Search

PDF file - 109K

PDF file - 428K

Antecedentes: Hay varias razones por las que las pruebas de PCR nasofaríngeas para la enfermedad por coronavirus 2019 (COVID-19) dan un resultado positivo tardío, no sólo en casos asintomáticos sino también en pacientes gravemente enfermos. Los atributos fisiopatológicos aún se desconocen. Exploramos esta posibilidad mediante el reporte de un caso. Presentación del caso: Un varón de 64 años con antecedentes de infección fúngica pulmonar, asma y enfermedad pulmonar obstructiva crónica (EPOC), diabetes y arteriopatía coronaria se presentó con dificultad respiratoria, fiebre e imágenes torácicas de opacidad esmerilada y engrosamiento intersticial reticular, altamente sospechoso de COVID-19. Sin embargo, las pruebas de hisopado nasofaríngeo fueron discordantemente negativas durante cuatro ocasiones en dos semanas, y los anticuerpos IgG para COVID-19 también fueron negativos. Sin embargo, el nivel de IgE en suero era elevado. No se identificaron otros patógenos. Sus síntomas se deterioraron a pesar del tratamiento con corticosteroides, antibióticos y broncodilatadores. Se realizaron un lavado broncoalveolar (LBA) y una biopsia en cuña a pulmón abierto para el diagnóstico de la etiología. Se demostró la presencia de neumonía organizativa fibrosante positiva para RNA viral COVID-19, y daños en las vías respiratorias caracterizados por un efecto citopático sospechoso de origen viral, inflamación mixta neutrofílica, linfoplasmática, histiocítica y eosinofílica, y fibrosis, además de los cambios esperados en el asma y la EPOC. Una semana después, las pruebas nasofaríngeas de COVID-19 repetidas en los días 40 y 49 resultaron positivas. Conclusión: Nuestro caso y la revisión de la literatura indican que el asma alérgica y el alto nivel de IgE asociado a ella, junto con la inhalación de corticosteroides, podrían contribuir a la positividad retardada del hisopado nasofaríngeo en las vías respiratorias superiores; la obstrucción crónica de las vías respiratorias relacionada con la EPOC y la adición de fibrosis provocaron dependencia del ventilador y un mal pronóstico en la neumonía por COVID-19, y deberían ser objeto de tratamiento junto con la terapia antiviral.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use., This study describes a new method that improves the sensitivity of viral detection compared with next-generation sequencing and enables the detection of emerging flaviviruses not specifically targeted a priori. Metagenomic sequencing with spiked primer enrichment is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making it applicable for diagnostic laboratory and field use.

Methionine (MET) is a general metabolic therapeutic target in cancer, whereby cancer cells have an elevated requirement for MET, termed MET dependence. We have developed recombinant L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase, EC 4.4.1.11]) as targeted therapy of all cancer types. Pharmacokinetics, MET depletion, antigenicity, and toxicity of rMETase were examined in macaque monkeys. Pharmacokinetic analysis showed that rMETase was eliminated with a T

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 μM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 μM, respectively, from the PLP baseline of 0.3 μM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.

Methionine (MET) is a general metabolic therapeutic target in cancer, whereby cancer cells have an elevated requirement for MET, termed MET dependence. We have developed recombinant L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase, EC 4.4.1.11]) as targeted therapy of all cancer types. Pharmacokinetics, MET depletion, antigenicity, and toxicity of rMETase were examined in macaque monkeys. Pharmacokinetic analysis showed that rMETase was eliminated with a T1/2 of 2.49 h. A 2-week i.v. administration of 4000 units/kg every 8 h/day for 2 weeks resulted in a steady-state depletion of plasma MET to less than 2 μM. The only manifest toxicity was decreased food intake and slight weight loss. Serum albumin and red-cell values declined transiently during treatment. Rechallenge on day 28 resulted in anaphylactic shock and death in one animal. Pretreatment with hydrocortisone prevented the anaphylactic reaction. Anti-rMETase antibodies (at 10-3) were found after the first challenge, increased to 10-6 after the fourth challenge, and decreased to 10-2 by 2 months post-therapy. Therefore, the therapeutic potential of rMETase is limited by its short plasma half-life and immunologic effects, including high antibody production in mice and anaphylactic reactions in monkeys. To overcome these limits, rMETase has been coupled to methoxypolyethylene glycol succinimidyl glutarate polyethylene glycol (MEGC-PEG-5000). The pharmacokinetics, antigenicity, and toxicity of MEGC-PEG-rMETase in macaque monkeys were evaluated using an escalating-dose strategy. In pharmacokinetic studies, a single 4000 units/kg dose showed that MEGC-PEG-rMETase holoenzyme activity was eliminated with a biological half-life of 1.3 h, and the MEGC-PEG-rMETase apoenzyme was eliminated with a biological half-life of 90 h, a 36-fold increase compared with non-PEGylated rMETase. The disparity in the T½ of the apoenzyme and the holoenzyme reflects the loss of co-factor pyridoxal-L-phosphate of the circulating MEGC-PEG-rMETase. A 7-day i.v. administration of 4000 units/kg every 12 h resulted in a steady-state depletion of plasma MET to

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 μM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 μM, respectively, from the PLP baseline of 0.3 μM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.

We report here two genome sequences of a newly designated rhinovirus genotype, RV-C56, which were obtained from respiratory specimens of three patients with acute respiratory illness in 2016 and 2017. To our knowledge, these sequences represent the first near-complete genomes for RV-C56 strains.

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16–18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.

Here, we report the full coding sequence of rhinovirus C47 (RV-C47), obtained from a patient respiratory sample collected during an acute respiratory illness investigation in Butte County, California, in January 2017. This is the first whole-genome sequence of RV-C47 to be reported.

A monkey model of Zika virus (ZIKV) infection is urgently needed to better understand transmission and pathogenesis, given its proven association with fetal brain defects in pregnant women and acute neurological illness. Here we experimentally infected 4 male marmosets with ZIKV (prototype 1947 African strain) and monitored them clinically with sampling of various body fluids and tissues for nearly 3 months. We show that the course of acute infection with ZIKV in these New World monkeys resembles the human illness in many respects, including (1) lack of apparent clinical symptoms in most cases, (2) persistence of the virus in body fluids such as semen and saliva for longer periods of time than in serum, and (3) generation of neutralizing antibodies as well as an antiviral immunological host response. Importantly, ZIKV-infected saliva samples (in addition to serum) were found to be infectious, suggesting potential capacity for viral transmission by the oral route. Re-challenge of a previously infected marmoset with a contemporary outbreak strain SPH2015 from Brazil resulted in continued protection against infection, no viral shedding, and boosting of the immune response. Given the key similarities to human infection, a marmoset model of ZIKV infection may be useful for testing of new drugs and vaccines.

In 2014, the United States experienced an epidemic of acute flaccid myelitis (AFM) cases in children coincident with a nationwide outbreak of enterovirus D68 (EV-D68) respiratory disease. Up to half of the 2014 AFM patients had EV-D68 RNA detected by RT-PCR in their respiratory secretions, although EV-D68 was only detected in cerebrospinal fluid (CSF) from one 2014 AFM patient. Given previously described molecular and epidemiologic associations between EV-D68 and AFM, we sought to develop an animal model by screening seven EV-D68 strains for the ability to induce neurological disease in neonatal mice. We found that four EV-D68 strains from the 2014 outbreak (out of five tested) produced a paralytic disease in mice resembling human AFM. The remaining 2014 strain, as well as 1962 prototype EV-D68 strains Fermon and Rhyne, did not produce, or rarely produced, paralysis in mice. In-depth examination of the paralysis caused by a representative 2014 strain, MO/14-18947, revealed infectious virus, virion particles, and viral genome in the spinal cords of paralyzed mice. Paralysis was elicited in mice following intramuscular, intracerebral, intraperitoneal, and intranasal infection, in descending frequency, and was associated with infection and loss of motor neurons in the anterior horns of spinal cord segments corresponding to paralyzed limbs. Virus isolated from spinal cords of infected mice transmitted disease when injected into naïve mice, fulfilling Koch’s postulates in this model. Finally, we found that EV-D68 immune sera, but not normal mouse sera, protected mice from development of paralysis and death when administered prior to viral challenge. These studies establish an experimental model to study EV-D68-induced myelitis and to better understand disease pathogenesis and develop potential therapies., Author summary Reports of polio-like paralysis, referred to as acute flaccid myelitis (AFM), have recently emerged in association with infections caused by enterovirus D68 (EV-D68). In the second half of 2014, 120 cases of AFM, mostly in young children, were reported during a nationwide outbreak of EV-D68 respiratory disease. The number of AFM cases has risen again in 2016. Although epidemiological evidence between EV-D68 infection and AFM is accumulating, a causal link has not been definitely established. Here we demonstrate that strains of EV-D68 recovered during the 2014 epidemic can cause a paralytic illness in mice that resembles human AFM. Evidence that EV-D68 causes paralysis in this mouse model include: (1) loss of spinal cord motor neurons innervating paralyzed limbs, (2) detection of virus in the spinal cord and, specifically, motor neurons, (3) transmission of neurological disease when injecting virus isolated from spinal cords of paralyzed mice into naïve mice, thus fulfilling Koch’s postulates, and (4) the ability to prevent AFM by pre-administering serum containing EV-D68 antibodies from previously infected mice. This experimental mouse model can be used to better understand the pathogenesis of EV-D68-induced CNS disease and to facilitate the development of potential therapies.

Human infections by Zika virus (ZIKV), a mosquito-borne flavivirus, are associated with a current widespread outbreak in the Americas, and have been associated with neurological complications and adverse fetal outcomes such as microcephaly in pregnant women. A suitable non-human primate model is urgently needed. To evaluate ZIKV infectivity, pathogenesis, and persistence, we inoculated 4 marmosets with ZIKV and followed them by clinical monitoring and serial sampling of body fluids for up to 11 weeks. We found that marmosets experimentally infected with ZIKV reproduced key features of the human disease, including (1) asymptomatic infection, (2) brief period of detectable virus in serum (

Primary amoebic meningoencephalitis (PAM) is a rare, often lethal, cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes.In this study, we sequenced a full draft assembly of the Balamuthia mandrillaris genome (44.2 Mb in size) from a rare survivor of PAM, and recovered the mitochondrial genome from six additional Balamuthia strains. We also used unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case of Balamuthia mandrillaris encephalitis in a 15-year-old girl.Comparative analysis of the mitochondrial genome and high-copy number genes from six additional Balamuthia mandrillaris strains demonstrated remarkable sequence variation, and the closest Balamuthia homologs corresponded to other amoebae, hydroids, algae, slime molds, and peat moss. Real-time NGS testing of hospital day 6 CSF and brain biopsy samples detected Balamuthia on the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. The presumptive diagnosis of PAM by visualization of amoebae on brain biopsy histopathology and NGS analysis was subsequently confirmed at the US Centers for Disease Control and Prevention (CDC) using a Balamuthia-specific PCR assay. Retrospective analysis of a day 1 CSF sample revealed that more timely identification of Balamuthia by metagenomic NGS, potentially resulting in a better clinical outcome, would have required availability of the complete genome sequence.These results underscore the diverse evolutionary origins of Balamuthia mandrillaris, provide new targets for diagnostic assay development, and will facilitate further investigations of the biology and pathogenesis of this eukaryotic pathogen. The failure to identify PAM from a day 1 sample without a fully sequenced Balamuthia genome in the database highlights the critical importance of whole-genome reference sequences for microbial detection by metagenomic NGS.

Importance There has been limited surveillance for acute flaccid paralysis in North America since the regional eradication of poliovirus. In 2012, the California Department of Public Health received several reports of acute flaccid paralysis cases of unknown etiology. Objective To quantify disease incidence and identify potential etiologies of acute flaccid paralysis cases with evidence of spinal motor neuron injury. Design, Setting, and Participants Case series of acute flaccid paralysis in patients with radiological or neurophysiological findings suggestive of spinal motor neuron involvement reported to the California Department of Public Health with symptom onset between June 2012 and July 2015. Patients meeting diagnostic criteria for other acute flaccid paralysis etiologies were excluded. Cerebrospinal fluid, serum samples, nasopharyngeal swab specimens, and stool specimens were submitted to the state laboratory for infectious agent testing. Main Outcomes and Measures Case incidence and infectious agent association. Results Fifty-nine cases were identified. Median age was 9 years (interquartile range [IQR], 4-14 years; 50 of the cases were younger than 21 years). Symptoms that preceded or were concurrent included respiratory or gastrointestinal illness (n = 54), fever (n = 47), and limb myalgia (n = 41). Fifty-six patients had T2 hyperintensity of spinal gray matter on magnetic resonance imaging and 43 patients had cerebrospinal fluid pleocytosis. During the course of the initial hospitalization, 42 patients received intravenous steroids; 43, intravenous immunoglobulin; and 13, plasma exchange; or a combination of these treatments. Among 45 patients with follow-up data, 38 had persistent weakness at a median follow-up of 9 months (IQR, 3-12 months). Two patients, both immunocompromised adults, died within 60 days of symptom onset. Enteroviruses were the most frequently detected pathogen in either nasopharynx swab specimens, stool specimens, serum samples (15 of 45 patients tested). No pathogens were isolated from the cerebrospinal fluid. The incidence of reported cases was significantly higher during a national enterovirus D68 outbreak occurring from August 2014 through January 2015 (0.16 cases per 100 000 person-years) compared with other monitoring periods (0.028 cases per 100 000 person-years;P Conclusions and Relevance In this series of patients identified in California from June 2012 through July 2015, clinical manifestations indicated a rare but distinct syndrome of acute flaccid paralysis with evidence of spinal motor neuron involvement. The etiology remains undetermined, most patients were children and young adults, and motor weakness was prolonged.

Background Primary amoebic meningoencephalitis (PAM) is a rare, often lethal, cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes. Methods In this study, we sequenced a full draft assembly of the Balamuthia mandrillaris genome (44.2 Mb in size) from a rare survivor of PAM, and recovered the mitochondrial genome from six additional Balamuthia strains. We also used unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case of Balamuthia mandrillaris encephalitis in a 15-year-old girl. Results and Discussion Comparative analysis of the mitochondrial genome and high-copy number genes from six additional Balamuthia mandrillaris strains demonstrated remarkable sequence variation, and the closest Balamuthia homologs corresponded to other amoebae, hydroids, algae, slime molds, and peat moss. Real-time NGS testing of hospital day 6 CSF and brain biopsy samples detected Balamuthia on the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. The presumptive diagnosis of PAM by visualization of amoebae on brain biopsy histopathology and NGS analysis was subsequently confirmed at the US Centers for Disease Control and Prevention (CDC) using a Balamuthia-specific PCR assay. Retrospective analysis of a day 1 CSF sample revealed that more timely identification of Balamuthia by metagenomic NGS, potentially resulting in a better clinical outcome, would have required availability of the complete genome sequence. Conclusions These results underscore the diverse evolutionary origins of Balamuthia mandrillaris, provide new targets for diagnostic assay development, and will facilitate further investigations of the biology and pathogenesis of this eukaryotic pathogen. The failure to identify PAM from a day 1 sample without a fully sequenced Balamuthia genome in the database highlights the critical importance of whole-genome reference sequences for microbial detection by metagenomic NGS. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0235-2) contains supplementary material, which is available to authorized users.

Tumor stroma drives the growth and progression of cancers. A heparin-binding epidermal growth factor–like growth factor, HB-EGF, is an EGF receptor ligand that stimulates cell growth in an autocrine or paracrine fashion. While elevated expression of HB-EGF in cancer cells and its contribution to tumor progression are well documented, the effects of HB-EGF expression in the tumor stroma have not been clarified. Here, we show that HB-EGF is expressed in stromal fibroblasts where it promotes cancer cell proliferation. In uterine cervical cancers, HB-EGF was detected immunohistochemically in the stroma proximal to the cancer epithelium. Proliferation of cervical cancer cells in vitro was enhanced by coculture with fibroblasts isolated from tumor tissues of patients with cervical cancer. Inhibition of HB-EGF function or treatment with platelet–derived growth factor (PDGF) inhibitors abrogated cancer cell growth enhanced by cervical cancer–associated fibroblast (CCF) coculture. Furthermore, tumor formation in a mouse xenograft model was enhanced by cotransplantation of CCF or mouse embryonic fibroblasts, but not with embryonic fibroblasts from HB-EGF–deficient mice. Conversely, conditioned medium from cancer cells induced HB-EGF expression in CCF. Mechanistic investigations established that PDGF was the primary factor responsible. Together, our findings indicate that HB-EGF and PDGF reciprocally mediate the interaction of cancer cells with cancer-associated fibroblasts, promoting cancer cell proliferation in a paracrine manner that has implications for novel combinatorial cancer therapies. Cancer Res; 71(21); 6633–42. ©2011 AACR.

The development of a quantitative real-time PCR (qPCR) assay for human rhinovirus serotype 16 (HRV16) is described using the plasmid pR16.11, which contains the full-length genome of HRV16. A standard curve was generated by plotting the critical threshold (Ct) against numbers of plasmid. The limit of sensitivity was less than10 cDNA copies, and the curve showed a high degree of linearity over a range of 101 to 106 cDNA copies with r2 ≥ 0.9989. Amplification efficiency of the qPCR was greater than 97.6 percent. The standard curve was highly reproducible with low intra- and inter-assay coefficients of variation. Standard curves were also generated from cDNA derived from two viral suspensions of known TCID50, and were exactly parallel to those generated from the plasmid. Comparison of the curves generated from the plasmid or viral cDNA showed that for the two suspensions, TCID50 corresponded to either 142 or 2088 viral particles. This new qPCR will permit quantitative assessments of interactions between virus and epithelium such as determinations of the affinity and number of viral binding sites or of the number of virus produced per infected cell.

Cardioviruses comprise a genus of picornaviruses that cause severe illnesses in rodents, but little is known about the prevalence, diversity, or spectrum of disease of such agents among humans. A single cardiovirus isolate, Saffold virus, was cultured in 1981 in stool from an infant with fever. Here, we describe the identification of a group of human cardioviruses that have been cloned directly from patient specimens, the first of which was detected using a pan-viral microarray in respiratory secretions from a child with influenza-like illness. Phylogenetic analysis of the nearly complete viral genome (7961 bp) revealed that this virus belongs to the Theiler's murine encephalomyelitis virus (TMEV) subgroup of cardioviruses and is most closely related to Saffold virus. Subsequent screening by RT-PCR of 719 additional respiratory specimens [637 (89%) from patients with acute respiratory illness] and 400 cerebrospinal fluid specimens from patients with neurological disease (aseptic meningitis, encephalitis, and multiple sclerosis) revealed no evidence of cardiovirus infection. However, screening of 751 stool specimens from 498 individuals in a gastroenteritis cohort resulted in the detection of 6 additional cardioviruses (1.2%). Although all 8 human cardioviruses (including Saffold virus) clustered together by phylogenetic analysis, significant sequence diversity was observed in the VP1 gene (66.9%–100% pairwise amino acid identities). These findings suggest that there exists a diverse group of novel human Theiler's murine encephalomyelitis virus-like cardioviruses that hitherto have gone largely undetected, are found primarily in the gastrointestinal tract, can be shed asymptomatically, and have potential links to enteric and extraintestinal disease.

This Case Study describes a 70-year-old immunocompetent male with rapidly-progressing dementia. Creutzfeldt–Jakob disease was suspected, but a diagnosis of enterovirus encephalitis was confirmed by brain biopsy. The authors discuss the diagnostic challenges presented by this case, and briefly review enteroviral infections of the brain. Background A 70-year-old immunocompetent male presented to a memory disorders clinic with a 7-month illness that had begun with somatic complaints including transient right temporal head pain, left buttock pain, and right conjunctival injection. About 3 months after the first signs of illness, the patient had begun to develop insidious cognitive and behavioral decline, which progressed most rapidly in the 2 months before presentation. An assessment completed during hospitalization for intermittent fevers and confusion had not revealed an infectious etiology, although mild pleocytosis in the cerebrospinal fluid had been noted. Upon presentation to the memory disorders clinic, the patient was disoriented, distractible, laughed at inappropriate moments, and followed only one-step commands. He had hypophonic speech and had mildly increased axial tone. He scored 5 out of 30 on the Mini Mental State Examination and was admitted for expedited evaluation. Investigations Physical examination, brain MRI, electroencephalogram, lumbar puncture, autoimmune and paraneoplastic testing, cerebral angiogram, cerebrospinal fluid analysis, enterovirus group-specific reverse transcriptase polymerase chain reaction assay, and RNA sequencing in brain biopsy samples. Diagnosis Enteroviral meningoencephalitis. Management Intravenous steroids with oral taper and intravenous immunoglobulin.

BACKGROUND. Encephalitis is a complex, debilitating, and sometimes fatal neurologic condition to which children are especially prone. Mycoplasma pneumoniae, a common respiratory pathogen, has been implicated as an etiology of encephalitis. Evidence for recent or acute M pneumoniae infection has been demonstrated in limited studies of both pediatric and adult patients with encephalitis. PATIENTS AND METHODS. Unexplained encephalitis cases are referred to the California Encephalitis Project for diagnostic testing. Serum, cerebrospinal fluid, and respiratory specimens are tested by polymerase chain reaction and serology methods for the presence of multiple pathogens, including M pneumoniae. M pneumonia–associated cases of encephalitis were compared with other bacterial agents, herpes simplex virus 1, and enterovirus. RESULTS. Of 1988 patients referred to the California Encephalitis Project, evidence of acute M pneumoniae infection was found in 111 patients, of which 84 (76%) were pediatric patients. Eighty percent of the 84 patients were positive for M pneumoniae by serology alone. Cerebrospinal fluid polymerase chain reaction for M pneumoniae was rarely positive (2%). Patients with M pneumoniae–associated pediatric encephalitis were a median of 11 years old, progressed rapidly (median: 2 days from onset to hospitalization), and were often in the ICU (55%). Symptoms included fever (70%), lethargy (68%), and altered consciousness (58%). Gastrointestinal (45%) and respiratory (44%) symptoms were less common. Compared with patients with other bacterial as well as viral agents, patients with M pneumoniae–associated encephalitis had fewer seizures and less-severe hospital courses. CONCLUSIONS. M pneumoniae is the most common agent implicated in the California Encephalitis Project. Patients with M pneumoniae–associated encephalitis are predominantly pediatric, and their presentations are clinically similar to enterovirus encephalitis, although they frequently require intensive care with prolonged hospitalizations. Given that M pneumoniae infection is found more than any other pathogen, increased emphasis should be placed on elucidating the role and mechanism of M pneumoniae in encephalitis.

A pan-virus DNA microarray (Virochip) was used to detect a human metapneumovirus (hMPV) strain associated with a critical respiratory tract infection in an elderly adult with chronic lymphocytic leukemia. This infection had previously eluded diagnosis despite extensive microbiological testing for possible etiologic agents. The patient's hMPV strain did not grow in viral culture, and only one of five specific reverse transcription-PCR assays for hMPV was positive.

Background Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infection, however, they are also known to replicate in the lower respiratory tract and associate with more severe respiratory illnesses. An outbreak of HRV occurred in a long-term facility in Santa Cruz, California with unusually high morbidity and mortality. Objectives To identify viral characteristics associated with this unique outbreak, genetic relationships between these clinical isolates (SCRVs) and prototype strains of rhinovirus were investigated. Study design Sequence homology and phylogenetic analyses of the SCRV VP4/VP2 region were performed in conjunction with all HRV prototypes. Due to the importance of the 5′noncoding region (NCR) and the structural genes to viral replication and host immune responses, respectively, we focused on a segment of the HRV genome which includes these regions. Molecular models of SCRV were also assessed. Results SCRV showed closest similarity to HRV82 with some divergence from the prototype. Amino acid differences were concentrated within predicted neutralization epitopes within VP2, VP3 and VP1. Conclusion Sequence analyses and differences in cell culture growth characteristics suggest that this virus is a variant of HRV which has distinctive properties from its respective prototype strain.

Primary amoebic meningoencephalitis (PAM) is a rare, often lethal cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes. In this study, we present the first draft assembly of theBalamuthia mandrillarisgenome recovered from a rare survivor of PAM, in total comprising 49 Mb of sequence. Comparative analysis of the mitochondrial genome and high-copy number genes from 6 additionalBalamuthia mandrillarisstrains demonstrated remarkable sequence variation, with the closest homologs corresponding to other amoebae, hydroids, algae, slime molds, and peat moss,. We also describe the use of unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case ofBalamuthia mandrillarisencephalitis in a 15-year old girl. Real-time NGS testing of a hospital day 6 CSF sample detectedBalamuthiaon the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. Retrospective analysis of a day 1 CSF sample revealed that more timely identification ofBalamuthiaby metagenomic NGS, potentially resulting in a better outcome, would have required availability of the complete genome sequence. These results underscore the diverse evolutionary origins underpinning this eukaryotic pathogen, and the critical importance of whole-genome reference sequences for microbial detection by NGS.

Summary Background Enterovirus D68 was implicated in a widespread outbreak of severe respiratory illness across the USA in 2014 and has also been reported sporadically in patients with acute flaccid myelitis. We aimed to investigate the association between enterovirus D68 infection and acute flaccid myelitis during the 2014 enterovirus D68 respiratory outbreak in the USA. Methods Patients with acute flaccid myelitis who presented to two hospitals in Colorado and California, USA, between Nov 24, 2013, and Oct 11, 2014, were included in the study. Additional cases identified from Jan 1, 2012, to Oct 4, 2014, via statewide surveillance were provided by the California Department of Public Health. We investigated the cause of these cases by metagenomic next-generation sequencing, viral genome recovery, and enterovirus D68 phylogenetic analysis. We compared patients with acute flaccid myelitis who were positive for enterovirus D68 with those with acute flaccid myelitis but negative for enterovirus D68 using the two-tailed Fisher's exact test, two-sample unpaired t test, and Mann-Whitney U test. Findings 48 patients were included: 25 with acute flaccid myelitis, two with enterovirus-associated encephalitis, five with enterovirus-D68-associated upper respiratory illness, and 16 with aseptic meningitis or encephalitis who tested positive for enterovirus. Enterovirus D68 was detected in respiratory secretions from seven (64%) of 11 patients comprising two temporally and geographically linked acute flaccid myelitis clusters at the height of the 2014 outbreak, and from 12 (48%) of 25 patients with acute flaccid myelitis overall. Phylogenetic analysis revealed that all enterovirus D68 sequences associated with acute flaccid myelitis grouped into a clade B1 strain that emerged in 2010. Of six coding polymorphisms in the clade B1 enterovirus D68 polyprotein, five were present in neuropathogenic poliovirus or enterovirus D70, or both. One child with acute flaccid myelitis and a sibling with only upper respiratory illness were both infected by identical enterovirus D68 strains. Enterovirus D68 viraemia was identified in a child experiencing acute neurological progression of his paralytic illness. Deep metagenomic sequencing of cerebrospinal fluid from 14 patients with acute flaccid myelitis did not reveal evidence of an alternative infectious cause to enterovirus D68. Interpretation These findings strengthen the putative association between enterovirus D68 and acute flaccid myelitis and the contention that acute flaccid myelitis is a rare yet severe clinical manifestation of enterovirus D68 infection in susceptible hosts. Funding National Institutes of Health, University of California, Abbott Laboratories, and the Centers for Disease Control and Prevention.

L-Methionine gamma-lyase is a pyridoxal 5'-phosphate-dependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.

A highly potent recombinant l-methionine γ-lyase (METase) conjugated with polyethylene glycol (PEG) was characterized physicochemically and pharmacokinetically in vivo and in vitro. Pegylated METase (PEG-METase), which contains pyridoxal 5′-phosphate (PLP) as a cofactor in the molecule, is a potent anticancer agent that can deplete l-methionine from plasma. Although pegylation decreased its specific activity, dithiothreitol (DTT) treatment increased it over three times with the detachment of one PEG moiety modified with a cysteine residue. We can produce DTT-treated PEG-METase on a large scale in sufficient quality for therapeutic use. The superiority of DTT-treated PEG-METase was confirmed by the enhancement of l-methionine depletion and amelioration of pharmacokinetics in mice. The holoenzyme of DTT-treated PEG-METase gave a several times larger area under the plasma concentration curve than that of DTT-untreated PEG-METase, not because of an increase of the half-life but because of high specific activity. Conversely, simultaneous PLP infusion led to a greatly increased half-life of the holoenzyme. DTT-treated PEG-METase administration with PLP infusion was the most useful combination for maximizing the potency of the enzyme. We showed that serum albumin interfered with holoenzyme activity in vitro. The decrease of holoenzyme activity was dependent on the type of serum albumin. We concluded that PLP was released from PEG-METase by serum albumin in vivo and in vitro. The deleterious effect of PLP dissociation from PEG-METase could be improved by supplementing PLP and oleic acid. Their synergistic effect in preventing a decrease of the holoenzyme activity was also observed. (Cancer Res 2006; 66(5): 2807-14)

Rhinovirus (RV) infection is the major cause of common colds and of asthma exacerbations. Because the epithelial cell layer is the primary target of RV infection, we hypothesize that RV-induced airway disease is associated with the perturbation of airway epithelial gene expression. In this study, well differentiated primary human airway epithelial cells were infected with either RV16 (major group) or RV1B (minor group). Transcriptional gene profiles from RV-infected and mock-infected control cells were analyzed by Affymetrix Genechip, and changes of the gene expression were confirmed by real-time RT-PCR analysis. At 24 h after infection, 48 genes induced by both viruses were identified. Most of these genes are related to the IFN pathway, and have been documented to have antiviral functions. Indeed, a significant stimulation of IFN-beta secretion was detected after RV16 infection. Neutralizing antibody specific to IFN-beta and a specific inhibitor of the Janus kinase pathway both significantly blocked the induction of RV-inducible genes. Further studies demonstrated that 2-aminopurine, a specific inhibitor double-stranded RNA-dependent protein kinase, could block both IFN-beta production and RV-induced gene expression. Thus, IFN-beta-dependent pathway is a part of the double-stranded RNA-initiated pathway that is responsible for RV-induced gene expression. Consistent with its indispensable role in the induction of antiviral genes, deactivation of this signaling pathway significantly enhanced viral production. Because increase of viral yield is associated with the severity of RV-induced airway illness, the discovery of an epithelial antiviral signaling pathway in this study will contribute to our understanding of the pathogenesis of RV-induced colds and asthma exacerbations.

An outbreak of myocarditis occurred among adults in Illinois in 2003. Diagnostic testing of myocardial tissues from 3 patients and comprehensive tests for enterovirus and adenovirus of other specimens from patients were inconclusive. Appropriate specimen collection from patients with idiopathic cardiomyopathy and further enhancement of diagnostic techniques are needed.

During a 6-week period in 2003, 56 residents and 26 staff developed respiratory illness in a long-term facility; 12 residents died. Seven of 13 respiratory specimens were culture-positive for rhinovirus; 6 of the isolates were serotype 82. In elderly populations, severe illness may be associated with organisms typically considered to be “benign,” such as rhinovirus.

Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in both immunocompromised and immunocompetent individuals. Because of a lack of pathognomonic symptoms and the difficulty in recognizing amebas in biopsied tissues, most cases are not diagnosed or effectively treated, leading to a >95% mortality. We report here on five cases of balamuthiasis that were diagnosed by indirect immunofluorescence (IIF) staining of serum for anti- Balamuthia antibodies (titer ≥ 1:128) and confirmed by IIF of unstained brain tissue sections and/or detection of amebas in hematoxylin-eosin-stained slides. Additionally, we have used the PCR for the detection of mitochondrial 16S rRNA gene DNA from the ameba in clinical specimens such as brain tissue and cerebrospinal fluid (CSF) from individuals with Balamuthia encephalitis. Balamuthia DNA was successfully detected by the PCR in clinical samples from all five individuals. It was detected in brain tissue from three cases, in CSF from three cases, and in one of two samples of lung tissue from two individuals, but not in two samples of kidney tissue tested. One sample of unfixed brain tissue was culture positive for Balamuthia . In order to test the sensitivity of the PCR for detection of Balamuthia DNA, CSF specimens from two individuals negative for amebic infection were spiked with Balamuthia amebas. We found that it was possible to detect Balamuthia DNA in the PCR mixtures containing mitochondrial DNA from 1 to as little as 0.2 ameba per reaction mixture. A single Balamuthia ameba contains multiple mitochondrial targets; thus, 0.2 ameba represents multiple targets for amplification and is not equivalent to 0.2 of an ameba as a target.

Methionine depletion by recombinant methioninase (rMETase) has been demonstrated previously to be highly effective in tumor-bearing mouse models. However, the therapeutic potential of rMETase has been limited by its short plasma half-life and immunologic effects, including high antibody production in mice and monkeys and anaphylactic reactions in monkeys. To overcome these limits of rMETase, the enzyme has been coupled to methoxypolyethylene glycol succinimidyl glutarate (MEGC-PEG-5000). In this study, we evaluated the pharmacokinetics, antigenicity and toxicity of MEGC-PEG-rMETase in Macaca fascicularis monkeys using an escalating-dose strategy. Dose ranging studies at 1,000, 4,000, and 8,000 units/kg i.v. determined that a single dose of 4,000 units/kg was sufficient to reduce plasma methionine to

Balamuthia mandrillarisamoebas are recognized as a causative agent of granulomatous amoebic encephalitis, a disease that is usually fatal. They were first recognized when isolated from the brain of a mandrill baboon that died in the San Diego Zoo Wild Life Animal Park. Subsequently, the amoebas have been found in a variety of animals, including humans (young and old, immunocompromised and immunocompetent persons), in countries around the world. Until recently, the amoebas had not been recovered from the environment and their free-living status was in question. The recovery of aBalamuthiaamoeba from a soil sample taken from a plant at the home of a child from California, USA, who died ofBalamuthiaamoebic encephalitis, was reported previously. In a continued investigation, a second amoeba was isolated from soil that was obtained from an outdoor potted plant in a spatially unrelated location. A comparison of these two environmental amoebas that were isolated from different soils with the amoeba that was obtained from the child's clinical specimen is reported here. Included are the isolation procedure for the amoebas, their growth requirements, their immunological response to anti-Balamuthiaserum, their sensitivity to a selection of antimicrobials and sequence analysis of their 16S rRNA gene. The evidence is consistent that the amoebas isolated from both soil samples and the clinical isolate obtained from the Californian child areB. mandrillaris.

Recombinant methioninase (rMETase) has been shown to target the elevated methionine (MET) dependence of tumor cells and arrest their growth as well as make tumors more sensitive to standard chemotherapy agents. Polyethylene glycol (PEG)-modified rMETase (PEG-rMETase) has reduced antigenicity compared with unmodified rMETase. However, PEG-rMETase has a limited active circulating half-life due to rapid in vivo dissociation of its cofactor pyridoxal-5′-phosphate (PLP), a surprising finding, because PLP is tightly bound to PEG-rMETase in buffer. The question asked in the current study was on the effect of increasing doses of PLP to extend the circulating half-life of active PEG-rMETase holoenzyme in vivo. rMETase was conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/ml PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 μm, respectively, from the PLP baseline of 0.3 μm. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. Pumps containing 500 mg/ml PLP increased the half-life of MEGC-PEG-rMETase holoenzyme 4.5-fold from 1.5 to 7 h. Infused PLP did not extend the half-life of MEGC-PEG-rMETase apoenzyme. With a dose of 4000 units/kg MEGC-PEG-rMETase in the mice infused with 5, 50, 200, and 500 mg/ml PLP, plasma MET was depleted from 50 μm to ≤5 μm for 8, 24, 72, and 72 h, respectively. Thus, PLP infusion could extend the period of MET depletion by MEGC-PEG-rMETase by ∼10-fold in a dose-dependent manner. The mice given 8000 units/kg MEGC-PEG-rMETase showed a similar plasma MET depletion time course, indicating that the limiting factor for MEGC-PEG-rMETase-mediated MET depletion in vivo was PLP. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP. The infused PLP either bound to apo-MEGC-PEG-rMETase and/or inhibited dissociation of PLP from holo-PEG-rMETase, thereby maintaining the holoenzyme form of MEGC-PEG-rMETase in vivo. The combination of MEGC-PEG-rMETase treatment with PLP infusion suggests an effective clinical strategy for long-term MET depletion to arrest cancer growth.

In California, molecular testing was useful in decreasing suspicion for severe acute respiratory syndrome (SARS), by detecting common respiratory pathogens (influenza A/B, human metapneumovirus, picornavirus, Mycoplasma pneumoniae, Chlamydia spp., parainfluenza virus, respiratory syncytial virus, and adenovirus) in 23 (45%) of 51 patients with suspected SARS and 9 (47%) of 19 patients with probable SARS.