Cole L. Irish

Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study

BACKGROUND Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA. METHODS At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR. FINDINGS 100 545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site. INTERPRETATION We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis. FUNDING US Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) Cooperative Agreement and Mayo Clinic Small Grant programme.

Direct Detection of Influenza A and B Viruses in Less Than 20 Minutes Using a Commercially Available Rapid PCR Assay

ABSTRACT We compared an FDA-cleared rapid (<20 min) PCR assay (Cobas Liat; Roche Diagnostics) to our routine influenza A and B real-time PCR assay (Simplexa Flu A/B & RSV Direct; Focus Diagnostics) using respiratory swabs (n = 197). The Cobas Liat influenza A and B assays demonstrated sensitivities of 99.2% (123/124) and 100% (23/23), respectively, while showing a specificity of 100% for each target.

Borrelia mayonii sp. nov., a member of the Borrelia burgdorferi sensu lato complex, detected in patients and ticks in the upper midwestern United States.

Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).

Rapid and Direct Detection of Herpes Simplex Virus in Cerebrospinal Fluid by Use of a Commercial Real-Time PCR Assay

ABSTRACT Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min.

Mutability in the matrix gene of novel influenza a H1N1 virus detected using a FRET probe-based real-time reverse transcriptase PCR assay

Influenza A viruses are well known for their genetic diversity. They are constantly evolving through point mutations in their hemagglutinin (H) and neuraminidase (N) genes and through genetic reassortment of their segmented RNA genome (2). The current pandemic/2009/novel H1N1 influenza A virus (called novel H1N1 herein) is a product of antigenic shift resulting from triple reassortment of human, avian, and swine viruses (7). Between 19 April 2009 and November 2009, 199 countries have reported nearly 6,000 novel influenza A H1N1-related deaths to the World Health Organization (WHO) (3).

Conversion to the COBAS AmpliPrep/COBAS TaqMan CMV Test for management of CMV disease in transplant recipients.

Testing of clinical plasma specimens by the COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV), COBAS AMPLICOR CMV MONITOR Test (CAM CMV), and a laboratory-developed assay using analyte-specific reagents (LC CMV) demonstrated substantial result bias for CAP/CTM CMV versus CAM CMV (r = 0.436) and CAP/CTM CMV versus LC CMV (r = 0.773).

Evidence for Amino Acid Changes in a 57-Base-Pair Region of the Highly Conserved Matrix Gene of Pandemic (H1N1) 2009 Influenza A Virus

Pandemic (H1N1) 2009 influenza A virus originated due to triple reassortment of human, avian, and swine viruses (6). The pandemic epicenter was in Mexico, where the first human case was reported on 12 April 2009 (1). Thereafter, 16,813 pandemic H1N1 influenza A-related deaths have been reported to the World Health Organization (WHO) by 213 countries between April 2009 and March 2010 (8). From a laboratory perspective, detection and discrimination of pandemic H1N1 from seasonal and swine H1N1 is crucial for accurate diagnosis and disease surveillance. We previously described a laboratory-developed influenza A virus real-time reverse transcription-PCR (rRT-PCR) assay (Mayo FLU A) for simultaneous identification and subtype discrimination of influenza A virus RNA using melting temperature (Tm) analysis (3, 9). As discussed in the paper by Dhiman et al., loss of subtype discriminatory ability occurred in just 3 months of testing for the pandemic virus, due to multiple point mutations in the targeted 242-base region of the highly conserved matrix (M) gene as determined on a subset of 12 isolates (3). In the current paper, we have extended our analysis to a significantly larger data set to conduct mutational and amino acid analysis and characterize the genetic changes in the M gene of the pandemic H1N1 influenza A virus. Between 1 May 2009 and 31 December 2009, we detected 1,731 influenza A virus clinical isolates out of 9,614 isolates tested (18% positivity rate) using the Mayo FLU A assay; the majority were identified as the pandemic H1N1 subtype based on predefined Tm criteria (50.5°C to 53.2°C). However, between 14 August and 31 December 2009, 48 clinical samples were identified with Tms outside the validated range for pandemic H1N1 (48 of 1,579 isolates [3.0%] during this period), with an overall lower mean Tm of 46.71 ± 1.73°C. Only one specimen, from Ann Arbor, MI, had a higher Tm of 55.5°C, which is unexpected, given that mutations typically cause a decrease rather than an increase in Tm. The significance of this result is unknown, as we did not have additional specimens from that geographical area to confirm if this result was an actual trend or an outlier. All atypical Tm samples were confirmed as pandemic H1N1 using the CDC swine flu assay. We sequenced the 242-base oligonucleotide amplicon in 37 isolates to identify the mutations responsible for the atypical Tms. Mutational analysis revealed multiple point mutations in the 57-bp region detected by the probes compared to the original pandemic H1N1 gene sequence published by the WHO on 28 April 2009 (2) (Table ​(Table1).1). Six of these were silent mutations, resulting in nucleotide changes GAG→GAA, ATC→ATT, GCG→GCA, AGA→AGG, CTG→TTG, and AAC→AAT at amino acid positions 23, 24, 25, 27, 28, and 36, respectively, compared to the original pandemic H1N1 gene sequence. Of interest, two additional nonsynonymous mutations, CTG→ATG and ACA→ATA, resulted in downstream changes in amino acid sequence at positions Leu28Met and Thr37Ile, respectively (Fig. ​(Fig.1).1). We also observed geographic clustering among the mutations in these analyses (Table ​(Table11). FIG. 1. Amino acid sequence of the 57-bp discriminatory region of pandemic (H1N1) 2009 influenza A virus. TABLE 1. Sequence alignment in the discriminatory region of the amplified matrix gene from 37 clinical isolates with atypical Tms using the Mayo FLU A assay Since the emergence of pandemic H1N1, several studies have tried to characterize the mutational trends of this new virus (4, 5, 7). Pan et al. described the emergence of a signature residue at the position of nucleoprotein 100 (NP-100) (valine to isoleucine) that exhibited a dominant change in as many as 93% of isolates by the later phases of the pandemic. In addition, four nonsignature residues, at positions neuraminidase 91 (NA-91), NA-233, hemagglutinin 206 (HA-206), and nonstructural protein 1 (NS1) to NS123, were observed during a short period of time within the epidemic (5). All of these mutant residues were characterized in the viral functional domains, suggesting potent roles in the human adaption and virulence. Mutations were also observed in M1/M2 genes, but they were of low frequency with an unknown role. Potdar et al. reported several mutations throughout the viral genome, including a predominant D225G mutation in the H gene present in the receptor binding domain of Indian novel H1N1 isolates (7). Nelson et al. conducted a whole-genome phylogenetic analysis on 290 isolates of pandemic H1N1 influenza virus collected globally and identified seven major clades that have cocirculated worldwide since April 2009 (4). Several amino acid changes were also identified, predominantly in the HA, NA, and NP genes. However, no mutations were observed in the matrix gene in the latter two studies. In our study, we found eight mutations in a very short sequence span of only 57 bp of the influenza A virus matrix gene, of which two were nonsynonymous (Leu28Met and Thr37Ile). This is an unexpected finding, as the influenza A virus matrix gene is relatively conserved and is not thought to be under the same genetic selective pressures as other genes, such as HA and NA, that have very high mutability rates. The implications of these findings for viral adaptation and virulence have yet to be determined. However, from a diagnostic perspective, these mutations resulted in the loss of viral subtype discriminatory ability using Tm analysis within just 3 months of the pandemic and hindered laboratory diagnosis. Evidence for mutability in the highly conserved M gene of influenza virus calls for pandemic as well as routine in silico monitoring of primers and probes for optimal target coverage.

Viral detection using a multiplex polymerase chain reaction–based assay in outpatients with upper respiratory infection

Abstract We evaluated a commercial multiplex polymerase chain reaction (PCR) assay in a cross-sectional study among 81 adult and pediatric outpatients—40 cases with upper respiratory infection symptoms and 41 asymptomatic controls—from February to April 2008. Two specimens (throat swab and nasal swab) from each participant were tested using the EraGen MultiCode-PLx Respiratory Virus Panel that detects 17 viral targets. Throat swabs were also tested for Group A Streptococcus (GAS) by PCR. Respiratory viruses were detected in 22/40 (55%) cases and in 3/41 (7%) controls (P < 0.001). GAS was detected in 10 (25%) cases; GAS and respiratory virus co-infection was found in 4 (10%). Agreement between nasal and throat swabs for viral detection was 69% in cases and 95% in controls. Of 22 cases with a detectable virus, 12 (54%) were picked up by only 1 (throat or nasal) specimen, and the detection rate was increased by combining results of nasal and throat swab testing.

Precision across the Analytical Measuring Range of a Quantitative Real-Time PCR Assay for Cytomegalovirus Detection among Three Clinical Laboratories

ABSTRACT This study measured the precision of a quantitative laboratory-developed real-time PCR test for cytomegalovirus performed at three different clinical laboratories that use the same methodology. The overall standard deviation (adjusted for analyte level) was 0.18 log10 copies/ml, and there was no significant relationship between standard deviation and analytical measuring range.

Testing for Herpes Simplex Virus in Low-Volume Cerebrospinal Fluid Samples: Comparison of Three Protocols To Optimize Detection

ABSTRACT Detection of herpes simplex virus 1 and 2 (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) is a medical emergency and requires rapid, sensitive testing. However, the volume of CSF received for microbiological studies may be limited, especially from young children. In this study, we compared three testing protocols to our routine real-time PCR method to determine the most sensitive approach for detecting HSV-1 and HSV-2 in low-volume (≤100 μl) CSF.