Homo sapiens) and great apes (including Pan troglodytes and Pongo abelii). Here we combined inter-species and intra-species genetic variations to discover genes involved in the evolution of human intelligence. Information was collected from published GWAS works on intelligence and a total of 549 genes located within the intelligence-associated loci were identified. The intelligence-related genes containing human-specific variations were detected based on the latest high-quality genome assemblies of three human’s closest species. Finally, we identified 40 strong candidates involved in human intelligence evolution. Expression analysis using RNA-Seq data revealed that most of the genes displayed a relatively high expression in the cerebral cortex. For these genes, there is a distinct expression pattern between humans and other species, especially in neocortex tissues. Our work provided a list of strong candidates for the evolution of human intelligence, and also implied that some intelligence-related genes may undergo inter-species evolution and contain intra-species variation.Understanding the evolution of human intelligence is an important undertaking in the science of human genetics. A great deal of biological research has been conducted to search for genes which are related to the significant increase in human brain volume and cerebral cortex complexity during hominid evolution. However, genetic changes affecting intelligence in hominid evolution have remained elusive. We supposed that a subset of intelligence-related genes, which harbored intra-species variations in human populations, may also be evolution-related genes which harbored inter-species variations between humans ( Humans have brains with significantly increased size and complexity compared to their ape counterparts . CorrespNOTCH2NL was functionally investigated—it was found that the gene was able to promote the expansion of cortical progenitors, serving as an important gene contributing to the evolution of the human brain  could provide key clues for understanding the process of the evolution of human intelligence. As the assembling gaps and errors in the previous reference genomes of the great apes , the previous genomes were not qualified enough for the detection of complex structural variations  such as Among a number of genomic differences between humans and apes, it is very important but technically difficult to know which variants are specific to intelligence. In order to further search out the candidate genes related to the evolution of human intelligence, we suppose that some intelligence genes may have both inter-species (between humans and the great apes) and intra-species (within humans) variations. For intra-species variations, we collected genomic loci identified by several sets of GWAS on human intelligence, and genes in these loci (termed as intelligence-associated-genes) could be regarded to contain intra-species intelligence differences. For inter-species variation, genomic differences between humans and the great apes, revealed by recent high-quality sequencing , were usTo exploit the genes related to human intelligence, six major works were collected from GWAS or GWAS meta-analyses on human intelligence with a large sample size from the last five years . A total of 271 loci associated with intelligence in the human genome  were iden = 53949, n = 112151, g or a primary measure of fluid intelligence  meta-analyses of general cognitive function  nearest genes (based on ANNOVAR annotations) were integrated with redundancies (the same gene identified in more than one study) removed. We took these 549 genes as “bait genes” , which wIt should be noted that some GWAS works underlying human intelligence that have been published very recently may be not included in this study. This would not affect our analyses because this study aimed to provide some candidate genes for human intelligence evolution and could not identify all related genes at one time. In addition, because genotype imputation was not performed for the X chromosome in some cohorts , the potential genes related to intelligence in the X chromosome were not included in our “bait genes”.The new genome assemblies of chimpanzees, gorillas and orangutans have become fully available recently through single-molecule, real-time (SMRT) long-read sequencing, which improved the resolution of large and complex genomic regions. We incorporated the human-specific structural variations (from intermediate size to large size) into a “variation pond”, including exon gain/loss, short tandem repeats (STRs), insertion/deletion (indels) of more than 50 bp, and inversions. Moreover, considering the important role of the human-specific segmental duplications , Pongo abelii (Orangutan) and Gorilla (Western Lowland Gorilla) were downloaded from: https://www.ncbi.nlm.nih.gov/assembly/GCF_002880755.1/, https://www.ncbi.nlm.nih.gov/assembly/GCF_002880775.1/ and https://www.ncbi.nlm.nih.gov/assembly/GCF_000151905.2/, respectively.The genome sequences of three great apes were downloaded from the NCBI database. The reference genome sequences from Localized alignments of the target gene sequences were performed to filter out the false positive human-specific variations from previous reports . The locMost “prey” genes contained the human-specific variation in introns, far away from the exon-intron junction site. These variants are less likely to affect the gene functions, so only genes that contained variations in the coding regions were considered, named human intelligence evolution genes (HIEGs). These HIEGs included all the genes containing exon-gain/loss (2 genes), hCONDEL (28 genes), or HSD (1 gene), and genes with exon-located indels (8 genes) or STRs (4 genes). 40 non-redundant genes were finally identified, which were associated with human intelligence and which carried significant human-specific variations.http://asia.ensembl.org/index.html). For exon alignment, all transcript isoforms of one gene were compared both intra-species (within human) and inter-species (between human and the great apes) in order to confirm the specificity of the new transcript in humans. The human principal isoform, the human variant isoform, the chimpanzee isoform, the gorilla isoform, and the orangutan isoform of the PCCB gene are ENST00000469217 (NM_001178014), ENST00000466072, ENSPTRT00000028811, ENSGGOT00000005484 and ENSPPYT00000016431, respectively. The human principal isoform, the human variant isoform, the chimpanzee isoform, the gorilla isoform, and the orangutan isoform of the STAU1 gene are ENST00000371856, ENST00000340954, ENSPTRT00000050938, ENSGGOT00000048652 and ENSPPYT00000012903, respectively.The gene transcript information was obtained from Ensembl Release 95 (http://smart.embl-heidelberg.de). The protein accession numbers in humans, chimpanzees, gorillas, and orangutans for KMT2D are NP_003473, XP_016778992, XP_018894141, XP_024112209, respectively. The protein accession numbers in human, chimpanzee, gorilla, and orangutan for TRIOBP are NP_001034230, XP_016794633, XP_004063488, and H2P4B5 (UniProt), respectively. We performed multiple alignments using the software Constraint-based Multiple Alignment Tool .The analysis for protein domain was performed using the Simple Modular Architecture Research Tool (SMART) in the normal mode (http://www.proteinatlas.org), including 102 samples of 37 tissues, in which TPM (transcripts per million) were used for the evaluation of expression level. In order to compare the expression levels between humans and great apes, the RNA-Seq data (NCBI ID: (100796) from 107 samples in 8 brain regions of humans, chimpanzees, gorillas, and gibbons were used (http://mev.tm4.org/).We assessed the expression patterns of the HIEGs using transcriptome data for humans and their closest relatives. The HPA RNA-seq data were downloaded from the Human Protein Atlas  but gained another new 60-bp exon . The gained 60-bp exon did not appear in any transcripts of the PCCB of the three great apes . However, the relative cerebral cortex expression level of the PCCB variational transcript was higher than that of the principal transcript (relative to the average expression level of the transcript in 37 tissues) . Neurological complications, such as intellectual disability, brain structural abnormalities, optic neuropathy, and cranial nerve abnormalities are significant symptoms of PA   and 3C. ms of PA . Moreovebilities .STAU1 was also located in a locus identified by GWAS for intelligence  in one transcript variant. The isoform, with the addition of a 123-bp exon, was not detected in any transcripts of chimpanzee, gorilla or orangutan , and one STR contraction was located within the 39th exon of the KMT2D gene.In the variation pond, there were a total of 1,465 human-specific STR contractions and 4,921 human-specific STR expansions. These STRs hit 100 “bait genes”  of the six genes. Moreover, GNB5 and TRIOBP carried an insertion in the 3′UTR region and an insertion in the exon region, respectively.Among 5,894 human-specific deletions and 11,899 human-specific insertions in the “variation pond”, we found 94 “prey genes” with 148 deletions and 144 genes with 298 insertions . FurtherTRIOBP resulted in a gain of 675-bp coding region (c.887-1560) in the 5th exon of the longest isoform when compared with that in the chimpanzee. This variation resulted in a 238-aa discontinuous insertion in the region p.296–811 of the human TRIOBP protein , and surprisingly, several causal mutations were located within the human-specific insertion regions  (The insertion in  protein . The GWAandidate . Biochemandidate . Mutatiod Q297X) .NRXN1, GRID2 and GRIA4, which were all involved in neurotransmission and the formation of synaptic contacts.We also searched for human conserved deletions (hCONDELs) near the “bait genes” that had been identified . These sGNB5 gene in the intelligence-associated locus on chromosome 15  of the gene  , which the gene . GNB5 waeceptor) . In humarhythmia . The humThe inversion variation is the rearrangement in which the genomic segment is reversed. Based on the previous report, there were a total of 625 inversions in our “variation pond” ranging from 9 kb to 8.4 Mb in size. Among these, 31 of them hit the “bait genes” . HoweverAFF3 gene. The AFF3 also contained an hCONDEL around the intron-exon junction regions (31-bp distance to the exon). The region around AFF3 has been identified to be an intelligence-associated locus in found in two distinct meta-analyses conducted on intelligence  the length of which were more than 5-kb . Among tFUT8, the remaining 11 genes are the hCONDEL-containing genes. The expression patterns of the 11 genes exhibited three types. Four genes, including GRIA4, NRXN1, CADM2, and CALN, showed the most significant high expressions in the cerebral cortex and low expressions in the other tissues. SGCZ, DCC, and GRID2 showed only relatively high expressions in the cerebral cortex compared with the low expressions in all other tissues. NCAM1, FBXL17, FUT8 and GNB5 were generally expressed in all tissues , while the expressions of STAB1 in the human neocortex were much higher than those in the great apes (fold change = 4.8). Hierarchical clustering was also performed for the 39 gene expressions in all samples. We found that 5 neocortex tissues in the same species could be always clustered , while CB, STR, and HIP were generally clustered based on their tissue types . This result suggested that the expression profiles of the HIEGs in the neocortex tissues displayed a strong species specificity, which was in contrast to the profiles in these non-neocortex tissues . Taken together, the intelligence-related genes, containing the human-specific variations, expressed differently in the neocortex tissues between the humans and the great apes.A transcriptome dataset sampling eight brain regions  of both humans and four primate species  was reported previously , which eHomo sapiens. However, it is still very challenging to identify the causative genes that are responsible for the intelligence difference between human and the other species in Hominidae. With both the human genome sequence and those of great apes becoming available, it is possible to pinpoint the genetic changes underlying the phenotypic differences between humans and the great apes. A subset of genes that were related to the human-ape intelligence differences may also contain intraspecific allelic variation underlying the variation of intelligence levels in human populations. Hence, in this work we integrated both the latest GWAS information on intelligence and the latest advances in the great ape genomics, aiming to mine the gene clues to understand the evolution of human intelligence. We found several strong candidates, for example, the genes TRIOBP and GNB5 contain human-specific variations and have the genetic evidence to be involved in the development of intelligence , were reported to be involved in the transport, relocation, translation of mRNA and mRNA decay  is often misleading. As a result, HIEGs must contain many unrelated genes, although the intelligence-related genes involved in human evolution have been partly enriched. Further experiments and analyses may include the validation of gene functions (whether and how these genes could influence the development of human brains) and the assessment of the effects of the human-specific variation (whether and how these sequence variations could influence the gene coding or the gene expression patterns).NOTCH2NL, FZD8, SRGAP2, ARHGAP11B, and TBC1D3) are only a small proportion of the whole gene set that encapsulates the vast differences in brain size and intelligence between great apes and humans, leaving many remaining gaps in our knowledge. More integrated approaches incorporating genetics, genomics, bioinformatics, and development biology will be needed in future works.There are already several findings of the human-chimpanzee differences altering the development of the neocortex to date. The knowledge from the works of gene functional studies and evolutionary genetics studies greatly enhanced our understanding of intelligence and the brain . CertainGWAS has identified hundreds of genes associated with intelligence variation in human populations. Through inter-species genome comparisons between human and great apes, we found that a small proportion of intelligence-related genes also contained human-specific variation. Through integrated approaches, especially the careful checking of sequence alignments and gene annotations, we identified 40 candidates in which human-specific variation may have effects on gene coding or expressions. Transcriptome-wide comparison between humans and four primate species for the 40 candidate genes suggests that several of them displayed a different expression pattern among these species. The results implied that at least a few of the intelligence-related genes may contain both intra-species variation and inter-species variation. The intra-species variation underlies the small variation of intelligence levels among different human individuals while the inter-species variation controlled the large genetic differences of intelligence between the great apes and humans. This work may provide a list of candidate genes to be used in subsequent studies.10.7717/peerj.8912/supp-1Table S1Click here for additional data file.10.7717/peerj.8912/supp-2Table S2Click here for additional data file.10.7717/peerj.8912/supp-3Table S3Click here for additional data file.10.7717/peerj.8912/supp-4Table S4Click here for additional data file.10.7717/peerj.8912/supp-5Table S5Click here for additional data file.10.7717/peerj.8912/supp-6Supplemental Information 1Raw code for mining the intelligence-associated genes which also harbored human specific variations. This perl script can be viewed in text file readers such as UltraEdit.Click here for additional data file.


Abnormalities of functional and structural connectivity in the amygdala-prefrontal circuit which involved with emotion processing have been implicated in adults with major depressive disorder (MDD). Adolescent MDD may have severer dysfunction of emotion processing than adult MDD. In this study, we used resting-state functional magnetic resonance imaging (rs-fMRI) and diffusion tensor imaging (DTI) to examine the potential functional and structural connectivity abnormalities within amygdala-prefrontal circuit in first-episode medication-naïve adolescents with MDD.Rs-fMRI and DTI data were acquired from 36 first-episode medication-naïve MDD adolescents and 37 healthy controls (HC). Functional connectivity between amygdala and the prefrontal cortex (PFC) and fractional anisotropy (FA) values of the uncinate fasciculus (UF) which connecting amygdala and PFC were compared between the MDD and HC groups. The correlation between the FA value of UF and the strength of the functional connectivity in the PFC showing significant differences between the two groups was identified.Compared with the HC group, decreased functional connectivity between left amygdala and left ventral PFC was detected in the adolescent MDD group. FA values were significant lower in the left UF within the adolescent MDD group compared to the HC group. There was no significant correlation between the UF and FA, and the strength of functional connectivity within the adolescent MDD group.First-episode medication-naïve adolescent MDD showed decreased functional and structural connectivity in the amygdala-prefrontal circuit. These findings suggest that both functional and structural abnormalities of the amygdala-prefrontal circuit may present in the early onset of adolescent MDD and play an important role in the neuropathophysiology of adolescent MDD. Major depressive disorder (MDD) is characterized by emotional dysregulation, implicating abnormalities of frontal-limbic neural circuits involved in emotional processing as the core feature. Convergent studies provide consistent evidence for functional and structural abnormalities in the prefrontal cortex (PFC) and amygdala, the key components of frontal-limbic neural circuits in adult MDD. Adolescent MDD is associated with the prominence of irritability, mood reactivity, and fluctuating symptoms , reflectDysfunction of amygdala-prefrontal circuits has been implicated in MDD through functional magnetic resonance imaging (fMRI). Hyperactivation of amygdala and hypoactivation of PFC were shown in task-related fMRI studies within adult MDD, as well as adolescent MDD. Recently, resting-state fMRI (rs-fMRI) was used to investigate resting state functional connectivity (rsFC), the correlation of low frequency blood oxygen level-dependent signal fluctuations between brain regions in MDD . Our prein vivo. Fractional anisotropy (FA) which measures the principal directionality of water diffusion is the commonly used parameter to assess whiter matter integrity in DTI studies. Decreased FA values were detected in the uncinate fasciculus, the superior longitudinal fasciculus, the cingulum, the corpus callosum  matched for sex, age and education by advertisements in the Frist Affiliated Hospital of China Medical University. A trained psychiatrist individually diagnosed all participants using the Schedule for Affective Disorders and Schizophrenia for School-Age Children (KSADS-PL). All MDD patients met the following inclusion criteria: fulfilling KSADS-PL criteria for MDD; first depressive episode; onset age between 13 and 17; no comorbid diagnosis of other affective and psychotic disorders; We used the 17-item Hamilton Depression Rating Scale (HAMD-17)  to scoreThe study was approved by the Institutional Review Board of the China Medical University. All participants and their parent/legal guardian received a detailed description of the study, after that, they provided written informed consent to make sure they fully understand and agree their involvement of the study.2, flip angle 90°. We used a spin-echo planar imaging sequence to collect the DTI data with the following scan parameters: 25 non-collinear directions, b = 1,000 s/mm2, TR 17,000 ms, TE 85.4 ms, 65 contiguous axial slices, 2 mm thickness, without gap, imaging matrix 120 × 120, FOV = 240 × 240 mm2.A GE Signa HDX 3.0T MRI scanner  with a standard head coil at the First Affiliated Hospital of China Medical University was used to perform the magnetic resonance imaging scan. Restraining foam pads and ear plugs were used for each participant to minimize the head motion and reduce the noise interference during the scan. Participants were asked to remain awake throughout the scan and keep their eyes closed. We used a gradient-echo planar imaging sequence to collect the rs-fMRI data with the following scan parameters: TR 2,000 ms, TE 30ms, 35 contiguous axial slices, 3 mm thickness, without gap, matrix 64 × 64, FOV 240 × 240mm3 voxels then smoothing with a Gaussian filter of 6 mm full-width at half-maximum (FWHM). Then REST software was used to remove linear drift through linear regression and temporal band-pass filtering (0.01–0.08 Hz) to reduce the effects of low-frequency drifts and physiological high-frequency noise. Linear regression of head motion parameters, global mean signal, white matter signal and cerebrospinal fluid signal was performed to remove the effects of the nuisance covariates.We used Resting-State fMRI Data Analysis Toolkit (REST) with Statistical Parametric Mapping 8 (SPM8) to processed the rs-fMRI data. The first 10 volumes of scanned data of each participant were deleted due to magnetic saturation effects. The remaining images were preprocessed with the following steps: First, slice timing and head motion correction: head motion parameters were computed by estimating translation in each direction and the angular rotation about each axis for each volume. The rs-fMRI data was excluded if their head motion was >2 mm maximum displacement in any of the x, y, or z directions or 2° of any angular motion throughout the course of the scan (no participants were excluded). Second, spatial normalization image to the standard Montreal Neurological Institute (MNI) space and resampled voxel size into 3 × 3 × 3 mmThe left and right amygdala ROIs were defined separately according to the automated anatomical labeling (AAL) template contained in REST . The tim3) via spatial normalization. The ICBM-DTI-81 WM labels atlas in the standard space allow for parcellation of the entire white matter into multiple regions of interest (ROI) (We processed DTI data using the PANDA toolbox  in FSL dst (ROI) . PANDA t2 tests compare demographic data and HAMD scores between the MDD and HC groups with SPSS 22.0. Two-tailed values of P < 0.05 were considered statistically significant.We used the independent two-sample t tests and χ3), corresponding to the p < 0.05 corrected by AlphaSim.The subject-specific maps of resting state correlations from the amygdala to all brain voxels of rs-fMRI data were combined across subjects within the MDD group and within the HC group. With age as covariate, Voxel-based 1-sample t-tests were used to produce group whole-brain composite maps. Then we created the contrast maps to assess between-group differences using voxel-based 2-sample (MDD vs. HC) t-tests. The contrast maps were corrected for multiple comparisons using Monte Carlo simulation within the PFC which was our hypothesized region. We defined the PFC ROI including 20 labels of AAL template, corresponding to Brodmann areas (BA) 9, 10, 11, 12, 24, 25, 32, 44, 45, 46, 47. The contrast map threshold was set at p < 0.05 for each voxel, with cluster size of at least 32 voxels .There were no significant differences in age (p = 0.972), gender (p = 0.236),or education (p = 0.228) between the adolescent MDD and the HC groups. MDD adolescents had significantly higher HAMD scores than the HC , T = 3.53]  rsFC from the left amygdala in the adolescent MDD group, compared with the HC group [peak MNI coordinates of the left VPFC region: x = -35, y = 21, z = -11, 45 voxels . There was also no significant correlation in the HC group .In this study, we reported decreased rsFC between left amygdala and left VPFC in adolescent MDD compared to HC. Furthermore, deficits of white matter integrity in the left uncinate fasciculus, fiber tracts connecting VPFC to temporal regions  were detected in adolescent MDD compared to HC. To our knowledge, this is the first study using differential MRI methods to explore functional and structural connectivity abnormalities in the amygdala-prefrontal circuits within adolescent MDD. Our findings provide primary evidence implicating abnormalities of amygdala-prefrontal circuits as the key components in the pathophysiology of adolescent MDD.Our results of decreased rsFC between left amygdala and left VPFC in adolescent MDD (age 13–17) compared to HC are consistent with our previous findings of both rs-fMRI and task-fMRI in adult MDD age 18–45) 25). AmygIn this study, adolescents with MDD showed decreased FA values in the left UF compared to HC. The UF connects the amygdala with inferior frontal regions including VPFC and ACC, which are key components of the frontal-limbic neural circuits involved in emotional processing , 28. DysThe major strength of our study is that multiple MRI methods (rs-fMRI and DTI) were used to detect functional connectivity, structural connectivity and their relationship in the same adolescent MDD sample. Our present results of decreased functional connectivity within amygdala-VPFC circuits as well as reduced structural connectivity between amygdala and VPFC in adolescent MDD suggest the potential association between functional and structural connectivity. Our explanation is that deficits of white matter integrity in the UF might contribute to the decrease of functional connectivity between amygdala and VPFC. We speculate that early abnormalities of brain development may be present in some adolescent MDD patients early in life, even before illness episodes. The unmaturation of brain development in adolescent may cause the inadequate compensatory mechanism, hence lead to the decrease of both functional and structural connectivity in adolescent MDD. The association between functional and structural disconnectivity in the amygdala-VPFC circuit may reflect the early onset mechanism of adolescent MDD, which need to be further investigated in future longitudinal studies including adolescents with high-risk of depression.The limitation of our findings is that as no significant correlation between strength of functional connectivity and white matter integrity detected, the direct relationship between functional and structural connectivity in the amygdala-VPFC circuit within adolescent is still unclear. Since a borderline significant correlation was detected in the MDD group, our speculation is that the relative small sample size may limit our ability to detect the statistical significance in current study. Future studies with large sample size is important to further understand the relationship between functional and structural connectivity and the neurodevelopmental mechanism of adolescent MDD.Our study present the primary evidence of both functional and structural connectivity abnormalities of amygdala-VPFC circuit in the sample of first-episode medication-naïve adolescent MDD. The abnormal white matter structural integrity in adolescent MDD may reflect the unmaturation of early brain development in adolescent, which may cause the functional dis connectivity in the same circuit. Both functional and structural abnormalities of amygdala-VPFC circuit may present in the early onset of adolescent MDD and play an important role in the neuropathophysiology of adolescent MDD.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the Medical Science Research Ethics Committee of the First Affiliated Hospital of China Medical University . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.FW, LK, and JS designed the study. JS, YZ, XJ, ZT, and HL acquired the data. LK, JS, and HG analyzed the data. FW, LK, JS, and ZT wrote the article.This study was supported by the National Natural Science Foundation of China , the Liaoning Scientific Foundation (2015020532 to FW and 201602833 to LK).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.


Recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive detection in infected but recovered individuals has been reported. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system. We sought to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better understand risks for prolonged infectivity and reinfection. A series of 414 patients with confirmed SARS-Cov-2 infection, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020. Statistical analyses were performed of the clinical, laboratory, radiologic image, medical treatment, and clinical course of admission/quarantine/readmission data, and a recurrence predictive algorithm was developed. 16.7% recovered patients with PCR positive recurring one to three times, despite being in strict quarantine. Younger patients with mild pulmonary respiratory syndrome had higher risk of PCR positivity recurrence. The recurrence prediction model had an area under the ROC curve of 0.786. This case series provides characteristics of patients with recurrent SARS-CoV-2 positivity. Use of a prediction algorithm may identify patients at high risk of recurrent SARS-CoV-2 positivity and help to establish protocols for health policy. The quarantining of patients testing positive for SARS-CoV-2 virus is considered mandatory in order to prevent continued viral spread (contagion). In last 6 months, many of COVID-19 patients have since clinically recovered, but it remains unclear the degree to which patients with COVID-19 clinical symptoms and polymerase chain reaction (PCR) test positivity remain contagious and or at risk for disease relapse.Given the sudden emergence and rapid community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) being observed worldwide, a strategy of social distancing and shelter in place has been widely adopted in an effort to curb the spread of COVID-19 across space and time3 that such individuals may be at risk of viral reactivation or reinfection and thus act as carriers and potentially reinfect others. In response, the World Health Organization (WHO) commented that there is currently “no evidence” demonstrating that people who have recovered from the coronavirus are not at risk of re-infection4. Thus, varying quarantine strategies have been implemented during the transition of COVID-19 recovering patients from healthcare to non-healthcare settings in this current pandemic.There is concern based on recent findings showing the recurrence of PCR positive results in 116 infected but recovered individuals5. Variability in PCR methodology will result in different thresholds of the assay for RNA detection, but in one study the SARS-CoV-2 RNA threshold upon PCR testing needs to be greater than 106 copies per sample6. To date, there have been no reports of live virus isolation from patients of PCR retest positivity. Importantly, though, pathological evidence of likely viable SARS-CoV-2 virus within pneumocytes was recently obtained from an individual who died unexpectedly from cardiac arrest after showing clinical recovery and three consecutive negative PCR nasopharyngeal (NP) swabs7.However, limited information is available regarding viral shedding kinetics and live virus isolationIn order to assist with pandemic management, a better understanding of the kinetics of SARS-CoV-2 positivity and associated potential (re)infectivity in recovered individuals is critical. Similarly, to assist in managing individuals, setting quarantine strategies and adjudicating limited healthcare resources, prediction models are needed to better define the risk, timing, and relevance of viral PCR positivity with (re)infectivity. Unanswered questions include the time between NP swab test negative and length of effective quarantine, and how infectious cana clinically recovered COVID-19 patient be with recurred SARS-CoV-2 positivity. Pragmatic models should seek to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better manage risks for prolonged infectivity and reinfection. Thus, limited resources can be concentrated on the isolation of these potential SARS-CoV-2 carriers, with immediate benefits for the patient, the population and the healthcare system.This study characterizes a single center cohort of consecutive patients with COVID-19 who were followed after recovery and PCR negativity and shown to have one or more recurrent positive PCR results despite strict quarantine. The primary objective was to describe the kinetics of SARS-CoV-2 PCR in a large cohort of infected but recovered individuals and better understand the relevance of recurrent SARS-CoV-2 positivity. The secondary objective was to develop a prediction algorithm to identify patients at high risk of recurrent SARS-CoV-2 positivity and help understand reactivation and reinfection possibilities to establish protocols for health policy.All methods were performed in accordance with relevant guidelines and regulations. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Southern University of Science and Technology. Written informed consent was obtained from the participants. For participants under the age of 18 years, written informed consent were obtained from their parents or legal guardians.8.The study cohort included consecutive COVID-19 patients admitted to The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, China since January 11, 2020. The last follow up date was April 23, 2020. All discharged COVID-19 patients were subjected to strict quarantine at a designated center for 14 days. SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNA testing was performed every 3–5 days during both hospitalization and quarantine. Follow-up at home quarantine was mandated for an additional 14 days with weekly SARS-CoV-2 qRT-PCR testing. Upon positive NP swab testing, according to the local health policy, these patients were immediately readmitted back to the hospital  history, typical clinical manifestations  and laboratory diagnosis. Pulmonary respiratory syndrome severity was classified into 4 categories: (1) mild: mild respiratory symptoms, no imaging findings of pneumonia; (2) moderate: fever, respiratory symptoms, imaging findings of pneumonia; (3) severe: shortness of breath, respiratory rate > 30 breaths/min, systemic oxygen saturation < 93% at rest on room air, ratio of the systemic arterial partial pressure of oxygen to the fraction of oxygen in inspired air ≤ 300 mmHg, or > 50% progression of radiologic pulmonary lesions over 24 to 48 h; (4) critical: needing mechanical ventilation, extracorporeal membrane oxygenation, or other organ support therapy in the ICU. Discharge criteria included: being afebrile for at least three days, improvement of respiratory symptoms and radiological abnormities in CT or X-ray, and two consecutive negative NP swab tests sampled > 1 day apart. All patients were discharged under strict monitoring conditions: patients were kept for 14 days at a designated center followed by another 14 days at home, in quarantine. All discharged patients were followed up and tested with repeated NP swab PCR analyses. According to the local policy, patients with a positive NP swab test during quarantine were immediately readmitted back to the hospital.Diagnosis, disease severity, treatment and follow-up criteria for COVID-19 infection were based on the preliminary diagnosis and treatment protocols (6th edition) from the National Health Commission of China10.Early morning NP swabs were analyzed every 3 days during the hospitalization, every 3–5 days during mandated quarantine at a designated center, and weekly during quarantine at home. Bronchoalveolar lavage washing was sampled from patients with severe illness or undergoing mechanical ventilation. Total RNA was extracted from the clinical specimens using the QIAamp RNA Viral Kit . A qRT-PCR Test Kit  targeting the ORF1ab and N genes of SARS-CoV-2 was used. A cycle threshold (Ct)value less than 37 was interpreted as positive for SARS-CoV-2 RNAEscherichia coli to be used as coating antigen of the indirect immunoassays in the IgG antibody kits.SARS-CoV-2 antibody Chemiluminescent microparticle immunoassay (CMIA) kit  was used to detect SARS-CoV-2 IgM and IgG in plasma. According to the manufacture’s protocol, total antibody detection was based on double-antigens sandwich methodology and the IgM antibody detection was based on μ-chain capture immunoassay. To develop total antibody and IgM antibody assays, recombinant antigens, including the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, were expressed in a mammalian cell system. A recombinant nucleoprotein of SARS-CoV-2 was expressed in Patients who had a positive NP swab test during post-discharge follow-up and were readmitted to hospital were defined as 'case'. Patients who did not have positive results of NP swab test after discharge were analyzed as ‘control’ patients.P values for feature filtering. For multivariate model building, a gradient boosting tree algorithm XGBoost was used for constructing a multivariable prediction model. The binary logistic function was used as the objective function. Parameters such as the learning rate (set to 0.3), the minimum child weight (set to 6.8), the maximum tree depth (set to 3), and the subsample fraction of a single tree (set to 0.6) were all tuned by cross validations to avoid over-fitting. The derived model score ranged from 0 to 100 describing the probability of recurrence after discharge. The recurrence prediction model was evaluated using area under the receiver-operating-characteristic curve (ROC AUC), sensitivity, and specificity from tenfold cross-validation. In the cross validation, all the samples were randomly split into 10 equal-sized subgroups, with ratio of cases and controls remaining consistent in each subgroup. Each time 9 out of the 10 folds were picked to train a model the remaining fold was for testing. The process was repeated 10 times so that every sample had a score. Statistical analyses were performed using R software (version 3.5.1).Features including demographics, comorbidities, symptoms, vital signs, laboratory findings, and treatments were assembled for modelling. Univariable analysis was performed on z-score-normalized features, and logistic regression was used to calculate the odds ratios and 9: typical clinical manifestations  and NP swab PCR positivity. These admitted patients were categorized to have mild (N = 16), moderate (N = 309), severe (N = 73), or critical (N = 19) conditions of pulmonary respiratory syndrome. Death occurred 3 of 417(0.7%) patients during initial hospitalization. Of the remaining 414 patients alive, 69  patients were with recurrence of NP swab PCR positivity and had ≥ 1 readmission(s). Demographics, clinical data, PCR data, and outcomes from the first hospitalization were summarized . The majority (93%) of case patients had mild or moderate pulmonary respiratory syndrome at the first admission, and had respiratory symptoms including cough (33.2%) and increased sputum (25%) at the recurrence of PCR positivity. Ct values measured at the 1st hospitalization were similar in the case and control patients  between the first NP swab test negative and test positive results  of the case group were IgG positive, and 30 (75%) were IgM positive  of both IgM and IgG against SARS-CoV-2. This suggests thatconvalescent patients, including aging populationsP values and utilized by the XGBoost algorithm for the final model  based on tenfold cross-validation , the observed overall rate and multiple recurrence of positive viral testing are unlikely to be from re-infection. This contrasts with the recently case reports in South Korea7. Importantly, it must be noted that the positive SARS-CoV-2 test does not equate with infectivity. Since the current standard for SARS-CoV-2 test positivity is predicated on viral load detection by PCR6, sample testing in the case cohort of < 106 copies per sample may not represent a live virus isolate. Unless equipped with a live virus isolate, we cannot be certain whether these retest positive patients were capable of infecting others (contagious) given that they were quarantined at a designated center. To address the possibility of false positive SARS-CoV-2 PCR results, every retest was validated by examinations at both laboratories of Center for Disease Control in Shenzhen and the Second Affiliated Hospital of Southern University of Science and Technology. 95% tests were consistent13. The PCR retest positivity cannot differentiate re-infectivity, relapse, and dead-viral RNA detection. Handoff note, a postmortem pathological study found that SARS-CoV-2-viruses still remained in pneumocytes of a recovered COVID-19 patient with a negative PCR test result7.COVID-19 patients with multiple recurrences of PCR positivity have not been previously reported. Thirteen and three cases recurred two and three times with PCR positivity, respectively, making it likely that the re-emergence of PCR positivity is due to low levels of viral RNA right at the level of detectability by PCR assay and therefore is at times below this threshold and other times above. The Ct values observed in the samples after the initial positive samples are all high suggesting potential non-viable viral RNA. However, the reimmigration of the virus from the lower to the upper respiratory tract cannot be ruled out6, e.g. temperature (Supplementary Fig. 14. The two readmitted febrile patients with typical clinical manifestations satisfying the COVID-19 diagnostic and admitting criteria9 may have been capable of transmission given the presence of positivity with both viral load testing and COVID-19 admission symptoms.Ninety-three percent of the retest positive patients had mild or moderate severity disease during their first hospitalization. No obvious trending was found in this case series between the initial viral load and first admission symptoms as has been previously reported28 and immunity.The key rationale of the local health policy for implementing the described strict center-based quarantine is to prevent transmission. Our findings demonstrate that the effectiveness of this quarantine strategy in the management of the pandemic may be crucial in minimizing late transmission. They also provide insight into the case management of retested positive patients with or without typical COVID-19 symptomsDetermining the kinetics of NP PCR and those at highest risk for recurrence would provide valuable information to understand if these individuals pose a public health risk for infecting others or individual risk for disease reactivation. Given that the majority of the individuals who were shown to have recurrent NP PCR positive had mild disease, this suggests that the amount of virus is at the threshold of detection and the discordant results may be a result of detectability of the assay.29. The algorithm described here enables different case management strategies upon risk stratification, and facilitates the incorporation of different assumptions about the impact of the intervention and quarantine (Supplementary Fig. Determining those at highest and lowest risks of recurrence of PCR positivity may be an essential component of any strategy to enable readmissions, guide interventions, prevent transmissions, and optimize limited care resource utilization. The analysis described herein builds on our previously validated machine-learning models to predict hospital readmission to develop COVID-19 readmission risk prediction toolThe driving factors for this model can be collected in any inpatient settings, and therefore it is portable internationally for immediate validation to provide clinical utility in the current COVID-19 pandemic.This study has several limitations. First, the study population only included COVID-19 patients within a single center in Southern China during COVID-19 pandemic. The imbalance between case and control numbers may compromise the comparability of both groups, limiting the translation of these real-world clinical observations into evidence. Second, the IgM/IgG tests were not performed on all patients due to the late introduction of the antibody test. Thirdly, the low specificities shown in Supplementary Table This case series demonstrates that recurrence of SARS-CoV-2 RNA positivity is relatively common, with a 16.7% rate, in recovered COVID-19 patients. Younger patients with less severe initial illnesses were more likely to retest positive. More information will be required to understand the relevance of these findings, which will be critical for informing the care management of the pandemic. Our prediction algorithm to identify patients at high risk of recurrence of SARS-CoV-2 positivity may establish protocols for better COVID-19 health policy.Supplementary Information.


AIP mutations lead to young-onset macroadenomas that are difficult to treat. Most are growth hormone secreting tumors, but all other secretory types can exist and the clinical profile of affected patients is variable. We present an overview of the current understanding of AIP mutation-related pituitary disease and illustrate various key clinical factors using examples from one of the largest AIP mutation-positive FIPA families identified to date, in which six mutation-affected members with pituitary disease have been diagnosed. We highlight various clinically significant features of FIPA and AIP mutations, including issues related to patients with acromegaly, prolactinoma, apoplexy and non-functioning pituitary adenomas. The challenges faced by these AIP mutation-positive patients due to their disease and the long-term outcomes in older patients are discussed. Similarly, the pitfalls encountered due to incomplete penetrance of pituitary adenomas in AIP-mutated kindreds are discussed.Clinically-relevant pituitary adenomas occur in about 1:1000 of the general population, but only about 5% occur in a known genetic or familial setting. Familial isolated pituitary adenomas (FIPA) are one of the most important inherited settings for pituitary adenomas and the most frequent genetic cause is a germline mutation in the  AIP) gene [AIP mutations lead to pituitary adenomas that have a distinctive clinical profile with a significantly younger onset, more aggressive tumor growth and larger tumor diameter at diagnosis, as compared with non-AIP-associated pituitary tumors [AIP mutations, the young onset and aggressive tumor growth mean that AIP mutations are the main genetic cause of pituitary gigantism (about 30% of cases) [AIP mutations are of great clinical relevance as they are difficult to treat due to large tumor size and high hormonal secretion levels; additionally, AIP mutations confer relative resistance to first-generation somatostatin analogs [Clinically relevant pituitary adenomas have a prevalence of about one per thousand in the general population and are an important cause of morbidity due to the combined impact of hormonal dysfunction and tumoral mass effects ,2. AboutIP) gene ,8; rare IP) gene ,10. AIP y tumors . As somaf cases) . Pituita analogs .AIP mutations and deletions affecting various families and individuals [AIP mutations can present in the FIPA setting, we describe and discuss the experiences of a large kindred with six affected individuals with AIP mutations and pituitary adenoma-related disease.Results from large international studies have identified many hundreds of FIPA kindreds and have characterized a wide variety of ividuals ,14. WithA 39-year-old man attended the endocrinology outpatient department for routine follow-up. He had chronic hypopituitarism involving the gonadal, thyroid and adrenal axes, for which he was receiving testosterone gel 50 mg/day, levothyroxine 200 μg/day and hydrocortisone 20 mg/day. He had developed hypopituitarism secondary to the treatment of a GH secreting macroadenoma diagnosed 23 years previously at the age of 16. He had been diagnosed at that time with acromegaly following a one-year history of frontal headaches and worsening visual disturbances accompanied by acral overgrowth and increasing height (183 cm). A CT scan at the time identified a pituitary macroadenoma with suprasellar extension. He was operated via a transcranial approach and, as he had increased GH and IGF-1 levels postoperatively, he was treated with external beam radiotherapy. His postoperative MRI showed a 10 mm residue. Over time, his GH and IGF-1 levels gradually came under control and at the last outpatient visit, his nadir GH after an oral glucose load was <0.1 ng/mL and his IGF-1 was 137.1 ng/mL . In keeping with the treatment and hormonal profile, the current MRI showed no evidence of pituitary adenoma and a small linear residue of pituitary tissue on the sellar floor, accompanied by retraction of the optic chiasma A. Commentary: The clinical history of the index patient typifies many aspects of AIP mutation-related pituitary adenomas. Patients with AIP mutations are at increased risk of developing early onset pituitary macroadenomas; the median age at diagnosis is 21 years and nearly 80% are diagnosed before the age of 30 [AIP mutated pituitary adenomas are male [AIP mutation-related pituitary adenomas are usually somatotropinomas, as in this patient’s case, or mixed GH and prolactin secreting tumors [AIP mutations [AIP mutation-related somatotropinomas have a significantly higher number of deficient axes compared with non-AIP mutated acromegaly patients [ge of 30 . A majorare male . AIP mutg tumors . These butations . The patpatients .The patient had a strong family history of pituitary disease. One uncle and one aunt were known to be affected with pituitary adenomas and the family had been investigated and were negative genetically for multiple endocrine neoplasia type 1 (MEN1). Therefore, the kindred were diagnosed with FIPA. AIP gene; sequencing was performed on peripheral blood leukocyte DNA as described previously [Investigations in the index case and the family were undertaken. The data collection was approved by the Ethics Committee of the CHU de Liège . A family tree was constructed, as shown in AIP gene in the index case revealed a heterozygotic c.543delT mutation. In the kindred, a total of 14 individuals agreed to testing for the c.543delT AIP mutation. Screening was performed, where possible, in a cascade manner from older to younger generations in order to focus testing on members with potential mutation carrier status. As noted above, there were two previously-identified members of the FIPA kindred with a diagnosis of pituitary adenomas.Genetic investigation of the AIP mutation-positive individuals (six with pituitary adenomas or a strong history of apoplexy) among 14 members of the kindred that were tested  and most are GH secreting or mixed GH/prolactin secreting tumors [AIP mutation carriers, as part of a comprehensive clinical assessment that focusses on signs and symptoms suggestive of a pituitary adenoma or hormonal dysregulation [AIP mutation in FIPA, kindred, clinical, hormonal and genetic assessments were undertaken throughout the family. The familial AIP mutation was confirmed in the two previously known pituitary adenoma family members and new affected cases and mutation carriers were also identified on screening:g tumors . Geneticgulation . Given tAIP mutation. He was diagnosed with a pituitary tumor at the age of 16, which had led to increased growth. His height was 170 cm, but this was complicated by severe scoliosis that occurred in adolescence. He underwent cranial surgery and radiotherapy >50 years previously overseas and detailed pathological records regarding tumor characteristics are unavailable. He had not attended for follow-up for many years and on his most recent hormonal profile he was found to have central hypothyroidism, which was corrected. His nadir GH was 0.3 ng/mL and his IGF-1 was low at 76.2 ng/mL ; his other hormonal axes were normal. MRI revealed a cystic sellar lesion measuring 19 × 14 × 7 mm , prolactin was normalized, her tumor shrank and her menses returned. After extended treatment and normal prolactin levels, bromocriptine was withdrawn at the age of 45 and she was later lost to follow-up.AIP mutation diagnosis in the index patient, she was re-contacted and when the prolactinoma history was learned of, she was offered hormonal and imaging screening in addition to genetic testing. In the intervening years she had been diagnosed with type II diabetes mellitus and hypertension. On examination, she had acromegalic features and complained of increased perspiration. Hormonal studies revealed hyperprolactinemia and an IGF-1 level of 373 ng/mL . Her GH was suppressed to <1 ng/mL on an oral glucose tolerance test, but was not below the limit of detection. A pituitary MRI revealed an intrasellar pituitary adenoma of 10 × 9 × 8 mm in size . Interestingly, her elevated IGF-1 also normalized with cabergoline treatment . After two years’ follow-up on cabergoline 2 mg/week, her prolactin and IGF-1 remained normal and her pituitary tumor shrank to 5 × 6 × 4.8 mm in size.During investigation of the family following the  in size C. The faCommentary: Prolactinomas are a known, if less frequent (14.5%), manifestation of AIP mutation-related pituitary disease [AIP-mutation-related prolactinomas—like all resistant prolactinoma types—more frequently affect males, as opposed to sporadic prolactinomas that are seen overwhelmingly in females. Individual III-7 was diagnosed at the age of 30 after some years of secondary amenorrhea and she responded well to bromocriptine. As such, her characteristics initially were those of a typical non-AIP mutation mutation-related prolactinoma in a female of childbearing age and her therapeutic response to a habitually-used dose of a dopamine agonist was excellent. After withdrawing bromocriptine following 15 years of therapy, she suffered a late recurrence of the pituitary adenoma, which, interestingly, was associated with both prolactin hypersecretion and elevated IGF-1. This suggests that she had a mixed GH- and prolactin-positive tumor at the time of recurrence, which is more typical of AIP mutation-related pituitary adenomas. Interestingly her prolactin and IGF-1 both responded to cabergoline at the upper end of the labeled dose range. The tumor, however, shrank by about 50% in size, unlike her original prolactinoma, which shrank completely on bromocriptine. Due to the lack of GH and IGF-1 results from the time of her original diagnosis, we do not know if there was always evidence of mixed hormone secretion or if this case represents an evolution from prolactinoma to mixed GH/prolactin-secreting adenoma. disease . While f disease ,14,15. AAIP mutation. She presented initially to a gynecologist at the age of 28 with a two-year history of amenorrhea. She was treated with estrogen therapy for 10 years but no other investigations were conducted at the time. On stopping treatment her amenorrhea returned but she did not seek further medical advice. At the age of 48 she complained of headaches and visual disturbances and a CT scan of the brain was performed at another institution; she was diagnosed at the time with a non-functioning pituitary macroadenoma. She underwent a transcranial resection that was accompanied by external beam pituitary irradiation. At the time of the current genetic analysis, she had not attended an endocrine follow-up for more than 20 years. On physical examination, she had no features of acromegaly. An MRI showed a pituitary tumor residue of <10 mm in diameter. Laboratory studies showed hypopituitarism affecting her thyroid axis, while her gonadotropins were low. She was started on levothyroxine and is currently well, having regular follow-up. Individual III-3 is the 74-year-old maternal aunt of the index case who was also heterozygous for the c.543delT Commentary: Non-functioning pituitary adenomas comprise a minority of AIP mutation mutation-related pituitary adenomas, they constituted 6.5% of cases in our comprehensive review [AIP mutation-positive FIPA families with homogenous presentation of non-functioning adenomas have been reported [AIP mutations share the same early onset of disease and large tumor size seen in AIP-associated acromegaly and prolactinoma [AIP mutations in that while she was not cured by initial surgery, following radiotherapy, and during long-term follow-up, the tumor size was controlled without regrowth [e review . They careported ,16. In tactinoma ,15,17. Iregrowth .AIP testing. On examination he was noted to be tall (197 cm) and he had a hypogonadal phenotype. He denied any past medical history but noted that he had been much taller than his peers during late childhood to mid-adolescence. He recalled that at the age of 16, his pubertal development stopped and he gradually lost his male pattern pubic hair distribution. Hormonal testing revealed hypopituitarism involving the somatotrope, thyroid and gonadal axes. Pituitary MRI showed an empty sella with erosion of the sellar floor. AIP testing revealed the familial c.543delT mutation. He received replacement therapy for his thyroid and gonadal insufficiencies (he declined GH replacement). Upon follow-up, at the age of 67, he was diagnosed with an adenocarcinoma of the transverse colon (pT3N2M1). This responded poorly to surgery and chemotherapy and he died due to metastatic progression.Individual III-9: As part of the family genetic study, this 62-year-old man was contacted and offered clinical assessment and Commentary: Patients with AIP mutation mutation-related pituitary adenomas have early disease onset and usually present with a macroadenoma. This implies that once tumorigenesis begins, tumor growth is rapid, as has been captured during serial MRI recently [AIP mutation-related pituitary adenomas grow rapidly comes from the reports of pituitary apoplexy seen in this population [AIP mutations can present with familial pituitary apoplexy as the only clinical disease manifestation [recently . Anotherpulation ,14,16,19estation . Individestation ,22,23. HAIP might be associated with tumor risk in other endocrine and non-endocrine organs. Unlike “classical” multiple endocrine neoplasia (MEN) risk genes such as MEN1, PRKAR1A or CDKN1B, which lead to multi-site tumorigenesis syndromes, AIP mutations only manifest to a reliable degree in the pituitary. Screening studies in other sporadic cancers like breast and colon or in non-MEN1-related MEN patients have shown no definitive link to AIP variants [AIP locus in tumoral tissue [AIP mutation-affected patients were seen, including metastatic colorectal cancer, bronchial carcinoid and a meningioma. In the case of the colorectal cancer in individual III-9, which occurred after the diagnosis of the AIP mutation, it was possible to study this cancer for AIP status. Study of the resected colorectal tumor specimen demonstrated low levels of AIP immunohistochemical staining and there was no loss of heterozygosity at the AIP local in colorectal tumor DNA. These data indicate strongly that the colorectal cancer was not associated with the germline AIP mutation; moreover, while AIP immunostaining is a valuable marker in colorectal cancer, increased AIP staining intensity is associated with poorer outcome [Since it was first identified in 2006 as a pituitary adenoma risk gene by Aaltonen’s group in Helsinki, much interest has been focused on whether variants ,25,26. Hl tissue ,27,28,29 outcome .AIP mutation. The first  was the 71-year-old mother of the index case. Clinical, biochemical and radiological screening were within normal limits initially and during long-term follow-up  was the 37-year-old brother of the index case. He was entirely asymptomatic and has normal clinical findings and hormonal testing. On MRI, a 6 mm diameter pituitary adenoma was identified. He remains asymptomatic on follow-up, without any change in size of his pituitary adenoma or hormonal abnormalities. Cascade screening studies were practically useful as they permitted the identification of one individual in generation II, five in generation III and one in generation IV who were wild-type for AIP. In total, a further 23 offspring of these unaffected non-carriers could be excluded from AIP mutation-related follow-up investigations, which has a number of advantages in terms of avoided stresses to individuals and unnecessary costs relating to hormonal and radiological testing.A further two individuals were found to be positive for the familial ollow-up D. The seCommentary: When an individual with an AIP mutation undergoes clinical and hormonal studies that are normal but nevertheless MRI reveals a pituitary adenoma, it raises a series of questions regarding etiology and follow-up. This is the case we encountered in the AIP mutation-positive individual IV-7, the brother of the index patient. His investigations demonstrated a pituitary microadenoma that was clinically and hormonally silent and did not grow over the course of some years of follow-up. The temptation is to classify the pituitary adenoma in this AIP mutation affected person as being AIP-related, however, several issues argue strongly against it. It must be recalled that pituitary adenomas occur at a high frequency in the general population, with one in five people having an unsuspected adenoma either at autopsy or on MRI performed for another reason . The overwhelming majority of these so-called incidentalomas are non-functioning adenomas of a few millimeters in diameter, which very rarely progress. MRI based screening studies of asymptomatic carriers of AIP or MEN1 mutations unsurprisingly encounter multiple individuals with such small adenomas that do not grow or secrete hormones. In AIP mutation carriers, as seen in individual IV-7, small non-functioning pituitary tumors in an adult are not the typical presentation, which is usually young and with large GH- or GH/prolactin-secreting adenomas. Clinical context is important. Hence, in MRI screening of AIP mutation carriers, the finding of a microadenoma in a child/adolescent/young adult or an adenoma with hormonal abnormalities or rapid growth argues for an AIP-related etiology. As typical aggressive AIP mutation-related pituitary adenomas are only exceptionally (if ever) diagnosed after the age of 30, small, non-progressing, non-functioning adenomas in adult AIP mutation carriers are potentially more likely to be incidentalomas. The molecular features of early pituitary adenoma formation in the setting of AIP mutations are not fully understood; hence it is possible that some smaller adenomas could form in mutation carriers but subsequently fail to develop aggressive characteristics, thereby contributing to the incomplete penetrance seen in this condition.AIP germline mutations in about 20% of cases, and chromosome Xq26.3 duplications in exceptionally rare cases of X-LAG [AIP mutations are associated with pituitary adenomas that usually present at a significantly younger age and with larger tumors than in non-AIP-associated adenomas [AIP mutations are the most frequent known cause of pituitary gigantism [AIP related-pathology come from large international series of hundreds of patients [AIP-mutated kindreds is incomplete and construction of a detailed family tree beyond the first-degree relatives of an index case is crucial to capture all possible cases (including apoplexy or hypopituitarism of unknown origin). The importance of mixed GH and prolactin secreting adenomas in AIP-mutated patients is also highlighted by the experience of one case in the kindred whose profile suggests that mild IGF-1 excess might have developed over time. Such instances of mild mixed IGF-1 and prolactin excess that are successfully treated with dopamine agonists have been noted previously in AIP-associated FIPA families [AIP-mutated FIPA kindreds are necessarily definitively linked to the genetic mutation. Small incidentally found non-secreting pituitary adenomas occur in up to one in five of the general population [AIP mutation carriers undergoing MRI screening, caution should be exercised before definitively labeling such tumors as being “AIP related” pituitary adenomas, as such cases may behave in a manner that is indistinguishable from other incidentalomas on follow-up. In support of this, recent data from screening analyses suggest that nearly half of prospectively diagnosed pituitary adenomas in AIP mutation carriers are non-functioning microadenomas; these non-functioning tumors are usually not associated with hormonal abnormalities and rarely receive any treatment during long-term follow-up [AIP mutation carriers from childhood to early adulthood. Finally, the clinical phenotype seen in FIPA related to AIP appears to be largely isolated to pituitary adenomas and their consequences. Despite a number of associated intracranial and other tumors being reported in the literature, they rarely have associated tumoral genetic studies supportive of an AIP-related role, as was confirmed in a case of metastatic colorectal carcinoma in the current FIPA family. Detailed studies of individual large kindreds like this one can be illustrative of the issues and choices faced by endocrinologists, geneticists and the family members themselves when an AIP mutation and pituitary adenomas are discovered.FIPA is one of the most frequent familial or inherited forms of pituitary adenomas and currently only two genetic causes have been identified: of X-LAG . AIP mutadenomas . They usigantism . These cpatients ,13,14; ifamilies . During pulation . Therefoollow-up . Screeni


The coronavirus disease 2019 (COVID-19) pandemic has emerged as one of the greatest challenges faced by humankind in the recent past. People with diabetes and related comorbidities are at increased risk of its complications and of COVID-19-related death. Older age, multi-morbidity, hyperglycaemia, cardiac injury and severe inflammatory response are predictors of poor outcome. The complex interplay between COVID-19, diabetes and the effects of related therapies is being explored. Most patients experience a mild illness with COVID-19, while people with diabetes are at increased risk of severe disease. Optimising glycaemic control and adopting measures to prevent disease spread are critical aspects. The management of mild disease is supportive, while very many immunomodulatory and antiviral therapies are being investigated for the treatment of severe disease. Several of these agents have specific considerations for use in people with diabetes. Since mass population lockdowns are considered a key step in controlling disease spread, it follows that, in addition to the direct vulnerability to severe COVID-19, people with diabetes can be affected by limited access to healthcare, insulin, other medications and blood glucose monitoring equipment. Measures to prevent disease spread at the individual and community level are the key to mitigating the rapidly escalating pandemic, while agents for chemoprophylaxis and vaccines are being explored. People with diabetes should be recognised as a vulnerable group for complicated disease and are at risk during times of disturbed social systems. Strategies are needed to safeguard the health of patients with diabetes during the pandemic. This review summarises the current knowledge and perceived challenges for prevention and management of COVID-19 in people with diabetes.The online version of this article (10.1007/s00125-020-05164-x) contains peer-reviewed but unedited supplementary material including a slideset of the figures for download, which is available to authorised users. Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) virus, has become a pandemic within a few months after it was first described in Hubei province in China. At the time of writing it had affected over 1,800,000 individuals in more than 200 countries and territories worldwide causing over 110,000 deaths [2:FiO2 < 300; and/or pulmonary infiltrates on >50% of lung fields on radiological imaging) [COVID-19 is highly transmissible from person to person through respiratory secretions. The virus enters through mucous membranes of the upper respiratory tract, later affecting lungs. . In the imaging) . A minorp = 0.005) [Both type 1 and type 2 diabetes increase the susceptibility to infections and their complications . Neutrop= 0.005) . The pre= 0.005) . In anot= 0.005) . Similar= 0.005) .According to the available evidence, people with diabetes do not have a higher susceptibility to SARS-CoV-2 infection . HoweverA larger study of 72,314 patients with COVID-19 in China indicated that patients with diabetes had a threefold higher mortality rate compared with the mortality rate in COVID-19 patients overall (7.3% vs 2.3%) .p = 0.037) [Older age, the presence of two or more comorbidities and obesity also predict poor prognosis among COVID-19 patients , 21. The= 0.037) .Similar to previous studies among patients with influenza and bacterial pneumonia, elevated serum ferritin, lactate dehydrogenase, C-reactive protein (CRP), procalcitonin and erythrocyte sedimentation rate (ESR) predicted severe disease among patients with COVID-19 , 24. ThiAmong 174 COVID-19 patients in Wuhan, China, people with diabetes had a greater inflammatory response , higher incidence of coagulopathy (higher D-dimer levels), metabolic derangements , severe pneumonia  and higher mortality rate, compared with those without . HoweverElevated N-terminal pro-brain-type natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI), were significantly correlated with severe disease, suggesting that COVID-19 may lead to myocardial injury and impair cardiac function . In peopThe receptor binding domain of SARS-CoV-2 uses host ACE2 for fusion of viral and host cell membranes . ACE2 isIn 2003, patients with SARS had higher rates of hyperglycaemia on admission than non-SARS pneumonia patients, irrespective of their pre-morbid glycaemic status, disease severity or glucocorticoid use . SubsequAlthough ACE2 shares some characteristics with ACE1, it is not inhibited by ACE inhibitors (ACEI). In fact, ACEI and angiotensin receptor blockers (ARBs) upregulate ACE2 expression . TherefoFollowing an incubation period of 2–14 days (median 5 days), the majority of people with COVID-19 will present with cough, fever, shortness of breath and, less commonly, nausea and diarrhoea . A late p = 0.02), and deterioration could occur rapidly in later stages [People with diabetes develop similar symptoms. However, the initial manifestation could be milder, fever may be less common  remains the gold standard for diagnosis of SARS-CoV-2 infection . Howeverhttps://easd-elearning.org/covid-19/.People with diabetes need timely integrated interventions to prevent them acquiring the disease. Enhanced self-management, supportive healthcare services and public health measures need to be in place Fig. . If the The COVID-19 pandemic is far from being solely a medical phenomenon. It disrupts personal and professional lives severely and affects people and societies on several levels. The key strategies promoted for containment of an outbreak such as isolation, social distancing and lockdown of cities can have a significant impact on the health and lifestyle of an individual. Management of a chronic disease such as diabetes, which requires dietary modification, regular exercise and good adherence to medications, poses many complex challenges.Maintaining a healthy diet may be challenging because of limited access to appropriate food. Careful portion selection and adjusting mealtime insulin according to carbohydrate intake continues to be the best strategy, particularly for patients on multiple-dose insulin regimens.Adopting a regular exercise plan might not be feasible owing to social distancing, restrictions on outdoor activities and concerns over the high risk of disease spread in gyms and sports centres . Activities such as indoor walking, gardening and stationary high intensity activities may be suitable alternatives to maintain an active lifestyle.Regular monitoring of blood glucose is important. Continuous glucose monitoring (CGM) and flash glucose monitoring systems are useful and allow remote monitoring by healthcare providers . For patients with type 1 diabetes, monitoring of ketone levels (particularly for people who are persistently hyperglycaemic) and vigilance for the development of symptoms of DKA are important.It is also important that people with diabetes have an adequate supply of their medications at home. In fact, increased incidence of DKA among children with type 1 diabetes has been observed due to a delay in seeking medical treatment and in providing routine healthcare for newly developed symptoms, as patients are afraid of contracting COVID-19 from healthcare settings .Routine clinic visits and overcrowding in hospitals should be minimised to reduce disease spread among people with diabetes. Measures such as telemedicine consultations or telephone advice, issue of medication to caregivers at lower risk, online coordination of drug delivery, and dispensing medications for extended periods should be considered by healthcare providers. Non-emergency routine evaluations  should be deferred, as close contact between the healthcare providers and patients may lead to increased risk of COVID-19 transmission. Patients with limb-threatening or vision-threatening complications should be triaged for immediate attention.During this time of uncertainty, fear, helplessness and strong emotions may increase stress in some patients. It is important to ensure psychological wellbeing, as stress may adversely affect glycaemic control. The WHO recommends minimising reading or listening to news that could cause distress and anxiety. Regular sleep routines are important. Relaxation techniques such as meditation can help people with stress and anxiety. Maintaining contact with relatives, friends and neighbours via telephone conversations or using online communication platforms can help to reduce the effects of social isolation .Employers should consider work from home strategies, or furlough, for employees with poorly controlled diabetes or those with cardiac or renal complications, particularly for those in high risk occupations such as frontline healthcare work or similar high risk jobs, and especially in areas with high COVID-19 prevalence.The majority of people with COVID-19 will develop a mild disease that can be managed at home according to local guidelines. Patients with diabetes developing symptoms suggestive of COVID-19 infection should immediately notify local healthcare services to determine the need for diagnostic evaluation, assessment of severity, isolation and the need for hospitalisation. Because there is a higher risk of adverse outcomes, patients with diabetes should be preferentially managed in hospitals or settings where close monitoring of disease progression is possible. For those managed at home, regular telephone contact with healthcare services and follow-up is crucial to recognise deterioration in glycaemic control, development of hyperglycaemic emergencies or deterioration of clinical status.Frequent glucose monitoring, healthy diet, adequate hydration and dose titration of glucose-lowering medication in liaison with healthcare providers should be prioritised. Patients may take symptomatic therapy, including paracetamol/acetaminophen, which is the preferred anti-pyretic agent . HoweverThe importance of good glycaemic control during the COVID-19 pandemic is emphasised . As thisAlthough there are no specific data on sodium–glucose cotransporter 2 (SGLT2) inhibitor use during COVID-19 infection, it is advisable to withhold these during acute illness because of the increased risk of dehydration and euglycaemic ketoacidosis , 53, as Existing evidence favours insulin over other glucose-lowering agents for glycaemic control in hospitalised patients. For non-critically ill hospitalised patients, subcutaneous insulin therapy with basal or intermediate-acting insulin given once or twice a day, along with mealtime boluses of short- or rapid-acting insulin, is the preferred strategy for glycaemic management . SlidingIn patients with type 1 diabetes with COVID-19 and hyperglycaemia, it is important to monitor the blood glucose and ketone levels, maintain hydration and continue insulin therapy.Strict infection control measures should be implemented for COVID-19 patients with diabetes irrespective of where they are treated, considering their vulnerability to acquiring secondary bacterial infections, with potential serious consequences.ACEI/ARBs are essential in management of hypertension, heart failure and diabetic nephropathy. Considering the contrasting effects of ACE2 on CoV infection and inflammatory lung injury  is thought to be central to the pathogenesis of rapid deterioration and multi-organ dysfunction in patients with COVID-19. Therefore, immunomodulatory agents are postulated to be of benefit.1. Chloroquine/hydroxychloroquine These two antimalarial agents have attracted much attention in the treatment of COVID-19 on the basis of limited clinical experience, as well as due to political reasons. Chloroquine has shown antiviral and anti-inflammatory properties in previous experimental studies [ studies . Hydroxy studies  and has  studies .A study from France, involving 36 virologically confirmed patients with COVID-19, showed that chloroquine, at a dose of 600 mg a day for 10 days, reduced the virus carriage significantly at 6 days of therapy compared with standard care . HoweverA systematic review of literature by Cortegiani et al. concluded that evidence is limited to experimental in vitro studies showing antiviral potency of chloroquine/hydroxychloroquine . More thThere are specific safety concerns for people with diabetes, as hypoglycaemia is a known adverse effect of chloroquine/hydroxychloroquine treatment. Suggested mechanisms are decreased intra-cellular insulin degradation, increased insulin-mediated glucose transport, increased insulin release and enhanced insulin sensitivity . TherefoProlongation of the QT interval and serious cardiac arrhythmias are known, albeit rare, dose-dependent adverse effects of chloroquine/hydroxychloroquine. The risk is greater in those with pre-existing cardiac disorders, especially comorbid coronary artery disease and diminished cardiac reserve. COVID-19 itself is known to induce myocardial injury, adding to the risk. Regular ECG monitoring before and while on therapy is recommended. Some guidelines recommend against combining with other QT prolonging agents such as azithromycin, lopinavir/ritonavir while others recommend cautious use (ESM Table 2. Glucocorticoids Although glucocorticoids are used in the treatment of severe acute respiratory distress syndrome (ARDS), data for their use in ARDS caused by viral pneumonia are minimal and therefore they are not recommended for routine use in COVID-19 [COVID-19 . The plaThe FDA has authorised emergency use of remdesivir, which, although not affecting mortality, significantly shortens the course of the disease . Further1. Metformin Yu et al. showed that metformin reversed lipopolysaccharide-induced pulmonary oedema, vascular leakage and neutrophil accumulation, and reduced the levels of TNF-α, IL-1β, IL-6 and IL-17 in an ARDS model [DS model . PatientDS model .2. Incretin based therapies DPP4 is a ubiquitous type II transmembrane glycoprotein expressed in many cells, including the alveolar epithelium and inflammatory cells. MERS-CoV uses DPP4 to gain entry into host cells [st cells . DPP4 inst cells . It is nst cells . Similarst cells . HoweverGeneral precautions are mandatory for patients and caregivers, to prevent contracting COVID-19 . Chemoprophylaxis (pre- and post-exposure) and vaccines are other strategies under evaluation.No agent had been approved so far for pre- or post-exposure chemoprophylaxis. Evidence from randomised clinical trials is urgently needed. Chloroquine has demonstrated antiviral activity against five out of seven known human coronaviruses, including COVID-19  and is aA safe and potent vaccine would obviously be very useful for high risk individuals, such as those with diabetes or cardiovascular disease and the elderly. Several vaccines are being investigated: the APICTH trial: recombinant novel coronavirus vaccine (adenovirus type 5 vector) (NCT04313127); mRNA-1273 vaccine (NCT04283461) and artificial antigen-presenting cells (aAPCs) as a vaccine (NCT04299724).COVID-19 has emerged as one of the greatest challenges for humankind after the Second World War. Identification of effective preventive and treatment strategies is urgently needed. People with diabetes and related comorbidities have been shown to fare worse, although the pathophysiological and molecular mechanisms behind this link are not yet fully understood. Researchers and authorities worldwide should take urgent steps to answer critical questions in the prevention and management of COVID-19 and the protection of people with diabetes (Text box: Unanswered questions).It is imperative to establish standard case definitions, data collection, recording and sharing strategies and operational guidelines to allow comparison and analysis of data. Standardisation of research protocols and identification of research priorities is essential to utilise time and resources productively. The role of pharmaceutical agents in the prevention and treatment of COVID-19, in terms of their efficacy, safety and cost effectiveness, should be evaluated as a priority. Further data are needed, especially looking at the effects of ACEI/ARBs and SGLT2 inhibitors in those infected, as well as in the severely ill.Healthcare systems should adopt strategies for case detection and treatment while maintaining care and supply of essential medicines for people with chronic diseases such as diabetes, to reduce morbidity and mortality risk due to such diseases during this period. The strategic utilisation of human resources in healthcare services and safeguarding their health is a timely need. The current challenge for healthcare systems should be an opportunity to improve service provision, learn from successful regional and global strategies and prepare for future challenges of greater magnitude. The pandemic also highlights the need for joined-up public health measures and care-for-all policies.ESM Tables(PDF 238 kb)Slideset of figures(PPTX 495 kb)


Without RI, median PFS was 12.7 months with Isa-Pd (n = 87) and 7.9 months with Pd . The overall response rate (ORR) with and without RI was higher with Isa-Pd (56 and 68%) than Pd (25 and 43%). Complete renal response rates were 71.9% (23/32) with Isa-Pd and 38.1% (8/21) with Pd; these lasted ≥60 days in 31.3% (10/32) and 19.0% (4/21) of patients, respectively. Isa pharmacokinetics were comparable between the subgroups, suggesting no need for dose adjustment in patients with RI. In summary, the addition of Isa to Pd improved PFS, ORR and renal response rates.The randomized, phase 3 ICARIA-MM study investigated isatuximab (Isa) with pomalidomide and dexamethasone (Pd) versus Pd in patients with relapsed/refractory multiple myeloma and ≥2 prior lines. This prespecified subgroup analysis examined efficacy in patients with renal impairment . Isa 10 mg/kg was given intravenously once weekly in cycle 1, and every 2 weeks in subsequent 28-day cycles. Patients received standard doses of Pd. Median progression-free survival (PFS) for patients with RI was 9.5 months with Isa-Pd ( Renal impairment (RI) affects up to 50% of patients with multiple myeloma (MM) . In mostPatients with RI are often excluded from or underrepresented in clinical trials , 5, and Based on its anti-myeloma capacity, and a route of metabolism that is unaffected by renal function, bortezomib-based regimens are currently recommended by IMWG as the first choice of treatment for patients with MM-related RI . BortezoCarfilzomib has also been shown to produce renal responses. Subgroup analysis of the ENDEAVOR study demonstrated complete renal response rates of 15.3% among patients with relapsed/refractory MM (RRMM) treated with carfilzomib and dexamethasone, with those achieving complete renal responses demonstrating longer median progression-free survival (PFS) and overall survival (OS) than patients who did not achieve renal response . HoweverIxazomib in combination with lenalidomide and dexamethasone has shown good antitumor activity in patients with RI , but no One of the most widely used immunomodulatory therapies, lenalidomide, is renally excreted, and so requires dose adjustment in patients with RI, but is still considered to be effective for the management of mild-to-moderate RI .Pomalidomide plus dexamethasone is well tolerated in patients with MM refractory to lenalidomide and bortezomib, including those with RI . Unlike P = 0.001)  75.6%) in patients with severe RI  plus Pd versus Pd in heavily pretreated patients with RRMM —shows thThe addition of Isa to the Pd backbone in patients with RI in ICARIA-MM resulted in a HR for PFS of 0.50  in favor of patients receiving Isa-Pd versus Pd, consistent with the observed PFS benefit of Isa-Pd in patients without RI  and the overall study population  . PFS obsThe ORR was greater with Isa-Pd than Pd in patients with RI (56.4% vs 24.5%) and without RI (67.8% vs 42.7%), again consistent with the overall study population  . Indeed,Although OS data for ICARIA-MM are not fully mature, a favorable HR of 0.53 can already be observed with the upper limit of the 95% CI not crossing 1 .Notably, compared with Pd, Isa-Pd increased the proportion of RRMM patients with RI who achieved both complete  and sustained  renal responses, supporting a role for Isa-Pd in achieving durable reversal of RI. Correspondingly, fewer patients in the Isa-Pd group compared with the Pd group experienced worsening of renal function to severe RI or ESRD. In these renally impaired patients, tumor response rates were higher in those with renal response than those without renal response. The higher rate of renal response experienced by patients in the Isa-Pd arm contributes importantly to the higher tumor response and hence benefit of this regimen over Pd.The results of the ICARIA-MM subgroup analysis provide the first evidence of improvement in renal function with a CD38-targeted therapy in patients with RRMM. Data on the anti-CD38 agent daratumumab in RRMM patients with RI are currently limited to small uncontrolled studies or case reports with single patients , 24. Dat2.In contrast with lenalidomide , pomalidThe addition of Isa to the Pd backbone had a manageable safety profile in RRMM patients with and without RI. The incidence of TEAEs of any grade or of grade ≥3 was broadly similar between the renal subgroups. TEAEs observed to occur at a more than 10% higher incidence in the Isa-Pd arm in patients with versus without RI were cardiac disorders (any grade), musculoskeletal disorders (grade ≥3), and infections (any grade and grade ≥3), with pneumonia identified as a driver behind the increased grade ≥3 infections. Grade ≥3 pneumonia was also observed to have a more than 10% higher incidence in patients with RI than those without RI in the Pd arm, suggesting that patients with RI should be carefully monitored for lower respiratory tract infections. Incidence of infusion-related reactions were similar between the RI and non-RI groups. Among patients with RI at baseline, the incidence of grade ≥3 and serious TEAEs was greater in the Isa-Pd versus Pd arm, but this did not translate to an increased incidence of grade 5 TEAEs or TEAEs leading to treatment discontinuation. Notably, patients in the Isa-Pd arm had increased treatment exposure compared with patients in the Pd arm, which may have led to a higher incidence of AEs in the Isa-Pd arm. When adjusted for difference in exposure, the event rate of serious TEAEs per patient year for patients with RI was similar in the Isa-Pd and Pd arms, and the event rate of serious infections per patient-year was similar in the two study arms.Rather than being renally excreted, protein therapeutics are eliminated from the body nearly exclusively by proteolysis, to which the kidneys may substantially contribute as a major site for protein catabolism . The PK The large study population, combined with the multicenter, prospective design, support the study findings as representative of Isa safety and efficacy in these patients. Furthermore, fixed-volume infusion (250 mL) of Isa may help with the management of fluid balance in patients with RI , 29. WhiIn summary, the addition of Isa to Pd improved PFS and ORR in patients with RI, consistent with the benefit observed in patients without RI and the overall RRMM study population. Isa-Pd also increased the number of patients with reversal of RI and sustained renal responses compared with Pd. Based on these findings, and the absence of any need for dose adjustments based on renal function, Isa-Pd represents a valuable treatment option for patients with RRMM presenting with renal dysfunction.Supplementary Table