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Background Shallow shotgun metagenomics (SSM) has been recently suggested as a promising strategy to study human microbiota, providing nearly identical taxonomic profiles to deep shotgun metagenomics but at a sequencing cost as low as that of metabarcoding. To help clinical researchers determine whether shallow sequencing is appropriate for their projects, it is crucial to ascertain the accuracy of the information it provides, compared to deep sequencing. Here, we design a mapping-based workflow to build taxonomic profiles from SSM data and assess its accuracy at varying sequencing depths at both sample and cohort levels using extensive simulations and several public data sets. Results To identify genuinely present species and spuriously identified ones, we propose a novel data-driven filtering method based on machine learning techniques that largely outperforms basic filtering strategies based on pre defined thresholds, resulting in reliable taxonomic profiles at different sequencing depths, ranging from 50K to 10M reads/samples. Up to 90% of species with relative abundances higher than 4.10−4 were recovered correctly at 500K reads/sample showing that only information about rare taxa is lost at shallow depths. Furthermore, our results clearly show that SSM is able to correctly recover relevant biological signal from the confidently identified taxa, such as differences between groups of patients and diagnosis-like classification. Conclusions This study confirms that SSM is suitable for clinical research on human gut microbiota. We recommend that researchers should consider moving from 16S to SSM to limit biases in taxonomic profiles, or moving from deep to shallow sequencing, when functional analyses are not the main focus, to reduce costs and be able to include more patients in research projects.

Due to the role of macrophages in glomerular inflammation, macrophage surface molecules such as CD163 and CD11b represent attractive biomarkers for monitoring Lupus Nephritis (LN). We hypothesize that their urinary levels may reflect kidney disease activity. Here, we first analyzed the levels of urinary soluble CD163 (U-sCD163) and CD11b (U-sCD11b) in a cohort of 40 patients with LN including 23 with active disease. U-sCD163 levels were significantly elevated in active LN and correlated with the renal activity score, in contrast to U-sCD11b. Then, we developed the pristane induced LN mouse model to analyze, in a longitudinal way, the evolution of U-sCD163 levels according to the glomerular inflammation progression and response to treatment. We showed an increase of U-sCD163 levels associated with glomerular immune-complex deposits on mouse kidney biopsies at an early stage of the disease and a correlation with inflammatory markers including interferon-α, C-reactive protein and tumor necrosis factor-α. In addition, we showed that U-sCD163 levels decreased following efficient hydroxychloroquine treatment. Altogether our results led us to conclude that U-sCD163 represent a non-invasive biomarker for LN reflecting glomerular inflammation. Although prospective study in patients with LN, active or not, are necessary, U-sCD163 could be proposed to monitor response to treatment.

Breath testing has become a widely used tool to diagnose small intestinal bacterial overgrowths (SIBO) and intestinal methanogen overgrowths (IMO) in clinical settings. Due to the heterogeneity in clinical manifestations and lack of standardization among centers performing breath testing, SIBO and IMO can be easily overlooked by the clinician.We studied the prevalence and symptoms of SIBO/IMO in French patients referred for breath testing after seeking medical advice.Breath test data and symptoms of 331 patients were assessed for SIBO/IMO using the H2/CH4 lactulose breath test (LBT). Wilcoxon test or χ2 test were used to compare patients with SIBO/IMO to patients without SIBO/IMO. LBT positive patients (H2+, CH4+, and CH4+/H2+) were compared using Kruskal-Wallis test for continuous data or χ2 test for categorical data.Among the 186 (68.1%) patients tested positive for an overgrowth with respectively 40.3%, 47.3%, 12.4% for H2+, CH4+ and CH4+/H2+, presence of diarrhea was significantly increased in hydrogen type overgrowths (p0.001). No significant difference according to age, gender and symptoms was associated with a positive test except for joint pain that was less prevalent among LBT positive patients (p=0.038). In 86.5% of IMOs, positivity with CH4 values ≥10ppm could be identified at baseline.There are little discriminating symptoms that can help the clinician to identify patients likely to have a SIBO/IMO. However, SIBO/IMOs remain a common disorder widely underdiagnosed that need further studies to better apprehend functional bowel disorders.

Background & aim Screening for genital infection (GI) such as bacterial vaginosis (BV) and yeast infection, for sexually transmitted infection (STI), and for asymptomatic carriage of group B streptococcus (GBS) in pregnant women are common reason for medical appointments. The diagnosis and control of GIs, STIs, and GBS are major issues, for fertility and overall well-being of affected women. Conventional testing is performed using vaginal/cervical classical sampling (VCS); this procedure requires pelvic examination performed by health care professionals which raises concerns among women. Vaginal-self-sampling (VSS), as an alternative to VCS, might capture more women. The aim was first to show non-inferiority of VSS compared with VCS to screen for GIs, STIs, and GBS; second to determine the feasibility of VSS. Methods VSS and VCS from 1027 women were collected by health care professionals and simultaneously carried out on each patient. GIs, STIs, and GBS were systematically screened in both paired VSS and VCS samples. Non-inferiority of VSS compared with VCS was assessed using z statistic for binomial proportions. Results Prevalence of GIs were 39.7% using VSS and 38.1% using VCS (p = 0.0016). Prevalence of STIs was 8.5% (VSS) vs 8.1% (VCS) (p = 0.0087). Prevalence of GBS was 13.4% (VSS) and 11.5% (VCS) (p = 0.0001). Most participants (84%) recommended the use of VSS. Conclusions This study shows that VSS was not inferior to VCS for the detection of GIs, STIs, and GBS. This study provides evidence that VSS can be used as a universal specimen for detection of lower genital tract infections in women. Study identification number ID-RCB 2014-A01250-4.

Background: Microbiotas play critical roles in human health, yet in most cases scientists lack standardized and reproducible methods. To date, stool sample preservation remains a source of technological bias in metagenomic sequencing, despite newly developed storage solutions. Studying those biases will help in identifying the appropriate stool preservation approach to promote the implementation of standard operating procedure and improve comparability across multiple microbiome studies. Results: Here, we conducted a comparative study of 10 storage methods for human stool over a 15-day period of storage at fluctuating temperatures. We first compared the performance of each stabilizer with observed bacterial composition variation within the same specimen. Then, we identified the nature of the observed variations to determine which bacterial populations were more impacted by the stabilizer. We found that DNA stabilizers display various stabilizing efficacies and affect the recovered bacterial profiles. Furthermore, our results showed that the bias associated with the stabilizers can be linked to the phenotypical characteristics of the bacterial populations present in the studied samples. Conclusions: Although newly developed storage solutions have improved our capacity to stabilize stool microbial content over time, they are nevertheless not devoid of biases. We therefore recommend considering the nature of these technological biases before claiming whether some microbes are beneficial or even deleterious to human health.

High-risk human papillomaviruses (HPVs) have been identified as the main contributors to cervical cancer. Despite various diagnostic tools available, including the predominant Papanicolaou test (Pap test), technical limitations affect the efficiency of cervical cancer screening. The aim of this study was to evaluate the diagnostic performance of spliced HPV16 E6/E7 mRNA viral loads (VL) for grade 2 or higher cervical intraepithelial neoplasia diagnosis. A new dedicated (quantitative reverse transcription polymerase chain reaction) qRT-PCR assay was developed, allowing selective quantification of several HPV16 E6/E7 mRNA: Full length (FL) with or without all or selected spliced forms (total E6/E7 mRNA corresponding to SP + E6^E7 mRNA (T), + spliced E6/E7 mRNA containing intact E7 ORF (SP), and E6/E7 mRNA containing disrupted E6 and E7 ORFs calculated by the following subtraction T-SP (E6^E7)). Twenty HPV16 DNA and mRNA positive uterine cervical smears representative of all cytological and histological stages of severity were tested. We have shown that all E6/E7 mRNA isoforms expression levels were significantly increased in high grade cervical lesions. Statistical analysis demonstrated that the SP-E6/E7 VL assay exhibited: (i) The best diagnostic performance for identification of both cervical intraepithelial neoplasia (CIN)2+ (90% (56&ndash, 100) sensitivity and specificity) and CIN3+ (100% (72&ndash, 100) sensitivity and 79% (49&ndash, 95) specificity) lesions, (ii) a greater sensitivity compared to the Pap test for CIN2+ lesions detection (80% (44&ndash, 97)), (iii) a predictive value of the histological grade of cervical lesions in 67% of atypical squamous cells of unknown significance (ASC-US) and 100% of low-grade (LSIL) patients. Overall, these results highlight the value of SP-E6/E7 mRNA VL as an innovative tool for improving cervical cancer screening.

Replication of the hepatitis C virus (HCV) is an error-prone process. This high error rate results in the emergence of viral populations (quasispecies) within hosts and contributes to interhost variability. Numerous studies have demonstrated that both viral and host factors contribute to this viral diversity, which can ultimately affect disease outcome. As the host’s immune response is an important correlate of infection outcome for HCV, many of these viral variations are strongly influenced by T-cell immune pressure and accordingly constitute an efficient strategy to subvert such pressures (viral adaptations). This paper will review the data on viral diversity observed between and within hosts infected with HCV from the acute to the chronic stage of infection and will focus on viral adaptation to the host’s T-cell immune response.

Genetic and cellular studies have shown that the host's innate and adaptive immune responses are an important correlate of viral infection outcome. The features of the host's immune response (host resistance) reflect the coevolution between hosts and pathogens that has occurred over millennia, and that has also resulted in a number of strategies developed by viruses to improve fitness and survival within the host (viral adaptation). In this review, we discuss viral adaptation to host immune pressure via protein-protein interactions and sequence-specific mutations. Specifically, we will present the "state of play" on viral escape mutations to host T-cell responses in the context of the hepatitis C virus, and their influence on infection outcome.