Self-Collected Anterior Nasal and Saliva Specimens versus Health Care Worker-Collected Nasopharyngeal Swabs for the Molecular Detection of SARS-CoV-2.

We prospectively compared health care worker-collected nasopharyngeal swabs (NPS) to self-collected anterior nasal swabs (ANS) and straight saliva for the diagnosis of coronavirus disease 2019 (COVID-19) in 354 patients. The percent positive agreement between NPS and ANS or saliva was 86.3% (95% confidence interval [CI], 76.7 to 92.9%) and 93.8% (95% CI, 86.0 to 97.9%), respectively. The percent negative agreement was 99.6% (95% CI, 98.0 to 100.0%) for NPS versus ANS and 97.8% (95% CI, 95.3 to 99.2%) for NPS versus saliva. More cases were detected by the use of NPS (n = 80) and saliva (n = 81) than by the use of ANS (n = 70), but no single specimen type detected all severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections.

Sequencing of the Hepatitis D Virus RNA WHO International Standard.

BACKGROUND: Well-characterized, stable calibration materials are essential to standardize quantitative viral reporting. The preferred calibration materials are the WHO International Standards and secondary standards derived from them. In 2013, the 1st WHO International Standard for Hepatitis D Virus (HDV) RNA became available. During the course of assay development in our laboratory, differences between the published sequence (GenBank ID: HQ005371) and sequence we generated from the WHO HDV Standard were identified. OBJECTIVES: We sought to sequence the entire genome of the WHO HDV Standard and compare the results to the published sequence. STUDY DESIGN: RNA extracted from the WHO HDV Standard was used to generate five overlapping PCR products, including one covering the entire HDV genome, which were Sanger sequenced using standard dye-terminator chemistry. Total RNA from the WHO HDV Standard was also converted to a cDNA library generating 2.1 million sequencing reads on a NextSeq500 instrument. RESULTS: Sanger sequencing produced 32 overlapping, partial sequences of the HDV genome. RNA-seq resulted in 8100 HDV sequences covering the viral genome an average of 645-fold. Sanger and RNA-seq consensus sequences had 100% agreement and showed 89.0% nucleotide identity with the published WHO HDV Standard sequence. BLAST analysis revealed HQ005369 as the closest match with 99.2% nucleotide identity. CONCLUSIONS: HQ005369 was deposited in GenBank along with HQ005371 and seven others from a study of nine Turkish patients. A sample mix-up or clerical error may have resulted in the incorrect association of identifier and sequence. The correct nucleic acid sequence for standards is critical for test accuracy, optimization, calibration, and troubleshooting.

Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards.

It has been hoped that the recent availability of WHO quantitative standards would improve interlaboratory agreement for viral load testing; however, insufficient data are available to evaluate whether this has been the case. Results from 554 laboratories participating in proficiency testing surveys for quantitative PCR assays of cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), adenovirus (ADV), and human herpesvirus 6 (HHV6) were evaluated to determine overall result variability and then were stratified by assay manufacturer. The impact of calibration to international units/ml (CMV and EBV) on variability was also determined. Viral loads showed a high degree of interlaboratory variability for all tested viruses, with interquartile ranges as high as 1.46 log10 copies/ml and the overall range for a given sample up to 5.66 log10 copies/ml. Some improvement in result variability was seen when international units were adopted. This was particularly the case for EBV viral load results. Variability in viral load results remains a challenge across all viruses tested here; introduction of international quantitative standards may help reduce variability and does so more or less markedly for certain viruses.

Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries.

Current methods for identification of Mycobacterium spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify Mycobacterium isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of Mycobacterium spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.

Evaluation of the Prodesse Hexaplex multiplex PCR assay for direct detection of seven respiratory viruses in clinical specimens.

We evaluated the Hexaplex assay (Prodesse, Waukesha, WI) for the detection of 7 respiratory viruses (influenza A and B, parainfluenza 1-3, and respiratory syncytial virus [RSV] A and B). The Hexaplex assay was performed on 300 respiratory samples during the 1999-2000 respiratory virus season. Results of this assay were compared with shell vial cell culture and/or direct fluorescent antibody stain. The overall sensitivity and specificity of the assay were 96.6% and 94.1%, respectively. The respective sensitivity and specificity of the Hexaplex assay for detection of specific virus groups were as follows: influenza A, 98.6% and 97.8%; influenza B, 100% and 100%; and for parainfluenza viruses (1-3), 100% and 99.1%. The assay did not perform as well with patients infected with RSV: sensitivity and specificity were 91.0% and 98.6%, respectively. There are 2 major drawbacks to this assay: it is technically demanding (3-4 hours hands-on time), and it is expensive ($80-$90 direct cost). Nevertheless, because of the excellent sensitivity and specificity, the Hexaplex assay may be valuable in the diagnosis of respiratory viral infections in immunocompromised patients.

Human immunodeficiency virus type 1 drug resistance testing: a comparison of three sequence-based methods.

The use of genotypic assays for determining drug resistance in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients is increasing. These tests lack standardization and validation. The aim of this study was to evaluate several tests used for the determination of HIV-1 drug resistance. Two genotypic tests, the Visible Genetics TruGene HIV-1 Genotyping Kit and the Applied Biosystems HIV Genotyping System, were compared using 22 clinical samples. Genotyping results were also obtained from an independent reference laboratory. The Visible Genetics and Applied Biosystems genotyping tests identified similar mutations when differences in the drug databases and reference strains were taken into account, and 19 of 21 samples were equivalent. The concordance between the two assays was 99% (249 of 252 mutation sites). Mutations identified by the reference laboratory varied the most among those identified by the three genotypic tests, possibly because of differences in the databases. The concordance of the reference laboratory results with the results of the other two assays was 80% (201 of 252). Samples with 500 to 750 HIV RNA copies/ml could be sequenced by the Visible Genetics and Applied Biosystems assays using 1 ml of input. The Visible Genetics and Applied Biosystems assays both generated an accurate sequence. However, the throughput of the Visible Genetics assay is more limited and may require additional instruments. The two assays differ technically but are similar in overall complexity. Data analysis in the two assays is straightforward, but only the reports provided by Visible Genetics contain information relating mutations to drug resistance. HIV drug resistance genotyping by sequencing is a complex technology which presents a challenge for analysis, interpretation, and reporting.

Performance characteristics of the COBAS AMPLICOR hepatitis C virus MONITOR Test, version 2.0.

We evaluated the performance characteristics of the COBAS AMPLICOR Hepatitis C Virus (HCV) MONITOR Test, version 2.0. Dilution studies using patient specimens demonstrated a lower limit of detection of 1,000 copies per milliliter. The assay was linear from 1,000 to 1 million HCV RNA copies per milliliter. Within-run precision and between-run precision were acceptable (approximately 0.100 and 0.14 SD for log10 [copies per milliliter]). A comparison of this version of the test (y), with the manual AMPLICOR HCV MONITOR Test, version 1.0 (x), yielded the following Deming regression equation: y = 1.004(+/- 0.04)x + 0.654(+/- 0.22); Sy/x¿D = 0.336; n = 92; r2 = 0.846; r = 0.920. Further comparison of the COBAS version 2.0 assay (x) with the QUANTIPLEX HCV bDNA Test (y) yielded the following Deming regression equation: y = 0.943 (+/- 0.130)x + 0.473 (+/- 0.717); Sy/x¿D = 0.194; n = 26; r2 = 0.600; r = 0.774. Version 2.0 detected the spectrum of HCV genotypes better than version 1.0.

Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.

Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.

Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation.

Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive for Legionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionella species by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.

Speciation of cone snails and interspecific hyperdivergence of their venom peptides. Potential evolutionary significance of introns.

All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50-200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10-35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro-region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment-specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.

A polymerase chain reaction-based epidemiologic investigation of the incidence of nonpolio enteroviral infections in febrile and afebrile infants 90 days and younger.

OBJECTIVE: Enteroviruses are important pathogens in infants, but their true contribution to febrile illness in infants

Evaluation of the ultrasensitive Roche Amplicor HIV-1 monitor assay for quantitation of human immunodeficiency virus type 1 RNA.

The ultrasensitive Amplicor HIV-1 Monitor test (Roche Diagnostic Systems) was evaluated for precision, linearity, and sensitivity and was compared to the standard Amplicor assay. The ultrasensitive assay reliably quantified samples in the range from 50 to 50,000 human immunodeficiency virus type 1 RNA copies/ml with acceptable correlation with the standard Amplicor test.

An O-glycosylated neuroexcitatory conus peptide.

We purified and characterized a novel peptide from the venom of the fish-hunting cone snail Conus striatus that inhibits voltage-gated K+ channels. The peptide, kappaA-conotoxin SIVA, causes characteristic spastic paralytic symptoms when injected into fish, and in frog nerve-muscle preparations exposed to the toxin, repetitive action potentials are seen in response to a single stimulus applied to the motor nerve. Other electrophysiological tests on diverse preparations provide evidence that is consistent with the peptide blocking K+ channels. The peptide has three disulfide bonds; the locations of Cys residues indicate that the spastic peptide may be the first and defining member of a new family of Conus peptides, the kappaA-conotoxins, which are structurally related to, but pharmacologically distinct from, the alphaA-conotoxins. This 30 AA tricyclic toxin has several characteristics not previously observed in Conus peptides. In addition to the distinctive biological and physiological activity, a novel biochemical feature is the unusually long linear N-terminal tail (11 residues) which contains one O-glycosylated serine at position 7. This is the first evidence for O-glycosylation as a posttranslational modification in a biologically active Conus peptide.

Enhancement of the AMPLICOR enterovirus PCR test with a coprecipitant.

The incorporation of a commercially available coprecipitant into the AMPLICOR enterovirus PCR test specimen preparation enhanced the sensitivity and reproducibility of this assay. Fifty-five previously tested archived cerebrospinal fluids (CSF) specimens were tested in a blind study in duplicate with and without Pellet Paint coprecipitant (Novagen, Inc., Madison, Wis.). Of these specimens, 26 had previously been determined to be positive and 29 had previously been determined to be negative. All previously positive CSF specimens were positive when Pellet Paint was used and only 18 were positive without Pellet Paint. No previously negative specimens were positive on repeat testing with or without Pellet Paint. The background signal was not affected by the addition of Pellet Paint. These data support the utility of a coprecipitant in minimizing false-negative results.

mu-Conotoxin PIIIA, a new peptide for discriminating among tetrodotoxin-sensitive Na channel subtypes.

We report the characterization of a new sodium channel blocker, mu-conotoxin PIIIA(mu-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: [sequence: see text] where Z = pyroglutamate and O = 4-trans-hydroxyproline. We demonstrate that Arginine-14 (Arg14) is a key residue; substitution by alanine significantly decreases affinity and results in a toxin unable to block channel conductance completely. Thus, like all toxins that block at Site I, mu-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized mu-conotoxin GIIIA (mu-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of mu-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to mu-PIIIA and mu-conotoxin GIIIA, (2) sensitive to mu-PIIIA but not to mu-GIIIA, and (3) resistant to mu-PIIIA and mu-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, mu-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

Conantokin-G precursor and its role in gamma-carboxylation by a vitamin K-dependent carboxylase from a Conus snail.

Conantokin-G isolated from the marine snail Conus geographus is a 17-amino acid gamma-carboxyglutamate (Gla)-containing peptide that inhibits the N-methyl-D-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of the propeptide sequence. The cDNA encodes a 100amino acid peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation, and a unique post-translational modification, gamma-carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent gamma-glutamyl carboxylase (apparent Km = 3.4 mM). Using peptides corresponding to different segments of the propeptide we investigated a potential role for the propeptide sequences in gamma-carboxylation. Propeptide segment -20 to -1 covalently linked to E.Con-G or the synthetic pentapeptide FLEEL increased their apparent affinities 2 orders of magnitude. These substrates are not efficiently carboxylated by the bovine microsomal gamma-glutamyl carboxylase, suggesting differences in specificities between the Conus and the mammalian enzyme. However, the role of propeptide in enhancing the efficiency of carboxylation is maintained.

kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel.

kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. kappa-Conotoxin PVIIA has the following structure. This is the first Conus peptide known to target K+ channels. [structure: see text] Although the Shaker K+ channel is sensitive to kappa-PVIIA, the rat brain Kv1.1 subtype is resistant. Chimeras between Shaker and the Kv1.1 K+ channels were constructed and expressed in Xenopus oocytes. Only channels containing the putative pore-forming region between the fifth and sixth transmembrane domains of Shaker retained toxin sensitivity, indicating that the toxin target site is in this region of the channel. Evidence is presented that kappa-PVIIA interacts with the external tetraethyl-ammonium binding site on the Shaker channel. Although both kappa-PVIIA and charybdotoxin inhibit the Shaker channel, they must interact differently. The F425G Shaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes kappa-PVIIA sensitivity. The precursor sequence of kappa-PVIIA was deduced from a cDNA clone, revealing a prepropeptide comprising 72 amino acids. The N-terminal region of the kappa-PVIIA prepropeptide exhibits striking homology to the omega-, muO-, and delta-conotoxins. Thus, at least four pharmacologically distinct superfamilies of Conus peptides belong to the same "O" superfamily, with the omega- and kappa-conotoxins forming one branch, and the delta- and muO-conotoxins forming a second major branch.

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom.

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).