Nineteen neurologically asymptomatic, aviremic, HIV-positive subjects, underwent multi-voxel 2D MRS on a 3 T MR unit and synchronous neurocognitive assessment in a five-year follow-up period. Twelve voxels were placed in prefrontal cortices, anterior and posterior cingulate gyrus, intraparietal sulci, and frontal centrum semiovale white matter, to identify peaks of N-acetyl-aspartate (NAA), creatine (Cr), choline (Cho), and myoinositol (mI). Ratios of NAA/Cr, NAA/Cho, NAA/mI, mI/Cr, and Cho/Cr were analyzed. Longitudinal differences in ratios and neurocognitive scores were tested with the Wilcoxon signed-rank-test. Statistical significance was set at p ≤ 0.004 significant, and 0.05 > p > 0.004 trending toward significance. A significant longitudinal increase in NAA/Cr ratio was observed in 5/12 voxels, while there was a trend toward significance in an additional three. The increase in Cho/Cr reached statistical significance in one voxel. Changes in the mI/Cr ratio demonstrated a significant increase in 4/12 voxels. A progressive increase in NAA/Cr, followed by better neurocognitive performance, may be an indicator of brain plasticity in the setting of chronic HIV-related neuronal injury. A progressive mI/Cr increase could be partly explained by glial proliferation due to functional compartment remodeling and partly attributable to insufficient control of persistent neuroinflammation by cART.There is a lack of data about the long-term follow-up changes in neurometabolic profile and neuropsychological performance of HIV-positive subjects under continuous antiretroviral therapy (cART). The aim of the study was to assess changes in neurometabolic profile in chronically-infected, HIV-positive subjects during a five-year follow-up period, using multi-voxel proton magnetic resonance spectroscopy ( Although no signs of neuronal injury can be detected on magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) in the early phase of HIV transmission, there are already prominent signs of ongoing inflammation. This inflammation is reflected by elevated levels of myoinositol (mI), a marker of microglial proliferation, and choline (Cho), a marker of membrane metabolism and gliosis. Soon after the initiation of cART, in the absence of neuronal dysfunction, normalization of inflammation markers can occur in several weeks1. The initial increase in inflammation markers will decline after cART initiation, although they do not reach the level of healthy controls3. Further in the course of disease, a decline in N-acetyl aspartate (NAA), a neuronal marker, occurs and becomes the dominant finding in chronically infected HIV-patients4.Several longitudinal studies have shown changes in the biochemical profile of the brain in human immunodeficiency virus (HIV) infection, depending on the phase of infection and the introduction of combination antiretroviral therapy (cART)2. The neurodegenerative process associated with long-standing, well-controlled HIV infection results in progressive neurocognitive dysfunction5. The explanation for this process is thought to be due to chronic neuroinflammation and persistent immune activation, driven by an uneradicated pool of HIV-particles6.In the first MRS study performed in our institution on 110 subjects (60 HIV-positive patients and 50 age- and gender-matched controls), the results clearly showed that HIV-associated neurodegeneration affects the whole volume of the brain. Furthermore, the results of that study, as well as other, smaller studies, confirmed ongoing inflammation under cART1H MRS). In addition, a relationship between longitudinal changes in neurocognitive performance and MRS findings was investigated.In this longitudinal study, our aim was to assess longitudinal changes in the neurometabolic profile of neurologically asymptomatic HIV-positive individuals treated with cART during the follow-up period of five years, using multi-voxel proton magnetic resonance spectroscopy . Plasma viral load remained undetectable during the follow-up period in all patients. Participants underwent standard magnetic resonance imaging (MRI) and an MRS study on a 3 T MR unit , in the follow-up period of five years . Demographic and basic clinical data for both time points  are summarized in Table Inclusion criteria for the follow-up study were: completion of two MR examinations and synchronous neuropsychological testing; chronic HIV infection at the baseline (over one year after transmission); stable on cART (over one year after initiation); no signs of manifest neurocognitive disorder at baseline; and a normal conventional MRI. Exclusion criteria were: detectable viral load over the follow-up period (>50 copies/mL), current or past confounding neurological disease ; brain tumors; traumatic brain injury; major psychiatric disorders; cardiovascular diseases ; active abuse of narcotic drugs ; and hepatitis B or C coinfection at the baseline and over the follow-up period.All selected patients were compliant with therapy, without any changes in the regimen of cART over the observed period of time. Three patients were excluded from follow-up imaging due to a change in the cART regimen (transient neuropsychiatric symptoms presented due to the introduction of efavirenz). A CONSORT study diagram showing patient selection in detail is shown in Fig. All subjects signed written, informed consent.Imaging was performed in 2011 and 2016, using an eight-channel head array coil. The MRI protocol consisted of a three-planar, multi-sequential study: sagittal T1-weighted spin echo ; axial T2-weighted turbo spin echo ; FLAIR (FLuid Attenuation Inversion Recovery) ; and coronal T2-weighted turbo spin echo  MR tomograms.Proton multi-voxel intermediate-  and short-echo-time  MRS data sets were acquired using a two-dimensional (2D) PRESS (Point RESolved Spectroscopy) technique, with automatic volume-selective shimming. Features of the spectroscopic slab were: volume of interest 80 × 80 × 10 mm; thickness 10 mm; and region of interest positioned in the supracallosal white and gray matter. The multi-voxel network consisted of 64 voxels, of which we chose 12 symmetrical voxels in both hemispheres:1, 2: the grey matter of the prefrontal cortex (PFC);3, 4: the ventral part of the anterior cingulate gyrus (ACG);5, 6: the frontal centrum semiovale white matter (FCSWM);7, 8: the dorsal part of the ACG;9, 10: the intraparietal sulcus (IPS); and11, 12: the posterior cingulate gyrus (PCG)  by two experienced neuroradiologists. The post-processing protocol consisted of baseline corrections, peak identification, and metabolite ratio calculations.In each subject, two experienced neuroradiologists in consensus, selected the voxels that best covered the primary regions of interest (ROI), based on their location on the matching images (high-resolution). The investigators were blinded to the time of acquisition of the data. The mean of the peak area for observed metabolites were computed form fitted spectral data.The peaks identified were: NAA; Cho; mI; and total creatine (Cr), used as a reference marker. Ratios of NAA/Cr and Cho/Cr on intermediate-echo time and mI/Cr on short-echo time were analyzed.7; the Trial making test form A and B 8; the Rey auditory-verbal learning test (RAVLT)9; the Rey-Osterrieth complex figure test (ROCF)10; the Verbal fluency test-phonemic and categorical fluency tests11; the Verbal and visuospatial memory span12; the Wisconsin Sorting Card test13; and the Beck depression inventory scale II14. The same experienced clinical psychologist conducted all the neuropsychological testing. The study was conducted in compliance with the Helsinki Convention principles, was ethically approved, and every subject signed a written consent to participate in the research.Synchronous follow-up neurocognitive testing was performed at the same time points as MRS. All participants underwent a 90-minute neuropsychological test battery that assessed multiple neurocognitive functions typically found to be impaired in HIV-positive subjects. This battery consisted of: the Mini Mental Score Examination (MMSE)Statistical analysis was performed using IBM SPSS software . Descriptive statistics included mean values, standard deviation, median, minimum and maximum for continuous and median values, range (minimum-maximum), and interquartile range (IQR) for variables that did not follow a normal distribution.Metabolite ratios of NAA/Cr, Cho/Cr, and mI/Cr that were obtained on the MRS scans in 2011 and in 2016 were compared using the Wilcoxon signed-rank test for repeated measurements. Due to the suspected instability of Cr, we performed longitudinal analysis calculations of NAA/Cho and NAA/mI ratios, in order to exclude the influence of Cr on metabolite ratios. The results of follow-up neurocognitive assessments performed at the same time as MRS were compared using the same test. We used this non-parametric test used because of the small sample size. For the same reason, we used Bonferroni corrections, designating p-values equal to or below 0.004 as significant , and 0.05 > p > 0.004 as indicative of a trend toward significance.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.A significant rise in the NAA/Cr levels in 5/12 analyzed voxels was observed in the following locations . However, in several additional voxels, we observed an increase in Cho/Cr levels that trended toward statistical significance in the left PFC p = 0.013), in the left FCSWM (p = 0.005), and in the left (p = 0.007) and right IPS (p = 0.009) , the right dorsal ACG (p = 0.001), the right IPS (p < 0.001), and the right PCG (p = 0.001). In the left IPS (p = 0.029), the increase trended toward significance. In the left dorsal ACG, and in the right and left FCSWM, we confirmed a decline in mI/Cr , but this only trended toward significance Table .Table 4LIn the right frontal ACG (p < 0.001), the left PFC (p < 0.001), the left FCSWM (p < 0.001), the right IPS (p = 0.001), and the right PCG (p = 0.001), we observed a significant increase in the NAA/Cho ratio, while, in the right PFC and the left PCG, the increase trended toward significance . However, in the right FCSWM and the dorsal ACG on the same side, a significant decline in this ratio was observed , as well as a decrease in the left PCG (p = 0.009) that trended toward significance.In the right ventral ACG, the left FCSWM, the right IPS, and the right PCG , a significant increase in NAA/mI was observed. A decrease in the NAA/mI ratio was significant in the right FCSWM (p = 0.003) and in the right dorsal ACG that trended toward significance (p = 0.009).In the five-year follow-up period, there was a trend toward a better performance for functions of delayed recall and recognition of verbal material , for tests of visuospatial orientation abilities (ROCF), and for visuospatial span  Table . The res1. Despite the clear signs of neuroinflammation in the early phase of HIV infection, no signs of a neurodegenerative process have been observed3. Prior to the cART era, Chong et al. were the first to report a progressive reduction of the NAA level in HIV-positive subjects in a short follow-up period (three to eight months)15. Furthermore, based on later longitudinal studies, long-term control of both processes, neuroinflammation and consequent neurodegeneration, seemed to be suboptimal despite cART and good peripheral viral suppression16.Short-term longitudinal changes on MRS in the HIV-infected brain are well documented, and show a reduction of immune activation and inflammation in the brain parenchyma after the introduction of cART4.To the best of our knowledge, this is the first five-year follow-up longitudinal MR spectroscopic study on chronic, neurologically asymptomatic, aviremic HIV-positive patients, stable on cART (>one year at the baseline), with a normal MRI. According to the published data on neuropathogenesis of HIV infection and the suboptimal efficacy of cART in the brain, we expected to find signs of further, steady HIV-associated neuronal damage despite the reduction of an ongoing inflammatory process in patients with undetectable plasma viremiaSurprisingly, the results showed the expected, ongoing, low-level inflammation (reflected by a mI/Cr increase), and a clear long-term increase in NAA/Cr, suggesting the possibility of partial functional brain remodeling under cART.17. The increase in NAA is rather an unusual finding; transiently, it occurs after acute brain injury, whereas a stable increase has been reported in moyamoya disease, amyotrophic lateral sclerosis, and Wernicke encephalopathy19. In all these conditions, the NAA increase suggested brain plasticity and partial functional recovery. Cysique et al. recently described longitudinal decrease in NAA in PCG and frontal white matter in HIV-positive persons with HAND (both mild neurocognitive disorder and asymptomatic neurocognitive disorder). However, stable intact subjects showed NAA levels closest to healthy controls, implying that HIV-positive persons with normal cognition are less affected by neurocognitive decline and decline in NAA levels in these two locations20. In our study sample, we observed not only that these levels did not decrease in 5-year period, but in some locations, they increased, which might speak in favor of plasticity in the light of concomitant neuropsychological testing.The development of HIV-associated neurodegeneration might be more complex than merely a pure consequence of a continuous and uncontrolled inflammatory process in the brain parenchyma. An increase in the NAA/Cr level was observed in almost all analyzed voxels in our study Fig. , with noet al., conducted as a two-year follow-up MR volumetric study, showed no significant volume loss or reduction of cortical thickness in aviremic, stable, HIV-positive patients on cART, compared to HIV-negative controls21. The results and the patient selection criteria are in accordance with our findings.A recent study by Sanford 3. However, all these patients were symptomatic with severe neurocognitive disorders. This was not true for our patient population.The fact that no statistically significant longitudinal changes in Cho/Cr were detected and statistical significance was reached in only one location  in our study indicates good, long-term control of neuroinflammation under cART. In recent studies in chronic HIV-infection, a progressive decline in Cho/Cr was observed over time and explained as a probable reflection of tissue atrophy and a loss of brain parenchyma1. Sailasuta and Young showed a reduction in mI/Cr soon after cART initiation; however, the levels of healthy controls were never reported3. Our finding of a dominant and stable increase in mI/Cr levels indicates a continuous microglial proliferation, obviously insufficiently controlled by highly penetrative cART. In addition, a neurotoxic effect from long-standing cART might also be implied, concordant with a recent study by Winston et al.22.A transient increase in mI/Cr was observed in acute (up to eight weeks after transmission) and primary HIV infection (up to six months after transmission)The increase in both Cho/Cr and mI/Cr ratios in our study may represent ongoing low-level inflammation and cell proliferation triggered by the presence of uneradicated HIV virus and its particles in the brain. This further suggests a suboptimal performance of cART in the brain, especially in the regions that have a significant number of microglial cells. This might explain the stable prevalence and incidence of milder forms of HIV-associated cognitive disorder in patients with cART.23. A rather global increase in NAA/Cr was observed in our five-year follow-up study. Nevertheless, the increase in mI/Cr was seen only in some locations . This raises the question of whether certain brain regions are more susceptible to HIV injury due to complex pathways of HIV-associated neurodegeneration. Microglial cells are not equally distributed in the brain, with a variable density in the cerebral cortices, the brain stem, and the fiber bundles. Banati observed that persistent subtle microglial activity modulates neuronal functioning directly or indirectly, via astrocytic interaction. Microglial activation mobilizes functional compartments in which signaling molecules  imply a more open cell-to-cell communication, not present in the healthy adult brain. These communications occur in neuronal pathways where this glial activation is under the influence of specific brain pathology, independent of disease etiology. This forms the framework for functional remodeling in the brain24.Since the first studies on HIV-related brain injury, the diffuseness of the neurodegenerative process has been emphasized25. Thus, microglial proliferation and membrane metabolism may be increased in those regions of the brain that constitute the brain circuits involved in attention and working memory, immediate and delayed recall, and visuospatial abilities, as a consequence of functional compartment remodeling in response to HIV-related chronic neuronal injury. The demonstrated increase in neurobiochemical markers could represent proliferation of radial glial cells that lead neuronal migration to the site of injury. The dynamics of neurobiochemical changes observed in the locations analyzed in this study were supported by the results obtained in some neurocognitive tests. Namely, we observed better performance for the functions of immediate and delayed recall, regulated mostly by the dorsal ACG and parts of the PFC27. Dysfunction in connections to the ACG results in apathy, impulsivity, and disinhibition - behavioral changes that occur in neurodegenerative diseases. Finally, we observed better achievement in visual attention and working memory, regulated by many networks in the brain, mainly PCG28. It comes as no surprise that previous studies on HIV-positive subjects confirmed the most prominent reduction in NAA/Cr levels exactly in this region. In light of these findings, a question could be raised about the functional remodeling of the brain pathways involved in cognition, predominantly in attention/working memory, visuospatial abilities, and delayed recall and recognition of verbal material. Sanford et al., in a recently published two-year follow-up study, showed better interval performance on several neurocognitive tasks, especially TmtA, concordant with our study. The authors commented on the result as a probable beneficial effect of the on-time introduction of cART and long-standing stable aviremia21.During pathological process in the brain, the proliferation of microglia forms a network with regional differences that are not random, but functionally dependent29, or changes in the brain synthesis of Cr.The major limitation of the present study is the small number of participants, partially due to strict inclusion criteria. However, we performed a power analysis calculation for all three metabolite ratios. For NAA/Cr levels, the power analysis showed a satisfactory sample size (0.84–0.99), so that differences in NAA/Cr levels can be considered relevant despite a small sample size. The results of the power analysis for Cho/Cr and mI/Cr levels also showed a satisfactory sample size . Additional limitation is the partial volume effect of the cerebrospinal fluid, white matter or blood vessels) that can be present in some voxels that include mostly grey matter, such as voxels placed in cingulate gyrus. However, investigators that performed the MRS data processing controlled for this contamination and selected only spectra that best covered the ROIs with the grey matter. Finally, a significant issue might be the use of metabolite ratios instead of absolute values, given that the level of Cr is unstable in chronic HIV infected population, due to decreased uptake in the gutIn summary, an unexpected longitudinal increase in NAA/Cr in neurologically asymptomatic HIV-positive patients on cART, observed on multivoxel MRS, followed by better performance on neurocognitive tests with regard to attention/working memory, delayed recall, recognition and visuospatial abilities, suggests functional remodeling of brain circuits in HIV-related neurodegeneration. Increased microglial proliferation and membrane metabolism might be implied in cell-to-cell communication and functional remodeling as a response to chronic neuronal injury. However, the latter may also reflect persistent inflammation and immune activation, probably due to the suboptimal efficacy of cART in the brain. Regional differences in of mI/Cr increases suggest that some brain regions are more prone to inflammation, either due to anatomical or neurophysiological features.


Drug-resistant tuberculosis is a global health problem that hinders the progress of tuberculosis eradication programs. Accurate and early detection of drug-resistant tuberculosis is essential for effective patient care, for preventing tuberculosis spread, and for limiting the development of drug-resistant strains. Culture-based drug susceptibility tests are the gold standard method for the detection of drug-resistant tuberculosis, but they are time-consuming and technically challenging, especially in low- and middle-income countries. Nowadays, different nucleic acid-based assays that detect gene mutations associated with resistance to drugs used to treat tuberculosis are available. These tests vary in type and number of targets and in sensitivity and specificity. In this review, we will describe the available molecular tests for drug-resistant tuberculosis detection and discuss their advantages and limitations. Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). In low and middle income countries, DST accessibility is limited by its high cost and complexity and by technical constraints . Such difficulties could result in the transmission and emergence of highly DR-MTB strains due to inappropriate treatments.Drug resistance is a major challenge for tuberculosis (TB) treatment and eradication. The number of drug-resistant TB (DR-TB) cases is increasing worldwide: the estimated number of new cases of multidrug-resistant TB  or rifampicin-resistant TB (RR-TB) was 600,000 in 2016 compared with 480,000 in 2014 ,b. The tTM) are barcoded for the automated processing of large numbers of samples (up to 960 tubes)1 with a turn-around time between 10 and 30 days  (rpoB show the highest frequency and confer high-level RIF resistance. Isoniazid (INH) resistance is acquired through mutations in the katG, inhA and its promoter, ahpC, ndh, and furA genes but mainly in katG, inhA and its promoter (katG mutations (50–90%) are found at codon 315 and confer high-level resistance to INH. In ethambutol (EMB)-resistant isolates, mutations at codon 306 of the embB gene are the most frequent (47–62%). Mutations associated with resistance to second-line drugs also have been described. In streptomycin (STR)-resistant MTB isolates, the most frequent mutations are found at codons 43 and 88 of the rpsL gene, and codon 514 of the rrs gene , kanamycin (KAN), and capreomycin (CAP), is mainly associated with rrs gene mutations. Approximately 70–80% of CAP resistance and about 60% of KAN resistance are caused by the rrs A1401G mutation. Although the mechanisms of drug resistance is not clear in about 10–40% of DR-MTB isolates without mutations, the detection of known mutations enables to identify a high proportion of DR-MTB , in the circular chromosome . For eacfor RIF) . Mutatiopromoter . The mosrrs gene . Resista, and 88 . Mutatiof DR-MTB .Therefore, many nucleic acid-based assays that detect mutations associated with anti-TB drug resistance have been developed recently, in order to provide affordable, accurate, simple, and rapid diagnostic tests for DR-TB detection. In this review, we will describe the commercially available molecular tests for DR-TB detection and discuss their advantages and limitations (see 2 (Line probe assays (LPAs) are basically DNA–DNA hybridization assays that allow the simultaneous detection of different mutations by using multiple probes2 . After D2 .rpoB hotspot region  for MTB identification and RIF resistance screening ; and module 3 analyzes gyrA and embB for FQ and EMB resistance detection. The three modules include wild type and mutant probes to cover the most common mutations. The AID TB Resistance LPA showed high sensitivity and specificity for the detection of RIF, INH, STR, FQs, and second-line injectable agents resistance (between 90 and 100%), but lower sensitivity for EMB resistance (72.9%)  analyzes only the creening . The AIDcreening  includesening (3 . Module  (72.9%) . However (72.9%) .plus, GenoType MTBDRsl  that cause the human disease  (sl VER2.0 displays high sensitivity and specificity (between 91 and 100%) for detecting FQ resistance, but variable sensitivity and specificity for the screening of resistance to second-line injectable drugs (SLIDs) (sl VER2.0 specificity (between 59 and 100%) and sensitivity (between 83 and 87%) for detecting XDR isolates differ among studies. Moreover, uninterpretable results were reported for drug resistance screening in sputum specimens, especially smear-negative samples (sl (both VER1.0 and VER2.0) is used for FQ resistance screening, some synonymous and non-synonymous mutations  in the gyrA gene prevent the hybridization of either the wild type or mutant probe, leading to false-resistance results. Although these mutations  are not frequent, they account for around 7% of all MDR-TB strains in some studied regions (GenoType MTBDRnd 100%) . The Nipnd 100%) . Regardi (SLIDs) . Therefo samples . In addi regions .In conclusion, LPAs are rapid, simple and easy to perform. Result analysis  is simple. However, LPAs require complex laboratory infrastructure and expensive equipment that is normally only available in reference laboratories . The num6. Each probe has a specific melting temperature (Tm), and a Tm change reflects the presence of mutations in the target. This feature has been used to develop real-time PCR tests for drug resistance screening.Real-time PCR is now broadly applied for the development of rapid diagnostic tests. Two main approaches are commonly used in real-time PCR: (i) the use of non-specific fluorescent dyes to detect any double-stranded DNA generated by PCR amplification, and (ii) the use of sequence-specific probes tagged with a fluorescent reporter for the specific detection of the hybridization between probes and amplicons5-fold excess of human DNA does not affect the results with SMB probes  and the shorter turn-around time (compared with LPAs because of the absence of the hybridization step) are among the advantages of real-time PCR. However, real-time PCR has some remarkable disadvantages. First, it requires expensive, specific equipment and skilled technicians. Second, the number of probes that can be used in one reaction is limited, resulting in limited target numbers. This is a major drawback for MTB, a pathogen that can harbor many mutations. Third, the size range of the amplified targets should be limited to 75–200 bp for efficiently detecting multiple mutations (see text foot note 6).The following sections present some examples of real-time PCR-based commercial kits and new strategies for DR-TB detection.Xpert MTB/RIF . This terpoB gene (codons 507–533) that is then hybridized to five molecular beacon probes . The platform can detect as low as five genome copies. It is user-friendly and fast (results in 75 min). Although the Genedrive system can analyze only eight samples per working day9, the low price ($4000 for the system and $10–$17 for the disposable cartridge) makes the assay affordable for low-income regions  and tagging-oligonucleotide cleavage and extension (TOCETM) and the real-time PCR CFX96TM apparatus for the highly specific detection of specific SNPs. The Anyplex kits can identify multiple targets in one reaction because the PCR CFX96TM system allows the simultaneous detection of five different fluorescent dyes. Specifically, Anyplex II MTB/MDR detects the 34 most frequent mutations in rpoB, katG, and inhA for MDR-TB screening, and the MTB/XDR kit detects the 13 main mutations in the gyrA and rrs genes and eis promoter associated with XDR-TB (see text foot note 10) (The Anyplex kits (note 10) . The entnote 10) .sl , an asymmetric PCR method with sets of Lights-On/Lights-Off probes, has been developed to analyze nucleotide substitutions . In eachrpoB, katG, and inhA in one single tube11. The kit includes probe pairs to target a set of known mutations in these genes , and can also detect unknown mutations, but not identify the exact type of mutation   are amplified  and sequenced. The AmpliSeq for Illumina TB Research Panel (Illumina) targets the same genome regions (see text foot note 12). Besides, WGS is now only valuable for cultured strains due to its requirement in terms of quantity and quality of DNA , the development of Next Generation Sequencing (NGS) has been a major breakthrough in molecular biology because it can rapidly provide whole genome data in a single run . This alne (PGM) . Eight gy of DNA . Althougy of DNA . The Oxfy of DNA . Though,The large-scale application of sequencing, especially in middle- and low-income countries is still difficult for the following reasons: (i) robust software and database tools need to be developed for the full exploitation of this technology in this specialized area of medicine; (ii) specialized personnel and bioinformatics facilities are required for the experiments, data acquisition and data analysis; (iii) the high cost of NGS platforms; (iv) need to determine whether new mutations confer anti-TB drug resistance; and (v) high amounts of high quality DNA are required for sequencing . Moreove13. Currently, these platforms are only available for surveillance and research use. Ultimately, they will be interoperable and transferred to the WHO to support global DR-TB surveillance in the short term and clinical diagnosis of DR-TB in the long term  is another kit based on a multiplexed PCR-microarray-based method and microfluidic chamber to detect MDR-TB, MTB and nine other Mycobacterium species15. Interestingly, embB gene and 12 probes for identification of the main lineages circulating in Russia. By using two separate asymmetrical PCRs and universal adapters, this array covers nine genes including rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, and embB genes and six SNPs for lineage identification. The turn-around time is about 19 h (3 h for PCR and 16 h for hybridization), that is quite long compared to other molecular-based methods but detecting both MDR-TB and XDR-TB is the greatest advantage of this microarray. The sensitivity and the specificity of TB-TEST are identical between clinical samples and clinical isolates. This microarray has high potential to apply directly in clinical samples.To simplify the numerous steps of the microarray-based workflow, 14 , and to 16.Currently, no microarray-based assay has been endorsed by WHO for the detection of DR-TB . Most teOverall, DNA microarray-based tests show high specificity and sensitivity for the detection of mutations associated with anti-TB drug resistance in clinical isolates: 100% sensitivity and specificity for INH and RIF detection ; and 90 DNA microarray could become an effective method for the rapid screening of MTB resistance. However, additional improvements, such as dedicated software to analyze and interpret the fluorescent signal intensity, are necessary to make it accessible to all TB point-of-care sites and promote its application in the clinical routine.Many molecular tools for DR-TB diagnosis have been developed and are employed worldwide. However, there are still many challenges and issues that need to be solved to provide efficient and affordable point-of-care diagnostic tests.Technically, most of the tests only focus on one or few genes, hotspot genomic regions, or frequent mutations . TherefoatpE, Rv0678, and pepQ. However, it is only a non-exhaustive list because a standardized DST for bedaquiline does not exist yet, and not all resistance genotypes are known. Due to MTB high capacity of drug resistance acquisition, the development of a standardized DST should be started simultaneously with the evaluation of a new drug in clinical trials. Thereby, DST would be available when the new drug will be introduced in treatment regimens. However, it is a long and difficult path. For instance, although pyrazinamide has been used for many years, the standardized DST for this drug is still “work in progress” due to its current not satisfactory accuracy and reproducibility  and the personnel skills required for running the test. Furthermore, to have a real impact, molecular diagnostic tests should be affordable for resource-poor countries, where TB and DR-TB are major problems. The turnaround time should be as short as possible to quickly prescribe the adapted treatment to the patients. As DR-TB is associated with mutations in different genes, an ideal test should simultaneously detect many mutations in one reaction. A simple testing procedure will increase the test accessibility at different laboratory levels.All authors contributed to the review, manuscript writing, critical review of the manuscript, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metabolic risk factors are poorly documented for the first generation of young adults who have lived with HIV since childhood. We compared their metabolic profile with that of adults of same age from the general population.We conducted a cross-sectional analysis of data from two populations: (1) COVERTE (ANRS-CO19), a French national cohort of 18 to 30-year-old patients HIV-infected since childhood, and (2) ENNS, a national cross-sectional population-based household survey on nutrition. Body mass index (BMI), blood pressure, waist circumference, fasting glucose, triglycerides, and HDL-, LDL- and total cholesterol were measured in both studies. Direct standardization on overweight and education level and logistic regression were used to compare the prevalence of metabolic abnormalities between the two populations.Data from 268 patients from COVERTE and 245 subjects from ENNS were analyzed. Tobacco use was similar in both groups. HIV-infected patients had increased mean waist-to-hip ratio and triglycerides to HDL-cholesterol ratio and decreased mean HDL-cholesterol as compared to their counterparts from the general population in both genders. In HIV-infected patients, metabolic syndrome was identified in 13.2% of men  and 10.4% (95% CI: 5.4–15.3) of women versus 10.6% (95%CI: 1.5–19.7) and 1.7% (95%CI: 0–4.1) in subjects from the general population, respectively.Young adults infected with HIV since childhood had a higher prevalence of dyslipidemia and metabolically detrimental fat distribution than adults of same age of the general population, supporting close monitoring for cardiometabolic diseases. Cardiovascular diseases are an important cause of non-AIDS-related morbidity and mortality in HIV-infected patients. They are the third cause of death of HIV-infected patients after AIDS events and hepatic diseases . In a prFew studies have examined the cardiovascular risks in the first generation of young adults living with HIV since birth or childhood. This is an emerging population, due to the improved prognosis of HIV infection treated with powerful ART. The cardiometabolic risk of HIV-infected patients has significantly declined over the last few years, due to broadened treatment indications, use of less toxic antiretrovirals, and better monitoring , but car. However, these studies were of small sample size and/or did not include non HIV-infected control groups.A study conducted in the United States reported that coronary artery vessel wall thickness was significantly higher in young adults aged 15–29 years who acquired HIV early in life than in sex- and race-matched uninfected controls, indicating that this population is likely to present subclinical coronary vascular disease . In addiThe French national cohort of young adults living with HIV since childhood was designed to study their living conditions and long-term prognosis. The availability of a national population-based survey on nutrition provided the opportunity to compare their metabolic profile to that of the adults of the same age living in France.The ANRS COVERTE-CO19  Files isThe ENNS . Participants in both studies provided written informed consent.COVERTE and ENNS received the approval of the Ethics Committee of the Ile de France III  was calculated as the ratio of the weight to the square of the height. Being overweight was defined as having a BMI ≥25. Metabolic parameters were measured using standard procedures. The metabolic abnormalities studied were those included in the Joint Interim Statement definition of metabolic syndrome : blood pThe main exposure was the HIV status: HIV-infected since childhood (patients enrolled in COVERTE) or not HIV-infected .The socio-demographic and behavioral characteristics collected in both studies were: gender, age (18–24 vs 25–30 years), marital status (married or living with a partner vs. being single), country of birth (metropolitan France vs. outside), employment status (working vs. not working or student), education level , and tobacco consumption (active vs. none or past), hormonal contraception, and psychotropic drug use.The immuno-virological status and ART of HIV-infected patients were described by the level of CD4 cell counts and viral load, the CDC clinical stage, the type of ART at study visit, and the duration of exposure to any antiretroviral drug, including those reported to have metabolic toxicity .The analysis was conducted separately for men and women  on the dFor HIV-specific parameters in COVERTE, the data for men and women were compared using Pearson Chi-Square or Fischer exact tests. Quantitative variables were compared using Kruskal-Wallis tests.The association of the HIV status (as main exposure) with each metabolic parameter (as dependent variable) was studied using linear regression models for continuous parameters and logistic regression models for qualitative parameters. The models were systematically adjusted for being overweight and education level in both genders. Further adjustment for the use of oral contraceptives was performed in women as they are known to modify lipid metabolism .A sensitivity analysis was conducted by excluding participants born outside France.Statistical analyses were performed using the survey procedure of SAS 9.4. The threshold for statistical significance was 0.05. No adjustment was used for multiple testing.Between 2010 and 2016, the COVERTE cohort enrolled 386 patients infected with HIV since childhood, of which 268 were included in the analyses , consistSeveral differences between HIV-infected patients and the ENNS general population of the same gender remained after standardization based on education level and BMI : HIV-inf10 copies/mL, when detectable.At the time of the study visit, less than 1.5% of HIV-infected patients had never been treated while, in the others, the median duration of ART was 19 years . ApproxiSix HIV-infected participants were treated with blood pressure-lowering drugs, two with lipid-lowering drugs, and one with insulin for type 1 diabetes, whereas no participant in the ENNS survey received any of these drugs.After adjustment for education level and being overweight, HIV-infected patients of both genders differed from the general population for several metabolic parameters . They haAn increased prevalence of several metabolic abnormalities was observed in COVERTE as compared to the ENNS population, after standardization or adjustment for education level and overweight.HIV-infected men had a higher prevalence of elevated triglycerides and reduced HDL-cholesterol levels than men of the general population . The preHIV-infected women were more likely to have reduced HDL-cholesterol levels and a high waist circumference, although they were less likely to have elevated total cholesterol than women from the general population . Their pWe conducted sensitivity analyses on the 412 subjects born in France . The results were quite similar to those obtained with the whole sample, except for a stronger association with reduced HDL-cholesterol in HIV-infected men and a weaker association with elevated total cholesterol in HIV-infected women .This study is the first to compare the metabolic parameters of young adults who acquired HIV infection during childhood and controls of the same age from the general population. It points to increased prevalence of cardiovascular and insulin resistance risk factors in HIV-infected young subjects.HIV-infected men had a lower BMI, but higher waist-to-hip ratio, higher triglycerides and lower HDL-cholesterol levels (without showing increased LDL-cholesterol and non-HDL cholesterol levels), than men of the same age in the general population. HIV-infected women had a similar BMI as women of the same age from the general population, but a higher waist-to-hip ratio associated with a larger waist circumference, and a higher systolic blood pressure. They also had lower HDL-cholesterol levels without increased LDL-cholesterol and non-HDL cholesterol levels. They had a higher mean triglycerides-to-HDL-cholesterol ratio and a higher prevalence of the metabolic syndrome than women in the general population. In addition, after further adjustment on the use of hormonal contraceptives, HIV-infected women showed significantly higher triglycerides mean levels than those from the general population. These results indicate that 18 to 30 year-olds with HIV infection display an atherogenic metabolic profile, as compared to uninfected adults of the same age, similar to the findings in French HIV-infected patients aged 35–44 years on protease inhibitors (APROCO study) . HoweverHigher waist-to-hip ratio and serum triglycerides, associated with lower HDL-cholesterol levels and higher triglycerides-to-HDL-cholesterol than in the general population, without difference in total or LDL cholesterol levels, are highly suggestive of a state of lower insulin sensitivity in young subjects with HIV infection since childhood . This meIn the aforementioned study of HIV-infected French adults aged 35–44 years , metabolOur study has several strengths. First, the availability of two national French studies that collected metabolic and behavioral data for a similar number of eligible subjects at comparable time periods made it possible to compare young HIV-infected patients with a general population of the same gender and age distribution. Second, it was possible to standardize prevalence and adjust models for BMI, strongly associated with metabolic syndrome, and education level, associated with behavioral factors, nutritional habits, and metabolic syndrome in women in a previous ENNS analysis . We did Our study has several limitations. First, the general population, as non-HIV-exposed group was assumed rather than systematic HIV testing, which might have underestimated the observed associations. The prevalence of HIV in France being about 4/1000 habitants, this bias may be low . Second,Our results show that, as compared to the general population of the same age living in France, young adults infected with HIV since childhood did not display increased tobacco consumption, which represent the main modifiable behavioral cardiovascular risk factor. We can also note that fasting glucose, and LDL-cholesterol did not differ between the two populations. The current treatment, care and support for vertically HIV-infected patients may have contributed to maintain these favourable metabolic parameters. However, young HIV-infected adults had a higher prevalence of dyslipidemia and android fat distribution, which are strongly associated with an increased risk of insulin resistance and further cardiometabolic disease, even in the youngest HIV-infected patients . This suS1 File(DOCX)Click here for additional data file.S2 File(DOC)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOC)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a widely used technique for the rapid and accurate identification of bacteria, mycobacteria and certain fungal pathogens in the clinical microbiology laboratory. Thus far, only few attempts have been made to apply the technique in clinical parasitology, particularly regarding helminth identification.We systematically reviewed the scientific literature on studies pertaining to MALDI-TOF MS as a diagnostic technique for helminths  of medical and veterinary importance. Readily available electronic databases  were searched from inception to 10 October 2018, without restriction on year of publication or language. The titles and abstracts of studies were screened for eligibility by two independent reviewers. Relevant articles were read in full and included in the systematic review.Caenorhabditis elegans, and the technique was primarily used for identification of specific proteins rather than entire pathogens. Since 2015, a small number of studies documented the successful use of MALDI-TOF MS for species-specific identification of nematodes of human and veterinary importance, such as Trichinella spp. and Dirofilaria spp. However, the quality of available data and the number of examined helminth samples was low.A total of 84 peer-reviewed articles were considered for the final analysis. Most papers reported on the application of MALDI-TOF for the study of Data on the use of MALDI-TOF MS for the diagnosis of helminths are scarce, but recent evidence suggests a potential role for a reliable identification of nematodes. Future research should explore the diagnostic accuracy of MALDI-TOF MS for identification of (i) adult helminths, larvae and eggs shed in faecal samples; and (ii) helminth-related proteins that are detectable in serum or body fluids of infected individuals.The online version of this article (10.1186/s13071-019-3493-9) contains supplementary material, which is available to authorized users. In clinical and laboratory diagnostic settings, mass spectrometry (MS) has been utilized for several decades as an approach for protein-centred analysis of samples in medical chemistry , 2 and hDuring the past decade, MALDI-TOF MS has been widely introduced as a diagnostic technique in microbiology laboratories, where it has replaced most other tools  as the first-line pathogen identification method due to its high diagnostic accuracy, robustness, reliability and rapid turn-around time . MALDI-TMALDI-TOF MS has several strengths if compared to other diagnostic tools, such as polymerase chain reaction (PCR) assays. Once the mass spectrometer and the corresponding databases are available in a laboratory, individual pathogen identification is inexpensive, and the sample preparation procedure does neither require highly skilled technicians nor complex additional laboratory infrastructure. Of note, MALDI-TOF MS is considerably less prone to contamination and results are available within a few minutes. However, constant power supply is a prerequisite, which limits the suitability of the technique in resource-constrained settings. Yet, it should be noted that MALDI-TOF MS is no longer restricted to high-income countries as it is increasingly available in reference laboratories in sub-Saharan Africa and elsewhere –19.MALDI-TOF does not always require culture-grown colonies of a given pathogen. Instead, it can also be employed to identify microorganisms directly from positive blood culture broths  with higLeishmania spp. [Giardia spp. [Cryptosporidium spp. [Trypanosoma spp. [Plasmodium spp. [Dientamoeba spp. [In contrast to clinical bacteriology, little research has been carried out pertaining to the application of MALDI-TOF MS for identification of parasites of human or veterinary importance . Severalnia spp. –26, Giardia spp. , Cryptosium spp. , Trypanooma spp. , Plasmodium spp. –32 and Deba spp. . These seba spp. –37, fleaeba spp. –41 and meba spp. –49. In ceba spp. .Ascaris lumbricoides, hookworm, Strongyloides stercoralis and Trichuris trichiura), cestodes (e.g. Taenia spp.) and trematodes (e.g. Fasciola spp. and Schistosoma spp.) account for a considerable global burden of disease and are among the most common infections in marginalized populations in the tropics and subtropics [Helminth infections caused by nematodes  Statement .The following electronic databases were systematically searched: MEDLINE/PubMed, ScienceDirect-Embase, Cochrane Library, Web of Science and Google Scholar. All studies published from inception to 10 October 2018 were eligible for inclusion without language restrictions. The bibliographies of all eligible documents were hand-searched for additional references. Conference abstracts or book chapters detected through these databases and additional library searches were also considered. The search strategy comprised keywords related to the MALDI-TOF MS technique (e.g. “MALDI-TOF” and “matrix-assisted laser desorption/ionization time-of-flight”) and helminthology . The full search strategies for every database are provided in Additional file Caenorhabditis elegans, as it is used as a model organism for biomedical research. Additionally, studies pertaining to MALDI-TOF/TOF tandem MS were excluded, as this is a different modification of the MALDI-TOF MS technique, which is not routinely employed in clinical microbiology laboratories, but rather in research laboratory use for accurate characterization or sequencing of components like amino acids, metabolites, saccharides, etc. [After the systematic literature search, all duplicates were removed. Titles and abstracts of potentially eligible studies were screened to identify manuscripts relevant to the research question. Scientific reports on helminths of either plants or insects as well as studies on symbiotic bacteria of helminths were excluded for this review. However, we kept all publications related to the soil nematode es, etc. –62.http://www.mendeley.com).The literature search was performed by the first author of this manuscript (MF). All titles and abstracts were then independently reviewed by the first and the last author (MF and SLB) for inclusion and any disagreement was discussed until consensus was reached. All extracted manuscripts were analysed using a reference manager software . These helminths could be differentiated by MALDI-TOF based on specific peaks and protein spectra patterns after a cell lysis using the Sepsityper Kit 50  and a protein extraction with 70% formic acid and acetonitrile. However, this study has several limitations, and it remains unclear whether calibration steps or assessments of the repeatability and reproducibility of the analyses were performed. An additional paper, published in 2017, reported on MALDI-TOF MS application for cyathostomin helminths, a very diverse group of intestinal parasites infecting horses [Coronocyclus coronatus, C. labiatus and C. labratus) and found distinct protein spectra among adult helminths of different species [The two studies aiming at an identification of entire pathogens provided evidence that MALDI-TOF MS could reliably differentiate between species within the genus chinella  and Diroofilaria , respectofilaria , nine spa et al. , which ha et al.  publisheg horses . These sg horses . The stu species . These f species .We systematically reviewed the available literature pertaining to the application of MALDI-TOF MS for identification of helminthic pathogens of human and veterinary importance. While the technique has been successfully employed for many major classes of pathogens , data on its use in diagnostic helminthology are scarce. Several studies reported on the differential analysis of specific components, such as proteins, peptides or lipids with MALDI-TOF MS techniques, but only two recent manuscripts and one conference abstract provided ‘proof-of-concept’ evidence of its potential utility in diagnosing and differentiating helminth species of medical or veterinary relevance.Meloidogyne spp. were detected in human faeces using a microscopic sedimentation method [Schistosoma japonicum [The majority of articles identified in this systematic review focused on protein-centred analyses of helminth samples. It is important to mention that some of the MALDI-TOF MS devices employed in these studies had been subjected to modifications that are not usually available in routine clinical laboratories. Additionally, these experiments frequently employed a complex sample pre-treatment comprising a protein separation by high pressure liquid chromatography (HPLC) or electrophoresis. Yet, some recent proof-of-concept studies have shown that MALDI-TOF MS is also capable of diagnosing entire helminthic pathogens and differentiating similar species within the same genus based on an analysis of their individual protein spectra , 67. Becn method . Future aponicum .Taenia [Taenia saginata or T. solium are orally ingested with meat of cattle or pig. While eggs of T. saginata are not infectious to humans, T. solium eggs can lead to the potentially fatal disease (neuro-)cysticercosis. While the correct diagnosis has important implications for treatment, patient management and potential contact screening , it is impossible to distinguish both species based on the identical morphology of their eggs under a microscope. Molecular tools can achieve an accurate differentiation of the two species, but are only available in research settings [Echinococcus species), novel species  and notIt is important to consider the fixative in which a parasitological sample is stored. Both formaldehyde and ethanol are commonly used to enable a long-term storage of biological specimens, but this may lead to profound changes of the protein structure , which iWuchereria bancrofti [The present review identified only a few successful studies that employed MALDI-TOF MS to diagnose helminths. Limitations include the complicated pre-treatment procedures employed in some studies and the rather incomplete data presentation in one of the more clinically oriented research projects . New resancrofti  or for tancrofti ).Yet, much research and rigorous validation is still needed before MALDI-TOF MS might be employed directly on stool samples, and priority should thus be given to (i) the establishment of in-house main spectra library databases to allow for species-specific identification of selected helminths; (ii) the subsequent development of sample treatment protocols; (iii) the validation of this technique on different clinical sample types; and (iv) the elaboration of MALDI-TOF MS to be employed on fixed samples.The present systematic review elucidated that MALDI-TOF MS, which is now routinely used in many clinical microbiology laboratories for identification of bacteria, fungi and mycobacteria, could potentially also be employed in the context of helminth diagnosis. Preliminary data suggest that MALDI-TOF MS might hold promise as a future diagnostic tool for direct and rapid identification of pathogenic helminths in clinical samples with sufficient diagnostic accuracy. Further studies are needed to evaluate these concepts and to develop specific databases for helminth identification, followed by rigorous validation on well characterised clinical specimens.Additional file 1. Search strategies employed for our systematic review pertaining to the application of MALDI-TOF mass spectrometry as a diagnostic tool in human and veterinary helminthology.Additional file 2. PRISMA checklist for a systematic review examining the application of MALDI-TOF mass spectrometry as potential tool in diagnostic human and veterinary helminthology.Additional file 3. List of references included in the final review (n = 84 articles).


Proton pump inhibitors (PPIs) are commonly used to lessen symptoms in patients with gastroesophageal reflux disease (GERD). However, the effects of PPI therapy on the gastrointestinal microbiota in GERD patients remain unclear. We examined the association between the PPI usage and the microbiota present in gastric mucosal and fecal samples from GERD patients and healthy controls (HCs) using 16S rRNA gene sequencing. GERD patients taking PPIs were further divided into short-term and long-term PPI user groups. We showed that PPI administration lowered the relative bacterial diversity of the gastric microbiota in GERD patients. Compared to the non-PPI-user and HC groups, higher abundances of Planococcaceae, Oxalobacteraceae, and Sphingomonadaceae were found in the gastric microbiota from the PPI-user group. In addition, the Methylophilus genus was more highly abundant in the long-term PPI user group than in the short-term PPI-user group. Despite the absence of differences in alpha diversity, there were significant differences in the fecal bacterial composition of between GERD patients taking PPIs and those not taking PPIs. There was a higher abundance of Streptococcaceae, Veillonellaceae, Acidaminococcaceae, Micrococcaceae, and Flavobacteriaceae present in the fecal microbiota from the PPI-user group than those from the non-PPI-user and HC groups. Additionally, a significantly higher abundance of Ruminococcus was found in GERD patients on long-term PPI medication than that on short-term PPI medication. Our study indicates that PPI administration in patients with GERD has a significant effect on the abundance and structure of the gastric mucosal microbiota but only on the composition of the fecal microbiota. The prevalence of GERD is 10%–20% in the Western world and 5.2%–18.3% in Asia Gastroesophageal reflux disease (GERD) is defined as the abnormal reflux of the contents of the stomach and duodenum that causes troublesome symptoms such as heartburn, regurgitation, or complications (Proton pump inhibitors (PPIs) are the most effective first-line treatment for GERD patients. PPIs do not effectively eradicate this disease, and some patients may require this treatment for life as continuous maintenance therapy or only when symptoms are present. Maintenance therapy is not a minor issue because symptom recurrence has been described when PPI therapy is discontinued, and these symptoms may severely impair quality of life.Clostridium difficile, Campylobacter spp., Shigella spp., and Salmonella spp. In general, PPI administration is considered safe, and there have been few reports of serious adverse events. However, long-term PPI use may result in several side effects, such as decreased bone metabolism, increased incidence of bone fractures, iron deficiency anemia, vitamin B12 deficiency, hypomagnesemia, and pneumonia Streptococcaceae is significantly increased in PPI-users Staphylococcus, Streptococcus, and Enterococcus as well as Escherichia coli species, were significantly increased compared with the abundances in samples from non-users. A study by Tsuda et al. H. pylori was found to be a minor bacterium in gastric luminal samples in a study by Tsuda et al. H. pylori-infected patients, as expected PPIs have been reported to substantially increase the abundance of commensals in the upper gastrointestinal (GI) tract, decrease microbial diversity and lower the abundance of commensals in the gut. At the family level, It is believed that PPI-mediated disruption of the normal gastric microenvironment and direct targeting of bacterial and fungal proton pumps are the potential mechanisms by which PPIs affect gastric bacterial composition. While the relationship between PPI use, especially continuous PPI use, and the gastric/gut microbiota is not fully understood, it is also unclear whether long-term PPI use affects the gastric and gut microbiota.In our study, we investigated the effects of PPIs on the microbiota of gastric mucosal and fecal specimens of GERD patients. We used 16S rRNA gene sequencing to evaluate the characteristics of the GERD microbiota and further clarify the association between PPI use and the gastric/fecal microbiota community.P > 0.05; Kruskal–Wallis test).To characterize the effects of PPIs on the gastric mucosal and fecal microbiota, we performed 16S rRNA gene sequencing analysis and compared the microbial community structures between healthy controls (HCs) and GERD patients. The clinical characteristics of the three groups, namely, GERD patients with and without PPI use and HCs, are shown in We obtained raw sequencing reads, and after filtering and removing the low-quality sequences, clean reads were retained for further analyses with an average of 44,254 and 45,632 sequences per sample for the mucosal and fecal microbiota, respectively. Rarefaction and rank abundance analyses suggested that sufficient sequencing information was achieved for all samples. Sequence clustering analysis yielded a total of 337 and 244 core operational taxonomic units (OTUs) (phylotypes) in the gastric mucosal and fecal microbiota, respectively.P = 0.076; observed species, P = 0.098; Wilcoxon test) and significantly higher community diversity  than that of non-PPI-users  and observed species  indexes than those of the PPI-user group  were found among the groups. Overall, there was no significant difference in the alpha diversity of the gastric mucosal microbiota between HCs and the PPI-user group   was performed to identify discrepancies in the gastric mucosal microbiome associated with PPI medication B. As exp= 0.001) .The alpha diversity of the fecal samples was assessed with the Shannon index, Chao1 index, and number of observed species. The HC group had significantly greater bacterial richness (Chao1 and observed species) than that of GERD patients with or without PPI use and had higher community diversity (Shannon and Simpson indexes) than that of the non-PPI-user and PPI-user groups A. The alHalomonas (10.7%), Helicobacter (7.7%), Rhodococcus (5.9%), Neisseria (5.4%), Streptococcus (5.2%), Pseudomonas (5.0%), Prevotella (4.9%), Brevundimonas (4.1%), Shewanella (3.5%), Lactobacillus (2.6%), Veillonella (2.0%), and Microbacterium (2.0%) were the 12 most abundant genera  analysis and cladograms were used to analyze the gastric mucosal bacterial community structure. Linear discriminant analysis (LDA) was used to estimate the difference in the effect size of each taxon among the HC, non-PPI-user, and PPI-user groups. The bacterial taxa with significantly higher abundances in the HC group were Caulobacteraceae and Porphyromonadaceae. In contrast, Desulfuromonadaceae, and Shewanellaceae were higher in the non-PPI-user group, whereas Planococcaceae, Oxalobacteraceae, and Sphingomonadaceae were higher in the PPI-user group A, B.FiguPelobacter, Desulfuromonas, Alcanlvorax, Kordiimonas, Desulfuromusa, Marinobacterium, and Marinobacter compared with the samples from GERD patients without PPI administration . The relative abundances of Luteimonas, Limonobacter, Herbaspirillum, Sphingobium, Phenylobacterium, Comamonas, Chryseobacterium, Duganella, Pedobacter, and SR1 genera incertae sedis were higher in the long-term PPI-user group than those in the non-PPI-user group  and those who received long-term PPI treatment (average time = 18 [12–120] months). There were no differences among the non-PPI-user, short-term PPI-user, and long-term PPI-user groups with respect to sex, age, and BMI (data not shown). Extended error bar plots were constructed to determine the mean gastric mucosal microbiota differences among the non-PPI-user, short-term PPI-user, and long-term PPI-user groups. At the genus level, samples from the short-term PPI-user group had significantly lower levels of er group C. The reer group C.The overrepresentation of fecal bacteria of GERD patients with or without PPI use, and HCs is depicted in a cladogram. LEfSe analysis (LDA ≥3) revealed a significantly higher relative abundance of Sutterellaceae, Pasteurellaceae, Methylobacteriaceae, Halomonadaceae, Comamonadaceae, Thermomonosporaceae, Nitrosopumilaceae, Vibrionaceae, Erysipelotrichaceae, Microbacteriaceae, Puniceicoccaceae, Nitrospiraceae, Colwelliaceae, Spirochaetaceae, Alteromonadaceae, Moraxellaceae, and Verrucomicrobiaceae in HC samples compared with GERD patient samples A, B. In Halomonas were significantly lower in the non-PPI-user group than those in the short- or long-term PPI-user groups using extended error bar plots analysis  and long-term PPI-user (average time = 24 [12–120] months) groups, to explore differences in the fecal microbiota in GERD patients. There were no differences in the microbiota in fecal samples among the non-PPI-user, short-term PPI-user, and long-term PPI-user groups with respect to sex, age, and BMI (data not shown). Our results showed that fecal microbes such as analysis C. The abanalysis C.To detect changes in microbiome metabolites induced by changes in microbiota abundance, the 16S rRNA data were annotated with metabolic pathways from the KEGG database using PICRUSt prediction analysis. KEGG metabolic pathways correlating with gastric mucosal and fecal microbiome differences among the non-PPI-user, PPI-user, and HC groups were revealed. According to the LEfSe analysis of metabolic function pathways, the gastric mucosal microbiota of the non-PPI-users included more relative methane metabolism, and terpenoid backbone biosynthesis pathways A. GlutatAs shown in With the development of high-throughput sequencing technology, 16S rRNA sequencing has been widely used to obtain large amounts of information regarding the stomach and fecal microbiota, but the defining characteristics of the gastric and gut microbiome associated with PPI use in GERD patients are still not fully understood.In recent years, numerous studies have consistently demonstrated that the inhibition of gastric acid secretion induces the dysbiosis of the gastric microbiota, including affecting the composition of the intestinal microbiota and promoting gastric bacterial overgrowth Prevotella, Streptococcus, Neisseria, Porphyromonas, and Haemophilus at the genus level. Furthermore, Streptococcus and Prevotella were shown to be the dominant genera in both the gastric lumen and gastric mucosa-associated microbiota Halomonas, Neisseria, Rhodococcus, Brevundimonas, Prevotella, and Streptococcus. The differing abundances at the genus level in our study and previous studies may be partially attributed to different geographic and ethnic differences in the study participants.Previous research revealed that the gastric mucosa-associated microbiota is strongly dominated by Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria at the phylum level Recent studies have indicated that contamination was present during microbiota sequencing experiments, occurring between sample collection and sequencing Halomonas, Shewanella, Marinobacter, and Mesorhizobium are present in the stomach, which may be due to the widely consumed traditional salted or fermented foods among Chinese populations. In 2017, Anubhav et al. Halomonas and Bradyrhizobium exist in gastric mucosal and negatively correlate with H. pylori infection. In addition, Avilés-Jiménez et al. Nesterenkonia, which can colonize human tissues and might also be associated with H. pylori infection. The presence of these microflora in the stomach may constitute colonized or translocation of upstream microflora, and the real composition and roles should be further confirmed.Interestingly, our study showed that certain halophilic bacteria and soil bacteria such as H. pylori colonization status in the gastric mucosal significantly alters the gastric microbiota H. pylori have been shown to have a protective effect on GERD. In our study, we did not find a difference in the abundance of H. pylori at the genus level between the patients and HCs. Therefore, we could not confirm the effects of H. pylori on the gastric mucosal microbiota; thus, the role of H. pylori in the pathogenesis of GERD patients requires further research.Numerous studies have demonstrated that H. pylori and increased the abundance of Fusobacteria and Firmicutes in the gastric mucosal microbiota of healthy dogs Most previous studies have confirmed that the composition of mucosal microbiota can be significantly affected by the use of PPI. It has been proposed that PPI use affects the gastric mucosal microbiota by directly targeting the bacteria’s proton pumps or increasing the pH to indirectly affect the microenvironment of the microbiota In previous reports, the gastric mucosa-associated microbiota demonstrated a higher alpha diversity in GERD patients than that in HCs, aligning with the results of the present study. The composition of the gastric mucosal microbiota differed between GERD patients and the HC group. At the family level, Porphyromonadaceae and Caulobacteraceae were more abundant in the HC group, while Desulfuromonadaceae and Shewanellaceae were more abundant in the non-PPI-user group. In addition, Planococcaceae, Sphingomonadaceae, and Oxalobacteraceae were more abundant in the PPI-user group.Pelobacter, Desulfuromonas, Alkanindiges, Koridiimonas, Marinobacterium and Marinobacter and higher relative abundances of Luteimonas, Limonobacter, Herbaspirillum, Sphingobium, Phenylobacterium, Comamonas, Chryseobacterium, Duganella, Pedobacter, and SR1 genera incertae sedis compared with the non-PPI-user group. The short-term PPI-user group had significantly lower levels of Pelobacter, Desulfuromonas, Alcanlvorax, Kordiimonas Desulfuromusa, Marinobacterium, and Marinobacter than did the non-PPI-user group. In addition, GERD patients with long-term PPI use had a higher abundance of Methylophilus than that of short-term PPI-users. Amir et al. We also found differences in bacterial richness and community diversity in the gastric mucosal microbiota between GERD patients with short- or long-term PPI use and non-PPI use. Extended error bar plots were generated to demonstrate that the long-term PPI-use group exhibited lower relative abundances of It has been reported that PPI can alter the composition of the gut microbiota in healthy twins or in patients with GI disease  C. difficile, Campylobacter, Shigella, Salmonella, and other enteric infections Lactobacillus and decreases in the abundance of Bacteroides and Clostridium cluster IV, which cause a further decrease in gut microbiota diversity. Jackson et al. Rothia and Streptococcus, were positively associated with PPI use. Imhann et al. Staphylococcus, Streptococcus, and Enterococcus as well as Escherichia coli at the species level, and there was an increase in oral bacteria, including the genus Rothia, in the fecal microbiota of PPI users Streptococcus at the genus level were significantly more abundant in short-term PPI users than nonusers. The genus Streptococcus causes severe damage to histiocytes and DNA by absorbing oxygen and releasing superoxide and hydrogen peroxide. Therefore, the family Streptococcaceae and genus Streptococcus are closely associated with PPI use. However, the real role of these bacterial taxa in GERD patients should be further investigated. In addition, changes in the microbiota associated with C. difficile in the PPI-use group of GERD patients were not detected. Although several studies have shown that PPI use is an independent risk factor for C. difficile infection C. difficile infection and PPI use should be further studied.The effects of PPI use on gut microbiota diversity have been investigated by several studies. The free use of gastric acid suppression is potentially associated with an increased risk of Halomonas in the gut microbiota was significantly higher in PPI-users (short-term and long-term PPI users) than that in non-PPI-user GERD patients. The relative abundance of Ruminococcus was significantly lower in GERD patients with short-term PPI use than non-PPI-users or long-term PPI-users. The abundance of Streptococcus was lower in GERD patients without PPI treatment than in those with short-term PPI administration.Clooney et al. Streptococcus is a diverse bacterial lineage that can occupy a myriad of environments; it has been found in the nasopharynx, oral cavity and esophagus Streptococcus in the gastric mucosal may be upstream community translocation or mucosal colonization. Therefore, whether Streptococcus colonizes the gastric mucosal still needs further verification, such as culturomics detection.The major limitation of our study was the relatively limited sample size, which may have affected our statistical analysis and subsequent conclusions regarding risk factors. Larger studies are warranted to validate our findings, and further studies are needed to investigate the long-term influence of PPIs on the gastric and gut microbiomes in GERD patients. In addition, a previous study demonstrated that the gastric fluid microbiota had higher diversity compared with that of the gastric mucosa-associated microbiota. During gastric mucosal biopsy sampling, it is unavoidable that gastric fluid may be included, which may impact the results of gastric mucosal microflora. For example, Methylophilus genus in the gastric mucosal microbiota and significantly higher abundances of the genera Ruminococcus in the fecal microbiota than that of short-term PPI users. Additional analyses with larger sample sizes investigating PPI use in GERD patients are needed to validate our results and to confirm the risk factors associated with PPI use in GERD patients, especially in long-term PPI users.Here, we report significant differences in the microbial community in the gastric mucosal and gut microbiota between GERD patients and HCs. In addition, GERD patients treated with PPI had significantly altered richness and structure of the gastric mucosal microbiota compared to non-PPI administration and only significantly altered the composition of the fecal microbiota. Indeed, long-term PPI users demonstrated a higher abundance of the H. pylori infections (assessed using the 13C breath test or the rapid urease test), and did not use PPIs or other medications for at least 30 days prior to the start of the study. Patients with GERD had endoscopic evidence of esophagitis based on the Los Angeles classification. GERD patients who were non-PPI-users (non-PPI-user) were defined as patients who did not use PPIs before enrolling in the study, while GERD patients with PPI use (PPI-user) were defined as patients who used PPIs . GERD patients with PPI use were classified into two patient subgroups based on the length of PPI therapy. GERD patients with short-term PPI use (short-term PPI-user) had taken PPIs for more than two months and less than one year at the time of study enrollment. GERD patients with long-term PPI use (long-term PPI-user) had taken PPIs for more than one year prior to study enrollment. Tissue biopsies from the gastric antrum were obtained during endoscopies, with fecal samples collected beforehand. The biopsies and fecal samples were immediately frozen in liquid nitrogen, then transferred to the laboratory and stored at −80 °C. All individuals were diagnosed by gastroendoscopy and did not receive any antibiotic treatment for one month prior to sample collection. The following exclusion criteria were applied: age below 18 years; a history of GI or hepatobiliary surgery; and suffering from organic GI lesions, such as ulcers and cancers.Gastric mucosal biopsy and fecal samples from HCs and GERD patients were collected by the Department of Gastroenterology of the Chinese PLA General Hospital from June 2013 to July 2014. A total of 40 GERD patients and 15 HCs were enrolled in this study. All participants provided their fecal samples, whereas 30 GERD patients and 5 HCs provided gastric mucosal samples as well. The HCs received clinical examinations with normal results. In addition, the HCs had normal endoscopy results, tested negative for This study was undertaken with the approval of the Chinese PLA General Hospital Ethics Service Committee. All experiments were performed in accordance with the approved guidelines. All individuals enrolled in this study provided their informed consent.The QIAamp DNA Mini Kit  combined with the bead-beating method was used to extract total DNA. After agitation with a bead beater , bacterial DNA was extracted according to the manufacturer’s instructions. Bacterial DNA samples were stored at −80 °C prior to sequencing.To characterize the taxonomic profiles of the gastric bacterial microbiota, we designed universal primers to sequence the V4 region of the 16S rRNA gene, targeting most bacteria . The following thermal cycling conditions were applied: initial denaturation at 98 °C for 1 minute; followed by 35 cycles of denaturation at 98 °C for 10 seconds, annealing at 50 °C for 30 seconds, and elongation at 72 °C for 60 seconds; with a final incubation at 72 °C for 5 minutes. Amplifications were performed in 25-μl reactions with 50 ng of template DNA. Normalized equimolar concentrations of PCR products were then pooled and sequenced using the Illumina MiSeq PE-300 platform . Barcodes and sequencing primers were trimmed before assembly.http://ccb.jhu.edu/software/FLASH/). QIIME (1.9.1) with the default setting was used for quality filtering http://drive5.com/uparse/) Paired-end sequence reads were assembled using FLASH . 16S rRNA data in the form of a BIOM format table were selected by mapping all 16S reads to references in the Greengenes tree with OTUs assigned at 97% identity. The obtained OTU table was normalized by 16S rRNA copy number, and metagenomes were predicted from the KEGG catalog. Moreover, the significant microbial functional properties associated with GERD patients and HCs were analyzed by the LEfSe (1.0) method PICRUSt (1.0.0) was performed using the online version of Galaxy http://stamp-software.com/). Using the STAMP program, we generated extended error bar plots to show that some properties differed in GERD patients using certain filter parameters .Statistical analyses of data were performed using the R packages Stats and Vegan. Significant differences in alpha diversity were assessed using the Wilcoxon rank sum test. The differences between microbial communities were determined by PCoA using the weighted UniFrac dissimilarity distance metric. The analysis of similarities (ANOSIM) test was performed with the Vegan package in R (version 2.15.3). The STAMP program is a statistical/econometric software package for time-series models with unobserved components, such as trends . A significant alpha at 0.05 based on the Kruskal–Wallis test and an LDA score ≥3 in LEfSe was employed to identify differing features between microbial communities The differentiating features of gastric mucosal and fecal microbiota composition and function were determined using the LEfSe algorithm method (available at https://www.ncbi.nlm.nih.gov/sra/SRP151219).All sequencing data are publicly available in the BioProject database under research ID PRJNA477423 and the SRA accession number SRP151219 (YCS analyzed the data and drafted the manuscript; STC collected the samples and analyzed the data; YPT analyzed the data; YBZ, JC, HJZ, XL, and RRR assisted with analysis; LHP and GS assisted with design and revised the manuscript for important intellectual content; and YSY designed and supervised the study. All authors read and approved the final manuscript.The authors have declared no competing interests.


To drive high-quality omics translational research using The Cancer Genome Atlas (TCGA) data, a TCGA Pan-Cancer Clinical Data Resource was proposed. However, there is an out-of-step issue between clinical outcomes and the omics data of TCGA for skin cutaneous melanoma (SKCM), due to the majority of metastatic samples. In clinical cases, the survival time started from the initial SKCM diagnosis, while the omics data were characterized at TCGA sampling. This study aimed to address this issue by proposing an observed survival interval (OBS), which was defined as the time interval from TCGA sampling to patient death or last follow-up. We compared the OBS with the usual recommended overall survival (OS) by associating them with both clinical data and microRNA sequencing data of TCGA-SKCM. We found that the OS of primary SKCM was significantly shorter than that of metastatic SKCM, while the opposite happened if OBS was compared. OS was associated with the pathological stage of both primary and metastatic SKCM, while OBS was associated with the pathological stage of primary SKCM but not that of metastatic SKCM. Five previously cross-validated survival-associated microRNAs were found to be associated with the OBS rather than OS in metastatic SKCM. Thus, the OBS was more appropriate for associating microRNA-omics data of TCGA-SKCM than OS, and it is a timely supplement to TCGA Pan-Cancer Clinical Data Resource. Skin cutaneous melanoma (SKCM) is the most common malignant skin cancer and its incidence, mortality, and disease burden have been increasing annually ,2,3. CliBy comprehensively characterizing molecular patterns in hundreds of SKCM samples, The Cancer Genome Atlas (TCGA) project has provided a comprehensive way to understand SKCM . Multi-oMethodologically, survival analysis  is now the dominant method to explore associations between outcome variables and the possible affecting factors. Therefore, the first step of survival analysis was to select an appropriate outcome variable. All previous studies adopted overall survival (OS), defined as the time interval from initial SKCM diagnosis to patient death or last follow-up , as the In this study, we aimed to address the out-of-step issue by proposing an observed survival interval and comparing it with OS in TCGA-SKCM dataset. Our findings prompted TCGA users to carefully select clinical outcomes when using TCGA-SKCM data for omics translational research.https://cancergenome.nih.gov/). The retrieval strategies, exclusion criteria, preprocessing of miRNA sequencing data and clinical data [Level 1 clinical data, level 3 miRNA isoform sequencing raw counts, and the corresponding meta-data of SKCM samples were retrieved and downloaded from TCGA repository  and metastatic SKCM (MCM) samples in TCGA-SKCM cohort. PCM samples with pathological stage I or II were defined as localized PCM (LPCM) samples and those with pathological stage III or IV were considered advanced PCM (APCM) samples . DiffereWe defined an observed survival interval (OBS) as the time interval from TCGA sampling to patient death or last follow-up A. UnlikeThe Kaplan-Meier survival analysis and log-rank test were applied to evaluate the prognostic effects of demographic and clinicopathological characteristics by considering both OS and OBS as clinical outcomes. The multivariate Cox regression model  was usedWe also inferred the pathological stage at the time of TCGA sampling for MCM patients based on the 8th edition of the AJCC melanoma staging system . SpecifiUnivariate Cox regression analyses and proportional hazards assumption tests  were usejth element of PI is the prognostic index of the jth patient, i.e.,iβ is the multivariate Cox regression coefficient of the ith miRNA and ijE is the expression value of the ith miRNA in the jth sample. The ability of SRS to predict the SKCM patient survival outcome was assessed by calculating the area under the curve (AUC) of the time dependent receiver operating characteristic (ROC) at 3 years, 5 years, and 10 years, respectively [The survival risk score (SRS), defined as the standard form of the prognostic index, was used as the synthetical index to represent the prognostic miRNA expression signature. The prognostic index was defined as a linear combination of the miRNA expression values weighted by the regression coefficients. Specifically,ectively .P values or adjusted p-values less than 0.05 were considered to be significant.All analyses were done using R 3.4.4 . MicroRNp-value of 0.472  samples, 352 were metastatic SKCM (MCM) samples, one was an additional MCM sample, and two were normal samples. We only analyzed the PCM and MCM samples due to the small number of normal and additional MCM samples. After preprocessing, 357 SKCM samples (82 PCM samples and 275 MCM samples) and 564 miRNAs were retained. To reproduce our analysis, the preprocessed clinical data and normalized miRNA sequencing data are presented in Datasets S1 and S2, respectively. Batch effect analyses of the normalized miRNA expression matrix showed that there was no discernible separation on the first two guided principal components A with a of 0.472 B. These Of the 357 patients in TCGA-SKCM cohort, 171 patients were deceased and 186 patients were alive at the time of last follow-up. The median OS time and OBS time of TCGA-SKCM cohort were 2184 days  and 986 days , respectively. For TCGA-PCM cohort, 18 patients were deceased and 64 patients were alive at the time of the last follow-up. The median OS time and OBS time of TCGA-PCM cohort were 1070 days  and 1276 days , respectively. For TCGA-MCM cohort, 122 patients were deceased and 153 patients were alive at the time of last follow-up. The median OS time and OBS time of TCGA-MCM cohort were 2402 days  and 896 days , respectively.p = 0.85) because the majority of the samples (80.49%) submitted to TCGA in this cohort were samples that were initially SKCM diagnosed. However, the OBS was significantly shorter than OS in TCGA-MCM cohort (p = 2.51×10−11) as the majority of the samples (90.55%) submitted to the TCGA in this cohort were not samples that were initially SKCM diagnosed, but samples excised from follow-up metastases . Furthermore, the median OS of TCGA-PCM cohort was significantly shorter than that of TCGA-MCM cohort . For TCGA-PCM cohort, OS and the OBS were usually the same, and indiscriminate usage of them will not result in a significant difference. Overall, these results indicated that the clinicopathological characteristics reasonably predicted OS, while they were not appropriate to predict the OBS due to the out-of-step issue.Differentially expressed miRNAs between PCM and MCM were mainly in the hsa-miR-205-5p, hsa-miR-203a-3p, and hsa-miR-200 family . Only hsSegura et al., Caramuta et al. and Tembe et al. proposed several miRNA expression signatures for MCM prognosis based on low sample size microarray data ,25,26. JUnivariate Cox regression analysis revealed that there was no miRNA associated with either OS or the OBS in TCGA-PCM cohort. Meanwhile, 27 and nine miRNAs were found to be significantly associated with the OBS and OS of patients in TCGA-MCM cohort, respectively . InteresUnlike the clinicopathological characteristics measured at the time of initial SKCM diagnosis, the molecular patterns were characterized at the time of TCGA sampling. Thus, the miRNA-omics data were in accordance with the start time point of the OBS, while they were out-of-step with respect to OS . Combined with the results from the above clinical analyses, OS and the OBS were usually the same in TCGA-PCM cohort, while they were usually different in TCGA-MCM cohort. Thus, for TCGA-MCM cohort, the OBS should be used for associating miRNA-omics data, and indiscriminate usage of OS and the OBS in TCGA-PCM cohort will not result in a significant difference. Overall, the OBS was more appropriate for identification of prognostic biomarkers than OS. Furthermore, to evaluate the independence and compare the prognostic power of clinicopathological characteristics and biomarkers, the time point of clinical data and omics data must be in accordance. As the time point of the omics data was fixed at TCGA sampling, it is wise to deduce the clinicopathological characteristics at the time point of TCGA sampling.Although Jayawardana et al. have cross-validated five miRNAs for MCM prognosis, the cross-validation was based on differential expression analysis between MCM patients with longer OS and MCM patients with poor OS . Thus, rp = 0.49) and all covariates in the model satisfied the proportional hazards assumption . The area under the curve of the receiver operating characteristic for SRS were 0.77 (95%CI: 0.71–0.84), 0.76 (95%CI: 0.68–0.83), and 0.79 (95%CI: 0.71–0.88) at three years, five years, and 10 years, respectively  was calculated for each MCM patient and Kaplan-Meier survival analysis showed that high-risk (SRS>0) MCM patients had shorter OBS compared with low-risk (SRS ≤ 0) MCM patients . Thus, TCGA-CDR prudently recommended using only the limited number of PCM samples for SKCM clinical outcome correlations [The recently proposed TCGA-CDR includes OS, PFI , DFI , DSS , and a list of outcome usage recommendations for each cancer type . As indielations . HoweverSome limitations of the current study should be noticed. (I) We addressed the out-of-step issue of survival outcome but various outcome endpoints based on relapses or metastases were not investigated. With the exception of OS, Liu et al. also recommended PFI and DSS for TCGA-SKCM data. However, there also existed the out-of-step issue for these outcomes in MCM. For example, all outcome endpoints of TCGA-W3-A825 in TCGA-CDR were 1917 days . AccordiIn conclusion, we defined the OBS, to supplement TCGA-CDR, and recommended it for TCGA-SKCMomics translational research. Our results could remind subsequent TCGA-SKCM data users to pay attention to the out-of-step issue of outcome endpoints. Although our analyses were based on associations between survival-based outcomes and TCGA-SKCM miRNA-omics data, they could be generalized to associate other TCGA-SKCMomics data and relapses, or metastasis-based outcomes. In addition, the five identified prognostic miRNAs may be of value in predicting the OBS of MCM patients and informing future experimental investigations.