The gut microbiome, resistome, and mycobiome in preterm newborn infants and mouse pups: lack of lasting effects by antimicrobial therapy or probiotic prophylaxis.

BACKGROUND: Enhancing our understanding of the underlying influences of medical interventions on the microbiome, resistome and mycobiome of preterm born infants holds significant potential for advancing infection prevention and treatment strategies. We conducted a prospective quasi-intervention study to better understand how antibiotics, and probiotics, and other medical factors influence the gut development of preterm infants. A controlled neonatal mice model was conducted in parallel, designed to closely reflect and predict exposures. Preterm infants and neonatal mice were stratified into four groups: antibiotics only, probiotics only, antibiotics followed by probiotics, and none of these interventions. Stool samples from both preterm infants and neonatal mice were collected at varying time points and analyzed by 16 S rRNA amplicon sequencing, ITS amplicon sequencing and whole genome shotgun sequencing. RESULTS: The human infant microbiomes showed an unexpectedly high degree of heterogeneity. Little impact from medical exposure (antibiotics/probiotics) was observed on the strain patterns, however, Bifidobacterium bifidum was found more abundant after exposure to probiotics, regardless of prior antibiotic administration. Twenty-seven antibiotic resistant genes were identified in the resistome. High intra-variability was evident within the different treatment groups. Lastly, we found significant effects of antibiotics and probiotics on the mycobiome but not on the microbiome and resistome of preterm infants. CONCLUSIONS: Although our analyses showed transient effects, these results provide positive motivation to continue the research on the effects of medical interventions on the microbiome, resistome and mycobiome of preterm infants.

Viral genome sequencing to decipher in-hospital SARS-CoV-2 transmission events.

The SARS-CoV-2 pandemic has highlighted the need to better define in-hospital transmissions, a need that extends to all other common infectious diseases encountered in clinical settings. To evaluate how whole viral genome sequencing can contribute to deciphering nosocomial SARS-CoV-2 transmission 926 SARS-CoV-2 viral genomes from 622 staff members and patients were collected between February 2020 and January 2021 at a university hospital in Munich, Germany, and analysed along with the place of work, duration of hospital stay, and ward transfers. Bioinformatically defined transmission clusters inferred from viral genome sequencing were compared to those inferred from interview-based contact tracing. An additional dataset collected at the same time at another university hospital in the same city was used to account for multiple independent introductions. Clustering analysis of 619 viral genomes generated 19 clusters ranging from 3 to 31 individuals. Sequencing-based transmission clusters showed little overlap with those based on contact tracing data. The viral genomes were significantly more closely related to each other than comparable genomes collected simultaneously at other hospitals in the same city (n = 829), suggesting nosocomial transmission. Longitudinal sampling from individual patients suggested possible cross-infection events during the hospital stay in 19.2% of individuals (14 of 73 individuals). Clustering analysis of SARS-CoV-2 whole genome sequences can reveal cryptic transmission events missed by classical, interview-based contact tracing, helping to decipher in-hospital transmissions. These results, in line with other studies, advocate for viral genome sequencing as a pathogen transmission surveillance tool in hospitals.

T-Cell-Dominated Immune Response Resolves Protracted SARS-CoV-2 Infection in the Absence of Neutralizing Antibodies in an Immunocompromised Individual.

Immunocompromised individuals are at higher risk of developing protracted and severe COVID-19, and understanding individual disease courses and SARS-CoV-2 immune responses in these individuals is of the utmost importance. For more than two years, we followed an immunocompromised individual with a protracted SARS-CoV-2 infection that was eventually cleared in the absence of a humoral neutralizing SARS-CoV-2 antibody response. By conducting an in-depth examination of this individual's immune response and comparing it to a large cohort of convalescents who spontaneously cleared a SARS-CoV-2 infection, we shed light on the interplay between B- and T-cell immunity and how they interact in clearing SARS-CoV-2 infection.

Transmission of SARS-CoV-2 among children and staff in German daycare centres.

In daycare centres, the close contact of children with other children and employees favours the transmission of infections. The majority of children <6 years attend daycare programmes in Germany, but the role of daycare centres in the SARS-CoV-2 pandemic is unclear. We investigated the transmission risk in daycare centres and the spread of SARS-CoV-2 to associated households. 30 daycare groups with at least one recent laboratory-confirmed SARS-CoV-2 case were enrolled in the study (10/2020-06/2021). Close contact persons within daycare and households were examined over a 12-day period (repeated SARS-CoV-2 PCR tests, genetic sequencing of viruses, symptom diary). Households were interviewed to gain comprehensive information on each outbreak. We determined primary cases for all daycare groups. The number of secondary cases varied considerably between daycare groups. The pooled secondary attack rate (SAR) across all 30 daycare centres was 9.6%. The SAR tended to be higher when the Alpha variant was detected (15.9% vs. 5.1% with evidence of wild type). The household SAR was 53.3%. Exposed daycare children were less likely to get infected with SARS-CoV-2 than employees (7.7% vs. 15.5%). Containment measures in daycare programmes are critical to reduce SARS-CoV-2 transmission, especially to avoid spread to associated households.

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

Effects of caloric restriction on the gut microbiome are linked with immune senescence.

BACKGROUND: Caloric restriction can delay the development of metabolic diseases ranging from insulin resistance to type 2 diabetes and is linked to both changes in the composition and metabolic function of the gut microbiota and immunological consequences. However, the interaction between dietary intake, the microbiome, and the immune system remains poorly described. RESULTS: We transplanted the gut microbiota from an obese female before (AdLib) and after (CalRes) an 8-week very-low-calorie diet (800 kcal/day) into germ-free mice. We used 16S rRNA sequencing to evaluate taxa with differential abundance between the AdLib- and CalRes-microbiota recipients and single-cell multidimensional mass cytometry to define immune signatures in murine colon, liver, and spleen. Recipients of the CalRes sample exhibited overall higher alpha diversity and restructuring of the gut microbiota with decreased abundance of several microbial taxa (e.g., Clostridium ramosum, Hungatella hathewayi, Alistipi obesi). Transplantation of CalRes-microbiota into mice decreased their body fat accumulation and improved glucose tolerance compared to AdLib-microbiota recipients. Finally, the CalRes-associated microbiota reduced the levels of intestinal effector memory CD8+ T cells, intestinal memory B cells, and hepatic effector memory CD4+ and CD8+ T cells. CONCLUSION: Caloric restriction shapes the gut microbiome which can improve metabolic health and may induce a shift towards the naïve T and B cell compartment and, thus, delay immune senescence. Understanding the role of the gut microbiome as mediator of beneficial effects of low calorie diets on inflammation and metabolism may enhance the development of new therapeutic treatment options for metabolic diseases. TRIAL REGISTRATION: NCT01105143 , "Effects of negative energy balance on muscle mass regulation," registered 16 April 2010. Video Abstract.

The virome of German bats: comparing virus discovery approaches.

Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies.

First detection of bat-borne Issyk-Kul virus in Europe.

Bats have been gaining attention as potential reservoir hosts of numerous viruses pathogenic to animals and man. Issyk-Kul virus, a member of the family Nairoviridae, was first isolated in the 1970s from vespertilionid bats in Central Asia. Issyk-Kul virus has been described as human-pathogenic virus, causing febrile outbreaks in humans with headaches, myalgia and nausea. Here we describe the detection of a novel strain of Issyk-Kul virus from Eptesicus nilssonii in Germany. This finding indicates for the first time the prevalence of these zoonotic viruses in Europe.

Microbiomes in the insectivorous bat species Mops condylurus rapidly converge in captivity.

Bats are well known reservoir hosts for RNA and DNA viruses. The use of captive bats in research has intensified over the past decade as researchers aim to examine the virus-reservoir host interface. In this study, we investigated the effects of captivity on the fecal bacterial microbiome of an insectivorous microbat, Mops condylurus, a species that roosts in close proximity to humans and has likely transmitted viral infections to humans. Using amplicon 16S rRNA gene sequencing, we characterized changes in fecal bacterial community composition for individual bats directly at the time of capture and again after six weeks in captivity. We found that microbial community richness by measure of the number of observed operational taxonomic units (OTUs) in bat feces increases in captivity. Importantly, we found the similarity of microbial community structures of fecal microbiomes between different bats to converge during captivity. We propose a six week-acclimatization period prior to carrying out infection studies or other research influenced by the microbiome composition, which may be advantageous to reduce variation in microbiome composition and minimize biological variation inherent to in vivo experimental studies.

Zwiesel bat banyangvirus, a potentially zoonotic Huaiyangshan banyangvirus (Formerly known as SFTS)-like banyangvirus in Northern bats from Germany.

Bats are reservoir hosts for several emerging and re-emerging viral pathogens causing morbidity and mortality in wildlife, animal stocks and humans. Various viruses within the family Phenuiviridae have been detected in bats, including the highly pathogenic Rift Valley fever virus and Malsoor virus, a novel Banyangvirus with close genetic relation to Huaiyangshan banyangvirus (BHAV)(former known as Severe fever with thrombocytopenia syndrome virus, SFTSV) and Heartland virus (HRTV), both of which have caused severe disease with fatal casualties in humans. In this study we present the whole genome of a novel Banyangvirus, named Zwiesel bat banyangvirus, revealed through deep sequencing of the Eptesicus nilssonii bat virome. The detection of the novel bat banyangvirus, which is in close phylogenetic relationship with the pathogenic HRTV and BHAV, underlines the possible impact of emerging phenuiviruses on public health.

Dendritic Cells Generated From Mops condylurus, a Likely Filovirus Reservoir Host, Are Susceptible to and Activated by Zaire Ebolavirus Infection.

Ebola virus infection of human dendritic cells (DCs) induces atypical adaptive immune responses and thereby exacerbates Ebola virus disease (EVD). Human DCs, infected with Ebola virus aberrantly express low levels of the DC activation markers CD80, CD86, and MHC class II. The T cell responses ensuing are commonly anergic rather than protective against EVD. We hypothesize that DCs derived from potential reservoir hosts such as bats, which do not develop disease signs in response to Ebola virus infection, would exhibit features associated with activation. In this study, we have examined Zaire ebolavirus (EBOV) infection of DCs derived from the Angolan free-tailed bat species, Mops condylurus. This species was previously identified as permissive to EBOV infection in vivo, in the absence of disease signs. M. condylurus has also been recently implicated as the reservoir host for Bombali ebolavirus, a virus species that is closely related to EBOV. Due to the absence of pre-existing M. condylurus species-specific reagents, we characterized its de novo assembled transcriptome and defined its phylogenetic similarity to other mammals, which enabled the identification of cross-reactive reagents for M. condylurus bone marrow-derived DC (bat-BMDC) differentiation and immune cell phenotyping. Our results reveal that bat-BMDCs are susceptible to EBOV infection as determined by detection of EBOV specific viral RNA (vRNA). vRNA increased significantly 72 h after EBOV-infection and was detected in both cells and in culture supernatants. Bat-BMDC infection was further confirmed by the observation of GFP expression in DC cultures infected with a recombinant GFP-EBOV. Bat-BMDCs upregulated CD80 and chemokine ligand 3 (CCL3) transcripts in response to EBOV infection, which positively correlated with the expression levels of EBOV vRNA. In contrast to the aberrant responses to EBOV infection that are typical for human-DC, our findings from bat-BMDCs provide evidence for an immunological basis of asymptomatic EBOV infection outcomes.

Genetic Diversity and Spatial Segregation of Francisella tularensis Subspecies holarctica in Germany.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%.

Molecular identification of the source of an uncommon tularaemia outbreak, Germany, autumn 2016.

BackgroundIn 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.AimWe describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.MethodsWe incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case's lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.ResultsNo bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica, belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case's isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.ConclusionThe discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

Parasitic Nematodes Exert Antimicrobial Activity and Benefit From Microbiota-Driven Support for Host Immune Regulation.

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram+/- bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3+RORγt+ regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice.

Silent Witness: Dual-Species Transcriptomics Reveals Epithelial Immunological Quiescence to Helminth Larval Encounter and Fostered Larval Development.

Gastrointestinal nematodes are among the most prevalent parasites infecting humans and livestock worldwide. Infective larvae of the soil-transmitted nematode Ascaris spp. enter the host and start tissue migration by crossing the intestinal epithelial barrier. The initial interaction of the intestinal epithelium with the parasite, however, has received little attention. In a time-resolved interaction model of porcine intestinal epithelial cells (IPEC-J2) and infective Ascaris suum larvae, we addressed the early transcriptional changes occurring simultaneously in both organisms using dual-species RNA-Seq. Functional analysis of the host response revealed an overall induction of metabolic activity, without induction of immune responsive genes or immune signaling pathways and showing suppression of chemotactic genes like CXCL8/IL-8 or CHI3L1. Ascaris larvae, when getting in contact with the epithelium, showed induction of genes that orchestrate motor activity and larval development, such as myosin, troponin, myoglobin, and protein disulfide isomerase 2 (PDI-2). In addition, excretory-secretory products that likely facilitate parasite invasion were increased, among them, aspartic protease 6 or hyaluronidase. Integration of host and pathogen data in an interspecies gene co-expression network indicated links between nematode fatty acid biosynthesis and host ribosome assembly/protein synthesis. In summary, our study provides new molecular insights into the early factors of parasite invasion, while at the same time revealing host immunological unresponsiveness. Reproducible software for dual RNA-Seq analysis of non-model organisms is available at https://gitlab.com/mkuhring/project_asuum and can be applied to similar studies.

PriLive: privacy-preserving real-time filtering for next-generation sequencing.

Motivation: In next-generation sequencing, re-identification of individuals and other privacy-breaching strategies can be applied even for anonymized data. This also holds true for applications in which human DNA is acquired as a by-product, e.g. for viral or metagenomic samples from a human host. Conventional data protection strategies including cryptography and post-hoc filtering are only appropriate for the final and processed sequencing data. This can result in an insufficient level of data protection and a considerable time delay in the further analysis workflow. Results: We present PriLive, a novel tool for the automated removal of sensitive data while the sequencing machine is running. Thereby, human sequence information can be detected and removed before being completely produced. This facilitates the compliance with strict data protection regulations. The unique characteristic to cause almost no time delay for further analyses is also a clear benefit for applications other than data protection. Especially if the sequencing data are dominated by known background signals, PriLive considerably accelerates consequent analyses by having only fractions of input data. Besides these conceptual advantages, PriLive achieves filtering results at least as accurate as conventional post-hoc filtering tools. Availability and implementation: PriLive is open-source software available at https://gitlab.com/rki_bioinformatics/PriLive. Supplementary information: Supplementary data are available at Bioinformatics online.

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

BACKGROUND: We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. METHODOLOGY: An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. PRINCIPAL FINDINGS: The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. CONCLUSIONS: Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome.

OBJECTIVE: Hepatitis E virus (HEV) infection can take chronic courses in immunocompromised patients potentially leading to liver cirrhosis and liver failure. Ribavirin (RBV) is currently the only treatment option for many patients, but treatment failure can occur which has been associated with the appearance of a distinct HEV polymerase mutant (G1634R). Here, we performed a detailed analysis of HEV viral intrahost evolution during chronic hepatitis E infections. DESIGN: Illumina deep sequencing was performed for the detection of intrahost variation in the HEV genome of chronically infected patients. Novel polymerase mutants were investigated in vitro using state-of-the-art HEV cell culture models. RESULTS: Together, these data revealed that (1) viral diversity differed markedly between patients but did not show major intraindividual short-term variations in untreated patients with chronic hepatitis E, (2) RBV therapy was associated with an increase in viral heterogeneity which was reversible when treatment was stopped, (3) the G1634R mutant was detectable as a minor population prior to therapy in patients who subsequently failed to achieve a sustained virological response to RBV therapy and (4) in addition to G1634R further dominant variants in the polymerase region emerged, impacting HEV replication efficiency in vitro. CONCLUSIONS: In summary, this first investigation of intrahost HEV population evolution indicates that RBV causes HEV mutagenesis in treated patients and that an emergence of distinct mutants within the viral population occurs during RBV therapy. We also suggest that next-generation sequencing could be useful to guide personalised antiviral strategies.

Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

BACKGROUND: Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). METHODS: Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. RESULTS: Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. CONCLUSIONS: Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

What caused the outbreak of ESBL-producing Klebsiella pneumoniae in a neonatal intensive care unit, Germany 2009 to 2012? Reconstructing transmission with epidemiological analysis and whole-genome sequencing.

OBJECTIVE: We aimed to retrospectively reconstruct the timing of transmission events and pathways in order to understand why extensive preventive measures and investigations were not sufficient to prevent new cases. METHODS: We extracted available information from patient charts to describe cases and to compare them to the normal population of the ward. We conducted a cohort study to identify risk factors for pathogen acquisition. We sequenced the available isolates to determine the phylogenetic relatedness of Klebsiella pneumoniae isolates on the basis of their genome sequences. RESULTS: The investigation comprises 37 cases and the 10 cases with ESBL (extended-spectrum beta-lactamase)-producing K. pneumoniae bloodstream infection. Descriptive epidemiology indicated that a continuous transmission from person to person was most likely. Results from the cohort study showed that 'frequent manipulation' (a proxy for increased exposure to medical procedures) was significantly associated with being a case (RR 1.44, 95% CI 1.02 to 2.19). Genome sequences revealed that all 48 bacterial isolates available for sequencing from 31 cases were closely related (maximum genetic distance, 12 single nucleotide polymorphisms). Based on our calculation of evolutionary rate and sequence diversity, we estimate that the outbreak strain was endemic since 2008. CONCLUSIONS: Epidemiological and phylogenetic analyses consistently indicated that there were additional, undiscovered cases prior to the onset of microbiological screening and that the spread of the pathogen remained undetected over several years, driven predominantly by person-to-person transmission. Whole-genome sequencing provided valuable information on the onset, course and size of the outbreak, and on possible ways of transmission.