Diversity of Pneumocystis jirovecii during Infection Revealed by Ultra-Deep Pyrosequencing

International audiencePneumocystis jirovecii is an uncultivable fungal pathogen responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, the physiopathology of which is only partially understood. The diversity of the Pneumocystis strains associated with acute infection has mainly been studied by Sanger sequencing techniques precluding any identification of rare genetic events (\textless 20% frequency). We used next-generation sequencing to detect minority variants causing infection, and analyzed the complexity of the genomes of infection-causing P. jirovecii. Ultra-deep pyrosequencing (UDPS) of PCR amplicons of two nuclear target region [internal transcribed spacer 2 (ITS2) and dihydrofolate reductase (DHFR)] and one mitochondrial DNA target region [the mitochondrial ribosomal RNA large subunit gene (mtLSU)] was performed on 31 samples from 25 patients. UDPS revealed that almost all patients (n = 23/25, 92%) were infected with mixtures of strains. An analysis of repeated samples from six patients showed that the proportion of each variant change significantly (by up to 30%) over time on treatment in three of these patients. A comparison of mitochondrial and nuclear UDPS data revealed heteroplasmy in P. jirovecii. The recognition site for the homing endonuclease I-SceI was recovered from the mtLSU gene, whereas its two conserved motifs of the enzyme were not. This suggests that heteroplasmy may result from recombination induced by unidentified homing endonucleases. This study sheds new light on the biology of P. jirovecii during infection. PCP results from infection not with a single microorganism, but with a complex mixture of different genotypes, the proportions of which change over time due to intricate selection and reinfection mechanisms that may differ between patients, treatments, and predisposing diseases

Analytical validation of hepatitis B core‐related antigen (HBcrAg) using dried blood spots (DBS)

International audienceLimited access to nucleic acid testing (NAT) to quantify HBV DNA levels, an essential tool to determine anti-HBV treatment eligibility, represents a significant barrier to scale up HBV diagnostic services in resource-limited countries. Hepatitis B core-related antigen (HBcrAg) has the potential to become an affordable alternative because of its low cost (US$ <15/assay) and strong correlation with HBV DNA levels in treatment-naïve patients. However, the current assay requires plasma or serum. To further facilitate its application to decentralized settings, we developed and evaluated a standardized procedure to quantify HBcrAg using dried blood spots as a tool to diagnose HBV-infected people with high viraemia. We evaluated the following elution method optimized to quantify HBcrAg: suspension of a punched blood-soaked disc (11 mm) of Whatman 903 Protein Saver Card in 450 µL of PBS 0.05% Tween 20, followed by an incubation for 4 h at room temperature and a centrifugation at 10,000 g for 10 minutes. 150 µL of DBS eluate was used to quantify HBcrAg using chemiluminescent enzyme immunoassay (LUMIPULSE® G600II, Fujirebio). The limit of detection of dried blood spot HBcrAg in relation with HBV DNA levels was 19,115 IU/mL across the five major HBV genotypes (A/B/C/D/E). A strong linear correlation was confirmed between dried blood spot HBcrAg and HBV DNA levels (r = 0.94, p < 0.0001) in samples with high viral loads (range: 3.7–7.0 log IU/mL). The coefficient of variation ranged between 4.0–11.2% for repeatability and 3.9–12.2% for reproducibility. Analytical specificity was 100% (95% CI: 83.9–100%) in HBV-negative samples. Using our elution method, it may be possible to identify HBV-infected patients with high viraemia who need antiviral therapy using dried blood spot and HBcrAg. A large-scale clinical validation is warranted in resource-limited countries

RESEARCH High level of susceptibility to human TRIM5α conferred by HIV-2 capsid sequences

Background: HIV-2, which was transmitted to humans from a distant primate species (sooty mangabey), differs remarkably from HIV-1 in its infectivity, transmissibility and pathogenicity. We have tested the possibility that a greater susceptibility of HIV-2 capsid (CA) to the human restriction factor TRIM5α (hTRIM5α) could contribute to these differences. Results: We constructed recombinant clones expressing CA from a variety of HIV-2 viruses in the context of HIV-1 NL4-3-luciferase. CA sequences were amplified from the plasma of HIV-2 infected patients, including 8 subtype A and 7 subtype B viruses. CA from 6 non-epidemic HIV-2 subtypes, 3 HIV-2 CRF01_AB recombinants and 4 SIVsmm viruses were also tested. Susceptibility to hTRIM5α was measured by comparing single-cycle infectivity in human target cells expressing hTRIM5α to that measured in cells in which hTRIM5α activity was inhibited by overexpression of hTRIM5γ. The insertion of HIV-2 CA sequences in the context of HIV-1 did not affect expression and maturation of the HIV-2 CA protein. The level of susceptibility hTRIM5α expressed by viruses carrying HIV-2 CA sequences was up to 9-fold higher than that of HIV-1 NL4-3 and markedly higher than a panel of primary HIV-1 CA sequences. This phenotype was found both for viruses carrying CA from primary HIV-2 sequences and viruses carrying CA from laboratoryadapte

Genetic diversity of the human adenovirus species C DNA polymerase

International audienceBackground: Human Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. Methods: We assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. Results: We identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (+/- 6.2) and 5.2 (+/- 2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. Conclusions: Our study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments

Severe Pneumonia Associated with Adenovirus Type 55 Infection, France, 2014

International audienceHuman adenoviruses (HAdVs) comprise 70 recognized genotypes (as of February 15, 2016; http://hadvwg.gmu.edu/) and are frequently associated with mild and acute upper respiratory tract infections, depending on virus type and host immune status (1). HAdV type 55 (HAdV-55) has recently reemerged as a highly virulent pathogen, causing severe and sometimes fatal pneumonia among immunocompetent adults, particularly in Asia (2–4). Formerly known as HAdV-11a, HAdV-55 is a genotype resulting from recombination between HAdV-11 and HAdV-14 (5). We report 2 cases of severe pneumonia associated with HAdV-55 infection in Franc

A multi-disciplinary comparison of great ape gut microbiota in a central African forest and European zoo

We are grateful to the inhabitants and authorities in southeastern Cameroon for their warm welcome and their support of this study. We also acknowledge the invaluable assistance of the European zoo and its employees where we conducted the investigation. We also greatly appreciate the contributions of Olivia Cheny of the Center for Translational Science at the Institut Pasteur.International audienceComparisons of mammalian gut microbiota across different environmental conditions shed light on the diversity and composition of gut bacteriome and suggest consequences for human and animal health. Gut bacteriome comparisons across different environments diverge in their results, showing no generalizable patterns linking habitat and dietary degradation with bacterial diversity. The challenge in drawing general conclusions from such studies lies in the broad terms describing diverse habitats ("wild", "captive", "pristine"). We conducted 16S ribosomal RNA gene sequencing to characterize intestinal microbiota of free-ranging sympatric chimpanzees and gorillas in southeastern Cameroon and sympatric chimpanzees and gorillas in a European zoo. We conducted participant-observation and semi-structured interviews among people living near these great apes to understand better their feeding habits and habitats. Unexpectedly, bacterial diversity (ASV, Faith PD and Shannon) was higher among zoo gorillas than among those in the Cameroonian forest, but zoo and Cameroonian chimpanzees showed no difference. Phylogeny was a strong driver of species-specific microbial composition. Surprisingly, zoo gorilla microbiota more closely resembled that of zoo chimpanzees than of Cameroonian gorillas. Zoo living conditions and dietary similarities may explain these results. We encourage multidisciplinary approach integrating environmental sampling and anthropological evaluation to characterize better diverse environmental conditions of such investigations. Over the last decade, numerous studies have demonstrated the importance of environmental changes on the mammalian gut microbiome, which is strongly associated with host metabolic, immune, and neurological functions 1. Broad-ranging influences, including host genetics, living conditions, diet, stress, and antibiotic use can affect gut microbial diversity 2-5. Among these influences, diet and living conditions have been evaluated for human and animal populations, entailing significant effects on gut microbiota and consequences for human and animal health. Adverse microbial profile shifts, for instance, have been associated with dysbiosis and wide-ranging diseases among human beings, from obesity to pediatric environmental enteropathy, and from autism to asthma 2,6,7. Outside of laboratory conditions, disentangling the effects of living and dietary conditions on gut microbial composition from other influences remains a complex question. In humans, such questions have catalyzed multiple studies comparing environmental and gut microbiota between "westernized" and "rural" peoples 8-10. Among other mammalian populations, degradation in habitat quality affects the diversity of available flora and fauna for consumption, and in some cases, is associated with declines in microbial gut composition 11-14. Microbiome OPE

Ecological, parasitological and individual determinants of plasma neopterin levels in a natural mandrill population

International audienceInvestigating how individuals adjust their investment into distinct components of the immune system under natural conditions necessitates to develop immune phenotyping tools that reflect the activation of specific immune components that can be measured directly in the field. Here, we examined individual variation of plasma neopterin, a biomarker of Th1 immunity in wild mandrills (Mandrillus sphinx), who are naturally exposed to a suite of parasites, including simian retroviruses and malaria agents. We analyzed a total of 201 plasma samples from 99 individuals and examined the effect of sex, age, social rank, reproductive state and disease status on neopterin levels. We found higher neopterin concentrations in males than females, but were unable to disentangle this effect from possible confounding effects of retroviral infections, which affect nearly all adult males, but hardly any females. We further detected a non-linear age effect with heightened neopterin levels in early and late life. In addition, adult males that harbored very high parasitaemia for Plasmodium gonderi also showed high neopterin levels. There was no effect of social rank in either male or female mandrills, and no effect of female reproductive state. Taken together, these results indicate that plasma neopterin may prove useful to investigate individual variation in investment into specific immune components, as well as to monitor the dynamics of immune responses to naturally occurring diseases that elicit a Th1 immune response

Dynamics of cytomegalovirus populations harbouring mutations in genes UL54 and UL97 in a haematopoietic stem cell transplant recipient

We characterised by pyrosequencing, the dynamics of cytomegalovirus populations harbouring mutations A594V in gene UL97 and A834P and Q578H in gene UL54 in a haematopoietic stem cell transplant recipient. Unexpected re-emergence of A594V and decrease of A834P under CMX001 were shown to depend on both the selection pressure exerted by the antiviral treatments and the immune response

I223R mutation in influenza A(H1N1)pdm09 neuraminidase confers reduced susceptibility to oseltamivir and zanamivir and enhanced resistance with H275Y.

BACKGROUND: Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility. METHODS: The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0-15) and zanamivir (days 15-25 and 70-80). RESULTS: Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6-69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin. CONCLUSIONS: Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised