New and old lessons from a devastating case of neonatal E coli meningitis.

BACKGROUND: Neonatal Escherichia coli (E coli) meningitis results in significant morbidity and mortality. We present a case of a premature infant with extensive central nervous system (CNS) injury from recurrent E coli infection and the non-traditional methods necessary to identify and clear the infection. CASE PRESENTATION: The infant was transferred to our institution's pediatric intensive care unit (PICU) after recurrence of E coli CNS infection requiring neurosurgical intervention. He had been treated for early onset sepsis (EOS) with ampicillin and gentamicin for 10 days followed by rapid development of ampicillin-resistant E coli septic shock and meningitis after discontinuation of antibiotics. Sterility of the CNS was not confirmed at the end of 21 days of cefepime therapy and was subsequently followed by recurrent ampicillin-resistant E coli septic shock and CNS infection. Despite 6 weeks of appropriate therapy with sterility of CSF by traditional methods, he suffered from intractable seizures with worsening hydrocephalus. Transferred to our institution, he underwent endoscopic 3rd ventriculostomy with cyst fenestration revealing purulent fluid and significant pleocytosis. An additional 3 weeks of systemic and intraventricular antibiotics with cefepime and tobramycin were given but a significant CNS neutrophil-predominant pleocytosis persisted (average of ∼ 21,000 cells/mm3). Repeated gram stains, cultures, polymerase chain reaction (PCR) testing, and metagenomic next generation sequencing (NGS) testing of CSF were negative for pathogens but acridine orange stain (AO) revealed numerous intact rod-shaped bacteria. After the addition of ciprofloxacin, sterility and resolution of CSF pleocytosis was finally achieved. CONCLUSION: Neonatal E coli meningitis is a well-known entity but unlike other bacterial infections, it has not proven amenable to shorter, more narrow-spectrum antibiotic courses or limiting invasive procedures such as lumbar punctures. Further, microbiologic techniques to determine CSF sterility suffer from poorly understood limitations leading to premature discontinuation of antibiotics risking further neurologic damage in vulnerable hosts.

Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures.

Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.

SARS-CoV-2 Serologic Assays in Control and Unknown Populations Demonstrate the Necessity of Virus Neutralization Testing.

BACKGROUND: To determine how serologic antibody testing outcome links with virus neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we evaluated individuals for SARS-CoV-2 antibody level and viral neutralization. METHODS: We compared serum Ig levels across platforms of viral antigens and antibodies with 15 positive and 30 negative SARS-CoV-2 controls followed by viral neutralization assessment. We then applied these platforms to a clinically relevant cohort of 114 individuals with unknown histories of SARS-CoV-2 infection. RESULTS: In controls, the best-performing virus-specific antibody detection platforms were SARS-CoV-2 receptor binding domain (RBD) IgG (sensitivity 87%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 94%), spike IgG3 (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%), and nucleocapsid protein (NP) IgG (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. Twenty individuals with unknown history had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. Spike IgG3 provided the highest accuracy for predicting serologically positive individuals with virus neutralization activity (misidentified 1/20 unknowns compared to 2/20 for RBD and NP IgG). CONCLUSIONS: The coupling of virus neutralization analysis to a spike IgG3 antibody test is optimal to categorize patients for correlates of SARS-CoV-2 immune protection status.

Atypical Severe Shock-like Reactions in Adolescents After Trimethoprim-Sulfamethoxazole Therapy.

Severe drug hypersensitivity reactions to antibiotics are rare but trimethoprim-sulfamethoxazole (TMP-SMX) is uniquely associated with numerous and varied manifestations including a reaction resembling septic shock, first observed in human immunodeficiency virus (HIV)/AIDS patients. Over the past 25 years about 20 cases have been reported and an association with the virus and related immune system dysregulation was assumed. However, recent reports in adults have recognized similar shock-like reactions in non-HIV infected individuals. Here we review severe TMP-SMX hypersensitivity reactions and within the context of these known reactions, describe three non-HIV infected adolescent patients with shock-like reactions to TMP-SMX observed in one institution over 1.5 years.

Congenital Parvovirus B19 Infection: Persistent Viremia and Red Blood Cell Aplasia.

We describe a case of fetal parvovirus B19 infection resulting in preterm birth and leading to hydrops fetalis requiring multiple in utero transfusions. The infant developed chronic postnatal anemia responsive to intravenous immunoglobulin therapy. Serum viral load decreased after immunoglobulin treatment but remained detectable for over 1 year.

Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C.

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.

Analysis of the complete genome sequences of human rhinovirus.

Human rhinovirus (HRV) infection is the cause of about one half of asthma and chronic obstructive pulmonary disease exacerbations. With more than 100 serotypes in the HRV reference set, an effort was undertaken to sequence their complete genomes so as to understand the diversity, structural variation, and evolution of the virus. Analysis revealed conserved motifs, hypervariable regions, a potential fourth HRV species, within-serotype variation in field isolates, a nonscanning internal ribosome entry site, and evidence for HRV recombination. Techniques have now been developed using next-generation sequencing to generate complete genomes from patient isolates with high throughput, deep coverage, and low costs. Thus relationships can now be sought between obstructive lung phenotypes and variation in HRV genomes in infected patients and potential novel therapeutic strategies developed based on HRV sequence.

Full-genome sequence and analysis of a novel human rhinovirus strain within a divergent HRV-A clade.

Genome sequences of human rhinoviruses (HRV) have primarily been from stocks collected in the 1960s, with genomes and phylogeny of modern HRVs remaining undefined. Here, two modern isolates (hrv-A101 and hrv-A101-v1) collected approximately 8 years apart were sequenced in their entirety. Incorporation into our full-genome HRV alignment with subsequent phylogenetic network inference indicated that these represent a unique HRV-A, localized within a distinct divergent clade. They appear to have resulted from recombination of the hrv-65 and hrv-78 lineages. These results support our contention that there are unrecognized distinct HRV-A strains, and that recombination is evident in currently circulating strains.

Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution.

Infection by human rhinovirus (HRV) is a major cause of upper and lower respiratory tract disease worldwide and displays considerable phenotypic variation. We examined diversity by completing the genome sequences for all known serotypes (n = 99). Superimposition of capsid crystal structure and optimal-energy RNA configurations established alignments and phylogeny. These revealed conserved motifs; clade-specific diversity, including a potential newly identified species (HRV-D); mutations in field isolates; and recombination. In analogy with poliovirus, a hypervariable 5' untranslated region tract may affect virulence. A configuration consistent with nonscanning internal ribosome entry was found in all HRVs and may account for rapid translation. The data density from complete sequences of the reference HRVs provided high resolution for this degree of modeling and serves as a platform for full genome-based epidemiologic studies and antiviral or vaccine development.

Gene-based SNP mapping of a psychotic bipolar affective disorder linkage region on 22q12.3: association with HMG2L1 and TOM1.

Genetic linkage studies in both bipolar affective disorder (BPAD) and schizophrenia have implicated overlapping regions of chromosome 22q. We previously reported that BPAD pedigrees containing multiple members with psychotic symptoms showed suggestive linkage to chromosome 22q12.3. Now we have tested 189 single nucleotide polymorphisms (SNPs) spanning a 3 Mb region around the linkage peak for association with BPAD in 305 families, unrelated cases, and controls. SNPs were selected in or near genes, resulting in coverage at a density of 1 SNP per 6.7 kb across the 22 annotated genes in the region. The strongest signal emerged from family-based association analysis of an 11-SNP, 54 kb haplotype straddling the gene HMG2L1 and part of TOM1. A 3-marker haplotype of SNPs within TOM1 was associated with BPAD (allele-wise P = 0.0011) and with psychotic BPAD (allele-wise P = 0.00049). As hypothesized, the mean odds ratio for the risk alleles across the region was 1.39 in the psychotic but only 0.96 in the non-psychotic subset. Genotype-wise analyses yielded similar results, but the psychotic/non-psychotic distinction was more pronounced with mean odds ratios of 1.91 versus 0.8. Permutation of genotype-wise results for rs2413338 in HMG2L1 showed an empirical P = 0.037 for the difference between subsets. HMG2L1 is a negative regulator of Wnt signaling, a pathway of interest in psychotic BPAD as it is activated by both mood stabilizer and anti-psychotic medications. Further work is needed to confirm these results and uncover the functional variation underlying the association signal.

Inactivation of BRCA1 and BRCA2 in ovarian cancer.

BACKGROUND: Although BRCA1 and BRCA2 play important roles in hereditary ovarian cancers, the extent of their role in sporadic ovarian cancers and their mechanisms of inactivation are not yet well understood. Our goal was to characterize BRCA2 mutations and mRNA expression in a group of ovarian tumors previously evaluated for BRCA1 mutations and mRNA expression. METHODS: The tumors of 92 unrelated women with "ovarian" cancer (i.e., ovarian, peritoneal, or fallopian tube cancer) were screened for BRCA2 null mutations using a protein truncation test. Methylation-specific polymerase chain reaction (PCR) was used to examine the BRCA2 promoter for hypermethylation in tumors that did not express BRCA2 mRNA. All statistical tests were two-sided. RESULTS: Nine tumors had a germline (n = 5) or somatic (n = 4) BRCA2 mutation; each was associated with loss of heterozygosity. All of the somatic (1445delC, E880X, 4286del8, and 5783delT) and one of the germline (5984ins4) mutations were unique to this study. One tumor had somatic mutations in both BRCA1 and BRCA2. Two tumors are, to our knowledge, the first cases of germline BRCA2-associated peritoneal cancer. Twelve additional tumors lacked detectable BRCA2 mRNA, but the BRCA2 promoter was hypermethylated in only one of them, suggesting that other mechanisms effect transcriptional silencing of BRCA2. Tumors lacking BRCA1 mRNA were more likely to lack BRCA2 mRNA than tumors expressing BRCA1 mRNA (P<.001). Overall, 82% (95% confidence interval [CI] = 74% to 90%) of the tumors contained alterations in BRCA1, BRCA2, or both genes. Of 41 informative tumors with some alteration in BRCA2, 36 also had an alteration in BRCA1. The frequency, but not the mechanism, of BRCA1 or BRCA2 dysfunction in ovarian cancer was independent of family history. CONCLUSIONS: Multiple mechanisms cause nearly universal dysfunction of BRCA1 and/or BRCA2 in hereditary and sporadic ovarian carcinoma. Ovarian cancers with BRCA2 dysfunction often have simultaneous BRCA1 dysfunction.

Frequency of BRCA1 dysfunction in ovarian cancer.

BACKGROUND: Ovarian cancer is one of the most common hereditary cancers in women. Mutations in the BRCA1 gene increase a woman's risk of ovarian cancer. Testing for BRCA1 mutations is cumbersome and impractical for large populations. Therefore, we developed an efficient strategy to detect various types of BRCA1 dysfunction and also determined the relative frequency of BRCA1 dysfunction in ovarian cancer. METHODS: Tumors from 221 patients with epithelial ovarian cancer were screened for loss of heterozygosity (LOH) at the BRCA1 locus. BRCA1 complementary DNA (cDNA) and genomic DNA from all cancers with BRCA1 LOH (106 tumors) or noninformative status (15 tumors) were polymerase chain reaction (PCR) amplified and analyzed for protein truncation in a coupled transcription/translation test. When truncated BRCA1 protein was detected, the BRCA1 gene from both the tumor and a paired blood sample was sequenced. When BRCA1 expression in tumor cDNA was not detected with a protein truncation test, a methylation-specific PCR was used to determine whether the promoter region of BRCA1 was methylated and thus inactivated. All statistical tests were two-sided. RESULTS: Fifty-one (23.1%) of 221 tumors had BRCA1 dysfunction, including 18 with germline mutations, 15 with somatic mutations, and 18 with monoallelic or biallelic hypermethylated promoters. By the consideration of only tumors with LOH or that were noninformative, the efficiency for detecting BRCA1 dysfunction improved to 45 (37.2%) of 121 tumors. Therefore, LOH/noninformative was a strong predictor of mutation status (Fisher's exact test, P<.001). However, this subset of tumors did not include those with BRCA1 missense mutations (estimated at six [2.7%] of 221 not detected by our method) or biallelic promoter methylation (estimated at six [2.7%] of 221). CONCLUSIONS: BRCA1 dysfunction in ovarian cancer is common and occurs via multiple mechanisms. The use of LOH, rather than a family history of ovarian cancer, as a first step in a screening strategy, followed by protein truncation testing, appears to increase the chance of identifying tumors with BRCA1 dysfunction.