Biology subjects

Makhsous, N.

Publications and source records attributed to Makhsous, N..

7 recordsLinked to original sources

bioRxiv2023

Within-host rhinovirus evolution in upper and lower respiratory tract highlights capsid variability and mutation-independent compartmentalization

BackgroundHuman rhinovirus (HRV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how HRV evolves within hosts during infection. MethodsWe sequenced HRV complete genomes from 12 hematopoietic cell transplant patients with prolonged infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL) specimens. Metagenomic (mNGS) and amplicon-based NGS were used to study the emergence and evolution of intra-host single nucleotide variants (iSNVs). ResultsIdentical HRV intra-host populations in matched NW and BAL specimens indicated no genetic adaptation is required for HRV to progress from URT to LRT. Microbial composition between matched NW and BAL confirmed no cross-contamination during sampling procedure. Coding iSNVs were 2.3-fold more prevalent in capsid over non-structural genes, adjusted for length. iSNVs modeled onto HRV capsid structures were significantly more likely to be found in surface residues, but were not preferentially located in known HRV neutralizing antibody epitopes. Newly emergent, serotype-matched iSNV haplotypes from immunocompromised individuals from 2008-2010 could be detected in Seattle-area community HRV sequences from 2020-2021. ConclusionHRV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, HRV sequences.

microbiology↗

bioRxiv2019

LAVA: a streamlined visualization tool for longitudinal analysis of viral alleles

With their small genomes, fast evolutionary rates, and clinical significance, viruses have long been fodder for studies of whole genome evolution. One common need in these studies is the analysis of viral evolution over time through longitudinal sampling. However, there exists no simple tool to automate such analyses. We created a simple command-line visualization tool called LAVA (Longitudinal Analysis of Viral Alleles). LAVA allows dynamic and interactive visualization of viral evolution across the genome and over time. Results are easily shared via a single HTML file that also allows interactive analysis based on read depth and allele frequency. LAVA requires minimal input and runs in minutes for most use cases. LAVA is programmed mainly in Python 3 and is compatible with Mac and Linux machines. LAVA is a user-friendly command-line tool for generating, visualizing, and sharing the results of longitudinal viral genome evolution analysis. Instructions for downloading, installing, and using LAVA can be found at https://github.com/michellejlin/lava.

bioinformatics↗

bioRxiv2018

A novel qacA allele results in an elevated chlorhexidine gluconate minimum inhibitory concentration in cutaneous Staphylococcus epidermidis isolates

Chlorhexidine gluconate (CHG) is a topical antiseptic widely used in healthcare settings. In Staphylococcus spp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneous Staphylococcus isolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed 63 (6%) of these isolates had elevated CHG MICs ([≥] 4 g/mL). Screening of all 1,050 isolates for qacA/B by restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novel qacA/B RFLP pattern, qacAB273. The CHG MIC was significantly higher for qacAB273-positive isolates (MIC50: 4 g/mL, [range: 0.5 - 4 g/mL]) compared to other qac groups: qacA-positive (n=559, 1 g/mL, [0.5 - 4 g/mL]), qacB-positive (n=17, 1 g/mL, [0.25 - 2 g/mL]), and qacA/B-negative (n=418, 1 g/mL, [0.125 - 2 g/mL], p=0.001). The qacAB273-positive isolates also displayed a high proportion of methicillin resistance (96.4%) compared to other qac groups (24.9 - 61.7%, p=0.001). Whole genome sequencing revealed that qacAB273-positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, named qacA4, was carried on the novel plasmid pAQZ1. The qacA4-carrying isolates belonged to the highly resistant S. epidermidis clone ST2 and were collected from multiple centers across the United States and Canada. Curing an isolate of qacA4 resulted in a four-fold decrease in the CHG MIC, confirming the role of qacA4 in the elevated CHG MIC. Our results highlight the importance of further studying qacA4 and its functional role in clinical staphylococci.\n\nImportanceStaphylococcus epidermidis is an important cause of infections in patients with implanted devices. Bathing with chlorhexidine gluconate (CHG), a topical antiseptic, has been shown to reduce rates of device-associated infections, especially those caused by S. epidermidis. In S. epidermidis, reduced susceptibility to CHG is associated with carriage of the qacA gene. As part of a multicenter CHG bathing trial, we obtained cutaneous Staphylococcus isolates from pediatric oncology patients across the United States and Canada. We identified a group of isolates capable of surviving in higher concentrations of CHG and determined a novel allele of qacA, termed qacA4 and carried on the novel plasmid pAQZ1, was responsible for the isolates survival in higher CHG concentrations. The qacA4-carrying S. epidermidis isolates belonged to the highly resistant and virulent ST2 clonal type. Our results highlight the need to understand the global distribution of novel qacA alleles, including qacA4, and their mechanistic effect on efflux.

microbiology↗

bioRxiv2018

Prospective real-time metagenomic sequencing during norovirus outbreak reveals discrete transmission clusters

BackgroundNorovirus outbreaks in hospital settings are a common challenge for infection prevention teams. Given the high burden of norovirus in most communities, it can be difficult to distinguish between on-going in-hospital transmission of virus and new introductions from the community and challenging to understand the long-term impacts of outbreak-associated viruses within medical systems using traditional epidemiological approaches alone.\n\nMethodsReal-time metagenomic sequencing during an on-going norovirus outbreak associated with a retrospective cohort study.\n\nResultsWe describe a hospital-associated norovirus outbreak that affected 13 patients over a 27-day period in a large tertiary pediatric hospital and was chronologically associated with a spike in self-reported gastrointestinal symptoms among staff. Real-time metagenomic next-generation sequencing (mNGS) of norovirus genomes demonstrated that 10 chronologically overlapping hospital-acquired norovirus cases were partitioned into three discrete transmission clusters. Sequencing data also revealed close genetic relationships between some hospital-acquired and some community-acquired cases. Finally, this data was used to demonstrate chronic viral shedding by an immunocompromised hospital-acquired case patient. Analysis of serial samples from this patient provided novel insights into the evolution of norovirus within an immunocompromised host.\n\nConclusionsThis study documents one of the first applications of real-time mNGS during a hospital-associated viral outbreak. Given its demonstrated ability to detect transmission patterns within outbreaks and elucidate the long-term impacts of outbreak-associated viral strains on patients and medical systems, mNGS constitutes a powerful resource to help infection control teams understand, prevent, and respond to viral outbreaks.\n\nSummary StatementReal-time metagenomic sequencing performed during a hospital-associated norovirus outbreak identified genetically-distinct, chronologically-overlapping case clusters. After the epidemiologically-defined outbreak had ceased, on-going transmission and shedding of outbreak-associated virus was also detected. These findings illustrate the value of genomics as a tool for infection control.

genomics↗

bioRxiv2018

VAPiD: a lightweight cross platform viral annotation pipeline and identification tool to facilitate virus genome submissions to NCBI GenBank

BackgroundWith sequencing technologies becoming cheaper and easier to use, more groups are able to obtain whole genome sequences of viruses of public health and scientific importance. Submission of genomic data to NCBI GenBank is a requirement prior to publication and plays a critical role in making scientific data publicly available.\n\nGenBank currently has automatic prokaryotic and eukaryotic genome annotation pipelines but has no viral annotation pipeline beyond influenza virus. Annotation and submission of viral genome sequence is a non-trivial task, especially for groups that do not routinely interact with GenBank for data submissions.\n\nResultsWe present Viral Annotation Pipeline and iDentification (VAPiD), a portable and lightweight command-line tool for annotation and GenBank deposition of viral genomes. VAPiD supports annotation of nearly all unsegmented viral genomes. The pipeline has been validated on human immunodeficiency virus, human parainfluenza virus 1-4, human metapneumovirus, human coronaviruses (229E/OC43/NL63/HKU1/SARS/MERS), human enteroviruses/rhinoviruses, measles virus, mumps virus, Hepatitis A-E Virus, Chikungunya virus, dengue virus, and West Nile virus, as well the human polyomaviruses BK/JC/MCV, human adenoviruses, and human papillomaviruses. The program can handle individual or batch submissions of different viruses to GenBank and correctly annotates multiple viruses, including those that contain ribosomal slippage or RNA editing without prior knowledge of the virus to be annotated. VAPiD is programmed in Python and is compatible with Windows, Linux, and Mac OS systems.\n\nConclusionsWe have created a portable, lightweight, user-friendly, internet-enabled, open-source, command-line genome annotation and submission package to facilitate virus genome submissions to NCBI GenBank. Instructions for downloading and installing VAPiD can be found at https://github.com/rcs333/VAPiD.

bioinformatics↗

bioRxiv2018

Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.\n\nImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

epidemiology↗

bioRxiv2017

Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains

Quantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6 is important for detection of viral reactivation and inherited chromosomally integrated HHV-6 in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV6 candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Copy number measured between 4-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6 strain with a HHV-6A GS backbone and >5.5kb region from HHV-6B Z29 from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at UL1/UL2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6 strains derived from cord blood mononuclear cells from different labs on different continents revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.\n\nImportanceAnything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very usefulness through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6 biology and the standard selection process.

genomics↗