Deep sequencing prompts the modification of a real-time RT-PCR for the serotype-specific detection of polioviruses.

Polioviruses are members of the Enterovirus C species and asymptomatic fecal shedding allows for their transmission and persistence in a community, as well as the emergence of vaccine-derived polioviruses. Using three serotype-specific real-time RT-PCR (rRT-PCR) assays, the shedding and circulation of oral poliovirus vaccine (OPV) strains was previously investigated in a prospective cohort of Mexican children, their contacts, and nearby sewage. Subsequently, a deep sequencing approach targeting the P1 genomic region was applied to characterize OPV strains previously detected by rRT-PCR. Amplifiable RNA was obtained for sequencing from 40.3% (58/144) of stool samples and 71.4% (15/21) of sewage using nucleic acids extracted directly from primary rRT-PCR-positive specimens. Sequencing detected one or more OPV serotypes in 62.1% (36/58) of stool and 53.3% (8/15) of sewage samples. All stool and sewage samples in which poliovirus was not detected by deep sequencing contained at least one non-polio enterovirus C (NPEV-C) strain. To improve screening specificity, a modified, two-step, OPV serotype-specific multiplex rRT-PCR was evaluated. In stool specimens, the overall agreement between the original assays and the multiplex was 70.3%. By serotype, the overall agreement was 95.7% for OPV serotype-1 (S1), 65.6% for S2, and 96.1% for S3. Furthermore, most original rRT-PCR positive/multiplex rRT-PCR negative results were collected in the summer and fall months, consistent with NPEV-C circulation patterns. In conclusion, this deep sequencing approach allowed for the characterization of OPV sequences directly from clinical samples and facilitated the implementation of a more specific multiplex rRT-PCR for OPV detection and serotyping.

Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection.

The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/µL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

Real-time RT-PCR for Mayaro virus detection in plasma and urine.

BACKGROUND: Mayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3-5days of illness, but only a single rRT-PCR has been fully evaluated in the literature. OBJECTIVES: To develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains. STUDY DESIGN: A MAYV rRT-PCR targeting a region of the 5'UTR and nsp1 gene was designed from the alignment of all complete-genome MAYV sequences to be compatible with existing laboratory protocols. The assay was evaluated using human samples spiked with six MAYV strains, including strains from each of the three genotypes. RESULTS: The linear range of the MAYV rRT-PCR extended from 1.0 to 8.0 log10copies/µL, and the lower limit of 95% detection was 8.2copies/µL. No detection was observed when the MAYV rRT-PCR was tested with genomic RNA from related arboviruses. The assay demonstrated linear amplification of all 6 MAYV strains when spiked into human plasma samples as well as 2 strains spiked into urine. CONCLUSIONS: We report the design and evaluation of an rRT-PCR for MAYV. Given the concern for MAYV emergence in the Americas and the few molecular tests that have been evaluated in the literature, this assay should provide a useful diagnostic for patients with an acute febrile illness.

Characterization of Dengue Virus Infections Among Febrile Children Clinically Diagnosed With a Non-Dengue Illness, Managua, Nicaragua.

Background: We sought to characterize dengue virus (DENV) infections among febrile children enrolled in a pediatric cohort study who were clinically diagnosed with a non-dengue illness ("C cases"). Methods: DENV infections were detected and viral load quantitated by real-time reverse transcription-polymerase chain reaction in C cases presenting between January 2007 and January 2013. Results: One hundred forty-one of 2892 C cases (4.88%) tested positive for DENV. Of all febrile cases in the study, DENV-positive C cases accounted for an estimated 52.0% of patients with DENV viremia at presentation. Compared with previously detected, symptomatic dengue cases, DENV-positive C cases were significantly less likely to develop long-lasting humoral immune responses to DENV, as measured in healthy annual serum samples (79.7% vs 47.8%; P < .001). Humoral immunity was associated with viral load at presentation: 40 of 43 patients (93.0%) with a viral load ≥7.0 log10 copies/mL serum developed the expected rise in anti-DENV antibodies in annual samples versus 13 of 68 (19.1%) patients with a viral load below this level (P < .001). Conclusions: Antibody responses to DENV-positive C cases differ from responses to classic symptomatic dengue. These findings have important implications for DENV transmission modeling, immunology, and epidemiologic surveillance.

Malaria and Chikungunya Detected Using Molecular Diagnostics Among Febrile Kenyan Children.

BACKGROUND: In sub-Saharan Africa, malaria is frequently overdiagnosed as the cause of an undifferentiated febrile illness, whereas arboviral illnesses are presumed to be underdiagnosed. METHODS: Sera from 385 febrile Kenyan children, who presented to 1 of 4 clinical sites, were tested using microscopy and real-time molecular assays for dengue virus (DENV), chikungunya virus (CHIKV), malaria, and Leptospira. RESULTS: Malaria was the primary clinical diagnosis for 254 patients, and an arboviral infection (DENV or CHIKV) was the primary diagnosis for 93 patients. In total, 158 patients (41.0%) had malaria and 32 patients (8.3%) had CHIKV infections. Compared with real-time polymerase chain reaction, microscopy demonstrated a percent positive agreement of 49.7%. The percentage of malaria cases detected by microscopy varied significantly between clinical sites. Arboviral infections were the clinical diagnosis for patients on the Indian Ocean coast (91 of 238, 38.2%) significantly more often than patients in the Lake Victoria region (2 of 145, 1.4%; P < .001). However, detection of CHIKV infections was significantly higher in the Lake Victoria region (19 of 145 [13.1%] vs 13 of 239 [5.4%]; P = .012). CONCLUSIONS: The clinical diagnosis of patients with an acute febrile illness, even when aided by microscopy, remains inaccurate in malaria-endemic areas, contributing to inappropriate management decisions.

Development of a Real-Time Reverse Transcription Polymerase Chain Reaction for O'nyong-nyong Virus and Evaluation with Clinical and Mosquito Specimens from Kenya.

O'nyong-nyong virus (ONNV), an alphavirus closely related to chikungunya virus (CHIKV), has been the documented cause of two large outbreaks in east Africa; however, little is known about the contribution of ONNV to cases of acute febrile illness during interepidemic periods. An ONNV real-time reverse transcription polymerase chain reaction (rRT-PCR) was developed and evaluated using clinical and mosquito pool samples. The ONNV rRT-PCR linear range extended from 8.0 to 2.0 log10 copies/µL, and the lower limit of 95% detection was 22.4 copies/µL. No cases of ONNV infection were identified in serum from 385 Kenyan children who presented with an acute febrile illness. Additionally, ONNV was not detected in 120 mosquito pools collected in coastal and western Kenya. The ONNV rRT-PCR demonstrated good analytical sensitivity when performed in monoplex or as a component of an ONNV-CHIKV duplex assay. This assay should provide a useful diagnostic for the detection of ONNV in surveillance studies.

Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication.

Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection.

BACKGROUND: Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection. OBJECTIVES: In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR). STUDY DESIGN: From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua. RESULTS: The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/µL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/µL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples). CONCLUSIONS: The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.

Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients.

Plasmodium nucleic acids have been detected in serum and plasma, but there is little published data describing the diagnostic performance of malaria nucleic acid amplification tests (NAATs) using these specimen types. Previously, our group described a multiplex NAAT for the detection of dengue virus, Leptospira, and Plasmodium species with a callout for P. falciparum (the DLM assay) that demonstrated sensitive detection of P. falciparum from plasma samples during initial evaluation. In this study, we evaluated the sensitivity and specificity of P. falciparum detection in febrile Nigerian patients using the DLM assay, microscopy, and a rapid diagnostic test (BinaxNOW Malaria). Assay performances were compared using a composite reference, which was considered positive if malaria was detected by two or more methods. Serum (n = 182) or plasma (n = 148) from 317 patients was tested; the average sample volume was 70 µl (range, 5 to 300 µl). The sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivity of the malaria rapid diagnostic test (98.1%) was similar to that of the DLM assay, and both proved significantly more sensitive than microscopy (79%; P < 0.0001). When analysis was limited to samples with ≥75 µl of serum or plasma, the sensitivity of the DLM assay improved to 99% and specificity was 97.5%. For P. falciparum cases, cycle threshold values in the DLM assay correlated with the parasite density detected by microscopy (Spearman's rank correlation coefficient, P < 0.0001). In conclusion, malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with P. falciparum malaria.

Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing.

BACKGROUND: Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens. METHODS/PRINCIPAL FINDINGS: 478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%). CONCLUSIONS/SIGNIFICANCE: This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.

Improved serotype-specific dengue virus detection in Trinidad and Tobago using a multiplex, real-time RT-PCR.

Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01).

Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

BACKGROUND: Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. METHODOLOGY/PRINCIPAL FINDINGS: For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. CONCLUSIONS/SIGNIFICANCE: The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

The impact of the metabotropic glutamate receptor and other gene family interaction networks on autism.

Although multiple reports show that defective genetic networks underlie the aetiology of autism, few have translated into pharmacotherapeutic opportunities. Since drugs compete with endogenous small molecules for protein binding, many successful drugs target large gene families with multiple drug binding sites. Here we search for defective gene family interaction networks (GFINs) in 6,742 patients with the ASDs relative to 12,544 neurologically normal controls, to find potentially druggable genetic targets. We find significant enrichment of structural defects (P ≤ 2.40E-09, 1.8-fold enrichment) in the metabotropic glutamate receptor (GRM) GFIN, previously observed to impact attention deficit hyperactivity disorder (ADHD) and schizophrenia. Also, the MXD-MYC-MAX network of genes, previously implicated in cancer, is significantly enriched (P ≤ 3.83E-23, 2.5-fold enrichment), as is the calmodulin 1 (CALM1) gene interaction network (P ≤ 4.16E-04, 14.4-fold enrichment), which regulates voltage-independent calcium-activated action potentials at the neuronal synapse. We find that multiple defective gene family interactions underlie autism, presenting new translational opportunities to explore for therapeutic interventions.

Gadd45 stress sensors in malignancy and leukemia.

Gadd45 proteins, including Gadd45a, Gadd45b, and Gadd45g, have been implicated in stress signaling in response to physiological and environmental stress, including oncogenic stress, which can result in cell cycle arrest, DNA repair, cell survival, senescence, and apoptosis. The function of Gadd45 as a stress sensor is mediated via a complex interplay of physical interactions with other cellular proteins implicated in cell cycle regulation and the response of cells to stress, notably PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. Altered expression of Gadd45 has been observed in multiple types of solid tumors as well as in hematopoietic malignancies. Using genetically engineered mouse models and bone-marrow transplantation, evidence has been obtained indicating that Gadd45 proteins can function to either promote or suppress tumor development and leukemia; this is dependent on the molecular nature of the activated oncogene and the cell type, via engagement of different signaling pathways.

Should tumor VEGF expression influence decisions on combining low-dose chemotherapy with antiangiogenic therapy? Preclinical modeling in ovarian cancer.

Because of its low toxicity, low-dose (LD) chemotherapy is ideally suited for combination with antiangiogenic drugs. We investigated the impact of tumor vascular endothelial growth factor A (VEGF-A) expression on the efficacy of LD paclitaxel chemotherapy and its interactions with the tyrosine kinase inhibitor SU5416 in the ID8 and ID8-Vegf models of ovarian cancer. Functional linear models using weighted penalized least squares were utilized to identify interactions between Vegf, LD paclitaxel and antiangiogenic therapy. LD paclitaxel yielded additive effects with antiangiogenic therapy against tumors with low Vegf expression, while it exhibited antagonism to antiangiogenic therapy in tumors with high Vegf expression. This is the first preclinical study that models interactions of LD paclitaxel chemotherapy with antiangiogenic therapy and tumor VEGF expression and offers important lessons for the rational design of clinical trials.

HSV oncolytic therapy upregulates interferon-inducible chemokines and recruits immune effector cells in ovarian cancer.

Cooperation between oncolytic herpes simplex virus (HSV) and host effector immune mechanisms has been previously described. In the present study, we investigated the mechanism underlying such cooperation in a murine syngeneic model of ovarian carcinoma. Therapeutic administration of HSV-1716, a replication-restricted mutant, resulted in significant reduction of tumor growth and a significant survival advantage. Intratumoral injection of HSV-1716 induced expression of IFN-gamma, MIG, and IP-10 in the tumor. This was accompanied by a significant increase in the number of tumor-associated NK and CD8+ T cells expressing CXCR3 and CD25. Ascites from HSV-1716-treated animals efficiently induced in vitro migration of NK and CD8+ T cells, which was dependent on the presence of MIG and IP-10. Murine monocytes and dendritic cells (DCs) were responsible for the production of MIG and IP-10 upon HSV-1716 infection. In monocytes, this was partially abrogated by neutralizing antibodies against IFN-alpha and -beta, thus indicating a role for type-1 IFNs in the reported effect. Human ovarian carcinomas showed high numbers of monocytes and DCs. Upon HSV-1716 infection, human monocyte-derived DCs produced large amounts of IFN-gamma and upregulated MIG and IP-10 expression. These results indicate that HSV-1716 induces an inflammatory response that may facilitate antitumor immune response upon oncolytic therapy.

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A.

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of beta-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse beta-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and beta-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow-derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that beta-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: beta-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.

Letal, A tumor-associated NKG2D immunoreceptor ligand, induces activation and expansion of effector immune cells.

NKG2D serves as one of the most potent activating receptors for effector lymphocytes. in peripheral tissues. Here we report the characterization of Letal, the first human trans-membrane NKG2D ligand lacking an immunoglobulin-like alpha-3 ectodomain. Letal is constitutively expressed by a variety of normal tissues, and is upregulated in tumor cells of different origins. Unlike other NKG2D ligands, Letal mRNA expression progressively decreased after treatment of tumor cells with retinoic acid. Simultaneous T-cell receptor activation and engagement of Letal stimulated proliferation of CD8(+) cells and dramatically increased IL-2 and IFNgamma secretion. In addition, Letal induced the killing of cancer cells by CD8(+) and NK cells. These results suggest that Letal delivers activating signals to NK cells and promotes tumor immune surveillance by inducing the expansion of anti-tumor cytotoxic lymphocytes.

Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer.

Vascular endothelial growth factor (VEGF) carries out multifaceted functions in tumor development, and it exists as at least five isoforms with distinct biologic activities and clinical implications. Several strategies have been developed to block VEGF for cancer therapy; however, the approach to target-specific VEGF isoform(s) has not been explored to date. In the present study, we show that DNA vector-based RNA interference (RNAi), in which RNAi sequences targeting murine VEGF isoforms are inserted downstream of an RNA polymerase III promoter, has potential applications in isoform-specific "knock-down" of VEGF. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF isoform-specific treatment in cancer.