a step that is bypassed by Tat and downregulated by c-Myc. Of interest, we find that SKIP and P-TEFb are dispensable for UV stress-induced HIV-1 transcription, which is strongly upregulated by treating cells with the CDK9 inhibitor flavopiridol. Thus, SKIP acts with c-Myc and Menin to promote HIV-1 Tat:P-TEFb transcription at an elongation step that is bypassed under stress.

High Incidence of Chikungunya Virus and Frequency of Viremic Blood Donations during Epidemic, Puerto Rico, USA, 2014.

Chikungunya virus (CHIKV) caused large epidemics throughout the Caribbean in 2014. We conducted nucleic acid amplification testing (NAAT) for CHIKV RNA (n = 29,695) and serologic testing for IgG against CHIKV (n = 1,232) in archived blood donor samples collected during and after an epidemic in Puerto Rico in 2014. NAAT yields peaked in October with 2.1% of donations positive for CHIKV RNA. A total of 14% of NAAT-reactive donations posed a high risk for virus transmission by transfusion because of high virus RNA copy numbers (10 (4) -10 (9) RNA copies/mL) and a lack of specific IgM and IgG responses. Testing of minipools of 16 donations would not have detected 62.5% of RNA-positive donations detectable by individual donor testing, including individual donations without IgM and IgG. Serosurveys before and after the epidemic demonstrated that nearly 25% of blood donors in Puerto Rico acquired CHIKV infections and seroconverted during the epidemic.

Genomic Assays for Identification of Chikungunya Virus in Blood Donors, Puerto Rico, 2014.

A newly developed transcription-mediated amplification assay was used to detect chikungunya virus infection in 3 of 557 asymptomatic donors (0.54%) from Puerto Rico during the 2014-2015 Caribbean epidemic. Viral detection was confirmed by using PCR, microarray, and next-generation sequencing. Molecular clock analysis dated the emergence of the Puerto Rico strains to early 2013.

The multi-tasking P-TEFb complex.

P-TEFb (CycT1:Cdk9), the metazoan RNA polymerase II Ser2 C-terminal domain (CTD) kinase, regulates transcription elongation at many genes and integrates mRNA synthesis with histone modification, pre-mRNA processing, and mRNA export. Recruitment of P-TEFb to target genes requires deubiquitination of H2Bub, phosphorylation of H3S10, and the bromodomain protein, Brd4. Brd4 activates growth-related genes in the G1 phase of the cell cycle and can also tether P-TEFb to mitotic chromosomes, possibly to mark sites of active transcription throughout cell division. P-TEFb co-operates with c-Myc during transactivation and cell transformation, and also requires SKIP (c-Ski-interacting protein), an mRNA elongation and splicing factor. Some functions of the P-TEFb/Ser2P CTD are executed by the Spt6 transcription elongation factor, which binds directly to the phosphorylated CTD and recruits the Iws1 ('interacts with Spt6') protein. Iws1, in turn, interacts with the REF1/Aly nuclear export adaptor and stimulates the kinetics of mRNA export. Given the prominent role of Spt6 in regulating chromatin structure, the CTD-bound Spt6:Iws1 complex may also control histone modifications during elongation. Following transcription, P-TEFb accompanies the mature mRNA to the cytoplasm to promote translation elongation.

Clinical evaluation of the Procleix SARS-CoV-2 assay, a sensitive, high-throughput test that runs on an automated system.

Testing is crucial in controlling COVID-19. The Procleix® SARS-CoV-2 assay, a transcription-mediated amplification nucleic acid test that runs on an automated system, was evaluated using inactivated virus and clinical samples. The sensitivity of the assay was assessed using heat-inactivated SARS-CoV-2 and compared to 3 other tests. Clinical validation utilized 2 sets of samples: (1) Nasal, nasopharyngeal and oropharyngeal samples (n = 963) from asymptomatic individuals, and (2) nasopharyngeal samples from symptomatic patients: 100 positive and 100 negative by RT-PCR. The Procleix assay had greater sensitivity (3-fold to 100-fold) than the comparators and had high specificity (100%) in asymptomatic subjects. In symptomatic patients, the Procleix assay detected 100% of PCR-positives and found 24 positives in the initial PCR-negatives. Eighteen of these were confirmed positive and 6 were inconclusive. These studies showed that the Procleix SARS-CoV-2 assay was a sensitive and specific tool for detecting COVID-19.

A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter.

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.

The Babesia observational antibody (BAOBAB) study: A cross-sectional evaluation of Babesia in two communities in Kilosa district, Tanzania.

BACKGROUND: Babesia, a tick-borne genus of intraerythrocytic parasites, is understudied in humans outside of established high-endemic areas. There is a paucity of data on Babesia in Africa, despite evidence that it is regionally present. A pilot study suggested that Babesia was present in a rural district of Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was conducted July-August 2017: residents in a case hamlet that had clustering of subjects with high signal-to-cut off (S/CO) ratios for antibodies against B. microti in the pilot study, and a control hamlet that had lacked significant signal, were evaluated for B. microti. Subjects aged ≥15yrs (n = 299) underwent clinical evaluation and household inspections; 10ml whole blood was drawn for Babesia transcription mediated amplification (TMA), B. microti indirect fluorescent antibody testing (IFA) and rapid diagnostic testing (RDT) for Plasmodium spp. Subjects aged <15yrs (n = 266) underwent a RDT for Plasmodium and assessment by ELISA for B. microti antibodies. A total of 570 subjects participated (mean age 22 [<1 to 90yrs]) of whom 50.7% were female and 145 (25.5%) subjects were Plasmodium RDT positive (+). In those <15yrs, the median ELISA S/CO was 1.11 (IQR 0.80-1.48); the median S/CO in the case (n = 120) and control (n = 146) hamlets was 1.19 (IQR 0.81-1.48) and 1.06 (IQR 0.80-1.50) respectively (p = 0.4). Children ≥5yrs old were more likely to have a higher S/CO ratio than those <5yrs old (p<0.001). One hundred (38%) subjects <15yrs were Plasmodium RDT+. The median S/CO ratio (children <15yrs) did not differ by RDT status (p = 0.15). In subjects ≥15yrs, no molecular test was positive for Babesia, but four subjects (1.4%) were IFA reactive (two each at titers of 128 and 256). CONCLUSIONS/SIGNIFICANCE: The findings offer further support for Babesia in rural Tanzania. However, low prevalence of seroreactivity questions its clinical significance.

Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis.

We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or laptop (MetaPORE). At titers ranging from 10(7)-10(8) copies per milliliter, reads to EBOV from two patients with acute hemorrhagic fever and CHIKV from an asymptomatic blood donor were detected within 4 to 10 min of data acquisition, while lower titer HCV virus (1 × 10(5) copies per milliliter) was detected within 40 min. Analysis of mapped nanopore reads alone, despite an average individual error rate of 24 % (range 8-49 %), permitted identification of the correct viral strain in all four isolates, and 90 % of the genome of CHIKV was recovered with 97-99 % accuracy. Using nanopore sequencing, metagenomic detection of viral pathogens directly from clinical samples was performed within an unprecedented <6 hr sample-to-answer turnaround time, and in a timeframe amenable to actionable clinical and public health diagnostics.

Atlas(®) Listeria monocytogenes LmG2 Detection Assay Using Transcription Mediated Amplification to Detect Listeria monocytogenes in Selected Foods and Stainless Steel Surface.

The Atlas Listeria monocytogenes LmG2 Detection Assay, developed by Roka Bioscience Inc., was compared to a reference culture method for seven food types (hot dogs, cured ham, deli turkey, chicken salad, vanilla ice cream, frozen chocolate cream pie, and frozen cheese pizza) and one surface (stainless steel, grade 316). A 125 g portion of deli turkey was tested using a 1:4 food:media dilution ratio, and a 25 g portion for all other foods was tested using 1:9 food:media dilution ratio. The enrichment time and media for Roka's method was 24 to 28 h for 25 g food samples and environmental surfaces, and 44 to 48 h for 125 g at 35 ± 2°C in PALCAM broth containing 0.02 g/L nalidixic acid. Comparison of the Atlas Listeria monocytogenes LmG2 Detection Assay to the reference method required an unpaired approach. For each matrix, 20 samples inoculated at a fractional level and five samples inoculated at a high level with a different strain of Listeria monocytogenes were tested by each method. The Atlas Listeria monocytogenes LmG2 Detection Assay was compared to the Official Methods of Analysis of AOAC INTERNATIONAL 993.12 method for dairy products, the U.S. Department of Agriculture, Food Safety and Inspection Service, Microbiology Laboratory Guidebook 8.08 method for ready-to-eat meat and environmental samples, and the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 10 method for frozen foods. In the method developer studies, Roka's method, at 24 h (or 44 h for 125 g food samples), had 126 positives out of 200 total inoculated samples, compared to 102 positives for the reference methods at 48 h. In the independent laboratory studies, vanilla ice cream, deli turkey and stainless steel grade 316 were evaluated. Roka's method, at 24 h (or 44 h for 125 g food samples), had 64 positives out of 75 total inoculated samples compared to 54 positives for the reference methods at 48 h. The Atlas Listeria monocytogenes LmG2 Detection Assay detected all 50 L. monocytogenes strains that encompassed 13 serotypes across the various lineages and none of the 30 exclusive organisms, including seven other Listeria species. The product consistency and kit stability studies revealed no statistical differences between the three lots tested or to the term of the shelf life. Finally, the robustness study demonstrated no statistical differences when samples were incubated at 33 ± 2°C or 37 ± 2°C, when enrichment aliquots were 1.3 mL or 1.7 mL, or when the samples were analyzed the same day or five days later. Overall the Atlas Listeria monocytogenes LmG2 Detection Assay is statistically equivalent to or better than the reference methods and is robust to the tested variations.

Requirement for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the HIV-1 promoter.

Activation of the human immunodeficiency virus type-1 (HIV-1) promoter in infected cells requires the sequential recruitment of several cellular factors to facilitate the formation of a processive elongation complex. The nucleosomal reorganization of the HIV-1 long terminal repeat (LTR) observed upon Tat stimulation suggests that chromatin-remodeling complexes could play a role during this process. Here, we reported that Tat interacts directly with Brm, a DNA-dependent ATPase subunit of the SWI/SNF chromatin-remodeling complex, to activate the HIV-1 LTR. Inhibition of Brm via small interfering RNAs impaired Tat-mediated transactivation of an integrated HIV-1 promoter. Furthermore, Brm is recruited in vivo to the HIV-1 LTR in a Tat-dependent manner. Interestingly, we found that Tat/Brm interaction is regulated by Tat lysine 50 acetylation. These data show the requirement of Tat-mediated recruitment of SWI/SNF chromatin-remodeling complex to HIV-1 promoter in the activation of the LTR.

A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat.

HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing.

Tat acetyl-acceptor lysines are important for human immunodeficiency virus type-1 replication.

The human immunodeficiency virus type-1 trans-activator Tat is a transcription factor that activates the HIV-1 promoter through binding to the trans-activation-responsive region (TAR) localized at the 5'-end of all viral transcripts. We and others have recently shown that Tat is directly acetylated at lysine 28, within the activation domain, and lysine 50, in the TAR RNA binding domain, by Tat-associated histone acetyltransferases p300, p300/CBP-associating factor, and hGCN5. Here, we show that mutation of acetyl-acceptor lysines to arginine or glutamine affects virus replication. Interestingly, mutation of lysine 28 and lysine 50 differentially affected Tat trans-activation of integrated versus nonintegrated long terminal repeat. Our results highlight the importance of lysine 28 and lysine 50 of Tat in virus replication and Tat-mediated trans-activation.

Differential acetylation of Tat coordinates its interaction with the co-activators cyclin T1 and PCAF.

The HIV-1 transactivator protein, Tat, is an atypical transcriptional activator that functions through binding, not to DNA, but to a short leader RNA, TAR. Although details of its functional mechanism are still unknown, emerging findings suggest that Tat serves primarily to adapt co-activator complexes such as p300, PCAF and P-TEFb to the HIV-1 long terminal repeat. Hence, an understanding of how Tat interacts with these cofactors is crucial. It has recently been shown that acetylation at a single lysine, residue 50, regulated the association of Tat with PCAF. Here, we report that in the absence of Tat acetylation, PCAF binds to amino acids 20-40 within Tat. Interestingly, acetylation of Tat at Lys28 abrogates Tat-PCAF interaction. Acetylation at Lys50 creates a new site for binding to PCAF and dictates the formation of a ternary complex of Tat-PCAF-P-TEFb. Thus, differential lysine acetylation of Tat coordinates the interactions with its co-activators, cyclin T1 and PCAF. Our results may help in understanding the ordered recruitment of Tat co-activators to the HIV-1 promoter.

Post-activation turn-off of NF-kappa B-dependent transcription is regulated by acetylation of p65.

NF-kappaB represents a family of eukaryotic transcription factors participating in the regulation of various cellular genes involved in the immediate early processes of immune, acute-phase, and inflammatory responses. Cellular localization and consequently the transcriptional activity of NF-kappaB is tightly regulated by its partner IkappaBalpha. Here, we show that the p65 subunit of NF-kappaB is acetylated by both p300 and PCAF on lysines 122 and 123. Both HDAC2 and HDAC3 interact with p65, although only HDAC3 was able to deacetylate p65. Acetylation of p65 reduces its ability to bind kappaBeta-DNA. Finally, acetylation of p65 facilitated its removal from DNA and consequently its IkappaBetaalpha-mediated export from the nucleus. We propose that acetylation of p65 plays a key role in IkappaBetaalpha-mediated attenuation of NF-kappaBeta transcriptional activity which is an important process that restores the latent state in post-induced cells.