The Centers for Disease Control and Prevention has recommended using a nucleic acid amplification test (NAAT) for diagnosing pulmonary tuberculosis (TB) but there is a lack of data on NAAT cost-effectiveness.We conducted a prospective cohort study that included all patients with an AFB smear-positive respiratory specimen at Grady Memorial Hospital in Atlanta, GA, USA between January 2002 and June 2008. We determined the sensitivity, specificity, and positive and negative predictive value of a commercially available and FDA-approved NAAT  compared to the gold standard of culture. A cost analysis was performed and included costs related to laboratory tests, hospital charges, anti-TB medications, and contact investigations. Average cost per patient was calculated under two conditions: (1) using a NAAT on all AFB smear-postive respiratory specimens and (2) not using a NAAT. One-way sensitivity analyses were conducted to determine sensitivity of cost difference to reasonable ranges of model inputs.During a 6 1/2 year study period, there were 1,009 patients with an AFB smear-positive respiratory specimen at our public urban hospital. We found the NAAT to be highly sensitive (99.6%) and specific (99.1%) on AFB smear-positive specimens compared to culture. Overall, the positive predictive value (PPV) of an AFB smear-positive respiratory specimen for culture-confirmed TB was 27%. The PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was significantly higher for HIV-uninfected persons compared to those who were HIV-seropositive . The cost savings of using the NAAT was $2,003 per AFB smear-positive case.Routine use of the NAAT on AFB smear-positive respiratory specimens was highly cost-saving in our setting at a U.S. urban public hospital with a high prevalence of TB and HIV because of the low PPV of an AFB smear for culture-confirmed TB. The U.S. Centers for Disease Control and Prevention (CDC) has recommended that a nucleic acid amplification test (NAAT) be used routinely for the diagnosis of pulmonary tuberculosis (TB) Grady Memorial Hospital (GMH) is 1,000-bed urban university-affiliated public teaching hospital located in a high TB incidence area in Atlanta, GA, USA. GMH provides care to a large number of persons living with HIV. The annual number of TB cases at GMH gradually decreased from >150 in the late 1990s Mycobacterium tuberculosis (MTB) culture results. As part of program evaluation, we conducted a cost analysis study to assess the impact of the routine use of a commercially available NAAT (amplified MTD) on AFB smear-positive respiratory specimens for early exclusion of TB in a "real-world" urban setting where HIV and TB are both prevalent diseases.In 1992, GMH implemented an enhanced respiratory isolation policy that requires airborne infection isolation (AII) of all patients with active TB, TB in the differential diagnosis, and HIV-infected patients presenting with respiratory symptoms and/or an abnormal chest radiograph M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae were used to identify mycobacterial isolates to the species level. The Amplified MTD (Mycobacterium Tuberculosis Direct) test (Hologic/Gen-Probe) was the NAAT used for M. tuberculosis; it was performed according to the manufacturer's recommendations. In the case of indeterminate results, the assay was repeated. For quality assurance the laboratory conducted quality control with positive and negative controls daily. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the NAAT performed on AFB smear-positive respiratory specimens were calculated using an AFB culture positive for MTB as the reference (gold) standard for diagnosis of TB disease.This study was approved by the Emory University Institutional Review Board (IRB) and the Grady Health System Research Oversight Committee. The Emory University IRB approved a request for a waiver of informed consent as the data analyzed for this study were originally collected for infection control and quality improvement purposes. The study took place at GMH and included all patients with an AFB smear-positive respiratory specimen between January 2002 and June 2008. Data on patients with an AFB smear-positive respiratory specimen were collected prospectively. AFB smear and culture were performed on patient respiratory specimens by the GMH Clinical Microbiology Laboratory using standard methodologies; AFB smears were performed using a fluorochrome technique and AFB cultures were performed using both solid and broth-based media A cost analysis of the routine use of NAAT at GMH was conducted using decision analysis models under two different diagnostic algorithms Figure 1A health care system perspective was used; costs that would have been incurred by the entire health care system for patients with an AFB smear-positive respiratory specimen were included. Costs (reported in 2008 U.S. dollars) were assumed to come from laboratory inputs , hospital room (either regular room or AII room), anti-TB medications, contact investigations, and hospital exposure investigations  Table 1.(1) “NAAT conditions”: Under this algorithm , all patients suspected of having pulmonary TB were placed on AII precautions and admitted to an AII room. Respiratory (sputum) specimens were collected and AFB smears and cultures were performed on respiratory specimens; a NAAT was automatically performed in all cases when a respiratory specimen was AFB smear-positive. The mean turn-around time for a NAAT was 3.8 days as specimens were batched and generally performed twice weekly. Patients were not reported as a TB case to their local health department and a contact investigation was not initiated unless the NAAT was positive.(a) NAAT positive: Those patients with a positive NAAT result were diagnosed with laboratory-confirmed pulmonary TB and kept in an AII room until hospital discharge. These patients were continued on anti-TB medications and were reported as a TB case to their local public health department which then initiated a contact investigation. These patients with pulmonary TB disease were treated for a minimum of 6 months. CDC guidelines state that contact investigations should be initiated no later than 7 business days after reporting of a TB case (b) NAAT negative: Those with an AFB smear-positive respiratory specimen but a negative NAAT were assumed not to have pulmonary TB. After the negative NAAT was reported, AII precautions were discontinued and patients were moved from an AII room to a regular room until hospital discharge. If patients with a negative NAAT were later found to have culture confirmed TB (based on a positive AFB culture for MTB) a hospital TB exposure investigation was conducted.(2) “No NAAT conditions”: Under this hypothetical algorithm (and the situation that existed before implementation of the NAAT in 2002), all patients suspected of having pulmonary TB were admitted to an AII room and had respiratory specimens (sputum) obtained; AFB smears and cultures were performed on respiratory specimens. Those patients with an AFB smear-positive respiratory specimen were kept in an AII room , continued on anti-TB medications throughout the hospitalization and at the time of discharge, and were reported as a TB case (or suspect) to their local public health department at the time of the positive AFB smear. The local public health department initiated a contact investigation on all such patients. After discharge, those found to have pulmonary TB on the basis of a positive culture result for M. tuberculosis were continued on anti-TB medications for the full course (minimum of 6 months for drug-susceptible TB). Those with a negative culture for M. tuberculosis (the culture may have been positive for a NTM) were assumed not to have TB. A contact investigation would have been initiated at the time of the positive AFB smear and carried out before the results of the culture were available based on CDC guidelines Among the 1,009 patients with an AFB smear-positive respiratory specimen between January 1, 2002 and June 30, 2008, 949 (94%) had a NAAT performed. Mean age was 44 years, 71% were male, and 84% were African-American. Four hundred forty-five (46.9%) patients were HIV-seropositive, 271 (28.6%) were HIV-seronegative, and 233 (24.5%) had unknown HIV status .M. tuberculosis)   of an AFB smear-positive respiratory specimen for culture-confirmed TB was 27% (252 of 949 respiratory specimens that were AFB smear-positive grew culosis) Table 2.Patients who had a respiratory specimen that was AFB smear-positive and NAAT-negative were removed from AII precautions and transferred to a regular hospital room. Prior to the availability of the NAAT, all persons who had respiratory specimens that were AFB smear-positive were kept in a AII room while hospitalized, started on empiric anti-TB medications pending culture results, and had their cases reported to the respective local county health department immediately which initiated a TB contact investigation. After the initiation of the NAAT, patients with an AFB smear-positive specimen were reported to their local county health department only if the NAAT was positive.The mean number of days in an AII room that was averted by a negative NAAT in a patient with an AFB smear-positive respiratory specimen was 5.3 . Additional cost savings were due to avoidance of first line anti-TB medications  among patients that were AFB smear positive, NAAT negative . There were also cost savings for a contact investigation that did not have to be performed on a patient with an AFB smear-positive respiratory specimen that was NAAT negative; the cost of a contact investigation was estimated to be $2,620 per index case (as described in the Methods section).Inputs for the cost analysis are shown in Our study is one of the first to demonstrate cost savings by routine use of a commercially available and FDA-approved NAAT (performed on an AFB smear-positive respiratory specimen from every patient who had a sputum/respiratory specimen that was AFB smear-positive) in a hospital setting in the U.S. The NAAT is recommended for routine use on respiratory specimens by the CDC We found a low PPV of an AFB smear for culture-confirmed TB at our institution (27%), especially among HIV-seropositive patients (<20%), which was due to the high prevalence of positive respiratory cultures for NTM among HIV-infected persons. TB infection control policies at our hospital are focused on detecting all TB cases and not missing any cases of TB among patients, including those with HIV infection. The low PPV of the AFB smear for TB was a major factor in the cost savings of the NAAT, although sensitivity analyses demonstrated cost savings even with a higher PPV. In addition, the utility of the NAAT improved the use of AII rooms by allowing patients admitted to AII rooms who had respiratory specimens that were AFB smear positive but did not have TB to be moved into regular rooms more rapidly. While the TB infection control program at GMH has been highly efficacious in protecting patient and health care worker safety by preventing nosocomial transmission of TB, it results in over-isolation of patients in AII rooms admitted with pulmonary symptoms and/or abnormal chest radiographs The NAAT performed extremely well among both HIV-infected and HIV-seronegative patients with extremely high sensitivity and specificity. Early exclusion of TB  allowed early discharge from AII rooms, avoidance of unnecessary anti-TB therapy, and avoidance of the initiation of contact investigations by local public health departments among those who were AFB smear-positive but turned out not to have TB . Routine use of NAAT on AFB smear-positive respiratory specimens was shown to be cost-savings in our setting (>$2000 per test performed).Our study is subject to several limitations. At our hospital, the NAAT is routinely performed only on AFB smear-positive respiratory specimens so we were not able to assess the cost effectiveness of the NAAT when performing the test on all specimens (i.e. including AFB smear-negative specimens). Performing the test on all respiratory specimens could potentially have led to different results in our cost analysis. In addition, our hospital cares for large numbers of HIV-infected patients and more cases of TB than any other hospital in Atlanta or the state of Georgia, so care must be employed in generalizing our findings to other settings. Because we used our patients as their own control (“no NAAT conditions”), we were not able to assess the impact of the NAAT on reducing hospital stay and therefore may have underestimated cost savings.In conclusion, we found the NAAT to be highly sensitive and specific (>99% for each) on AFB smear-positive specimens compared to culture. The overall PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was low at our hospital (27%), in large part because of a low PPV among HIV-infected patients. Routine use of the NAAT on AFB smear-positive respiratory specimens was highly cost-saving (saved >$2000 per AFB smear-positive case) in our setting at a U.S. urban public hospital with a high prevalence of TB and HIV.


As the secretory source of vitamins, peptides and hormones for neurons, the choroid plexus (CP) epithelium critically provides substances for brain homeostasis. This distributive process of cerebrospinal fluid (CSF) volume transmission reaches many cellular targets in the CNS. In ageing and ageing-related dementias, the CP-CSF system is less able to regulate brain interstitial fluid. CP primarily generates CSF bulk flow, and so its malfunctioning exacerbates Alzheimers disease (AD). Considerable attention has been devoted to the blood-brain barrier in AD, but more insight is needed on regulatory systems at the human blood-CSF barrier in order to improve epithelial function in severe disease. Using autopsied CP specimens from AD patients, we immunocytochemically examined expression of heat shock proteins (HSP90 and GRP94), fibroblast growth factor receptors (FGFr) and a fluid-regulatory protein (NaK2Cl cotransporter isoform 1 or NKCC1). CP upregulated HSP90, FGFr and NKCC1, even in end-stage AD. These CP adjustments involve growth factors and neuropeptides that help to buffer perturbations in CNS water balance and metabolism. They shed light on CP-CSF system responses to ventriculomegaly and the altered intracranial pressure that occurs in AD and normal pressure hydrocephalus. The ability of injured CP to express key regulatory proteins even at Braak stage V/VI, points to plasticity and function that may be boosted by drug treatment to expedite CSF dynamics. The enhanced expression of human CP 'homeostatic proteins' in AD dementia is discussed in relation to brain deficits and pharmacology. Accumulating evidence supports the idea that continually decreasing choroid plexus (CP) function in advanced ageing exacerbates Alzheimer's disease (AD). As part of a new paradigm to explain brain interstitium deterioration in age-related dementias, increasingly more attention is being paid to the role of compromised blood-CSF  and blooEfficient CSF turnover is essential for a healthy brain. It depends upon an exquisite balance between CSF formation and reabsorption . CompromIn serving the brain's metabolic needs by supplying 'biochemical goods', the CP uses CSF as a conduit for convecting substances . NumerouReduced formation of CSF and stagnated flow in ageing and AD  limits tStructure intimately relates to function in various epithelia. Therefore it is useful to analyze histopathological damage to CP at various stages of AD in order to clarify the onset of functional losses at the blood-CSF barrier. Ageing and AD cause similar degeneration in CP. Structural changes in AD though are usually greater than in non-demented control counterparts . Serot aSeveral features characterizing the CP in AD are highlighted in Table Due to the functional nexus of the CSF with brain interstitial fluid, the neuronal microenvironment in AD is impacted by markedly altered transport and permeability in CP. When neurodegenerative diseases, ischemia ,28 and e+] and immune molecule content of the CNS extracellular fluid [The CP is multifunctional, performing a wide range of homeostatic functions for the CNS . CSF homar fluid . AccordiMoreover to provide steady neuroprotection by regulating the extracellular milieu, the CP must protect itself against various stressor agents that build up during ageing and disease. There have been few investigations of the homeostatic systems within CP that stabilize choroidal functions in the face of ageing and AD. We have explored some candidate systems for compensatory responses by CP. Cytoplasmic heat shock proteins chaperone and perform housekeeping to maintain a healthy steady-state intraepithelial milieu ,32. GrowIt is difficult to functionally assess the CP in vivo ,33, partInformation about functional proteins in human CP is scarce. However protein expression was recently analyzed in autopsied lateral ventricle CP from normal adult brains and in those with confirmed AD ,37. The Ageing and AD dementia tax the CP and other CNS transport interfaces. In late life the deteriorating brain presents many potentially-destructive metabolites to the CSF for multi-site excretion into blood. The greater burden of macromolecule disposal in AD occurs when CP and arachnoid membrane, due to ageing debilities, are less able to transfer solutes. Nevertheless the CP seemingly attempts to maintain its 'epithelial soundness' when challenged to perform additional cleansing acts for the brain extracellular fluid (CSF).In healthly, young adults the CP epithelium sensitively acclimates to chemical and physical distortions in blood, CSF and parenchyma. Following cortical stabbing in rats, the CP upregulates TGFβ presumably to provide this CSF-borne growth factor for repairing the injury . A similAccordingly to assess pathophysiologic consequences of AD we investigated human CP's ability to upregulate certain functional proteins  in advanced states of AD dementia. With advancing knowledge about intricate cell physiology  it is relevant to evaluate components of cellular homeostasis. The term 'homeostatic proteins'refers to chaperones, receptors and transporters that stabilize the internal environment of the cell. CSF stability depends in large part upon CP epithelial homeostasis. Our group has analyzed several 'homeostatic proteins' involved in CP intracellular milieu stabilization:A wide spectrum of protection against neurodegeneration is provided by HSPs. The list of protective effects bestowed by HSP molecules is diverse and includes: accelerated degradation of misfolded proteins, maintenance of membrane lipid integrity, prevention of deleterious protein aggregation, and preclusion of damage to the translational apparatus ,42. HSPsTo evaluate human CP expression, we selected HSPs overexpressed in AD brains: GRP94 and HSP90. In contrast to previously found upregulation in brain, there was downregulated GRP94 in lateral ventricle CP. In aged controls there was abundant staining in the epithelial cytoplasm and stroma  ontogeny, ii) normal adult maintenance, and iii) neurodegeneration.FGF and FGFr are also fundamentally important in fostering neuron generation from stem cells in the subventricular zone (SVZ). This requires coordination between the CP-CSF and periventricular regions ,48. Pharnd but not 3rd gestational week [Receptor plasticity in aging and AD is better seen in light of information on FGF/FGFr expression dynamics in early life. In the fetus the formation of CP, neuronal stem cells and brain is promoted by CP growth factor secretion and CSF distribution ,49. InteFGF2 derived from CP is also conveyed by CSF bulk flow to the fetal germinal matrix where it acts on stem cell FGFr to promote neuronal maturation . By thisThe FGF/FGFr system also has a key role in adult CNS fluid homeostasis. CP helps the brain adapt to alterations in blood composition and flow. In otherwise healthy young adults, the imposition of dehydration or sudden ischemia upon the CNS elicits striking adaptive changes in CP epithelium. To endure insults by chemical or physical stressors , it is cDehydration seriously threatens CNS functions. Adjustments to dehydration in the healthy adult brain feature ion and water redistribution among fluid compartments. These compensatory responses to plasma hyperosmolality stabilize neuronal and interstitial volumes. Brain 'barriers' or transport interfaces are sites for the fluid homeostatic mechanisms. A working model for the restoration of fluid balance is offered: Dehydration or hyperosmolality upregulates the FGF2 and AVP peptides in CP ,52. FGF2Ischemia is another disorder with neuropathological consequences that are mitigated by growth factor upregulation or administration . BilaterFGF2 titers and FGFr receptor densities in degenerating CNS compartments provide insight on adaptive responses. FGF2 concentration is augmented in the AD brain . Moreove1-43 [FGF2 has an interesting relationship with amyloid. A worthwhile goal is to probe mechanisms of FGF2 interaction with amyloid in neuronal networks, extracellular matrix and CP-CSF. FGF2 co-localizes with several chemical forms of amyloid. Neuronal coexistence of FGF2 and amyloid precursor protein (APP) ,65 intim1-43  and atte1-43 . In the 1-43 ,70. ThisFGF2-mediated protective regulation of CP transport phenomena potentially affects the course of neurodegeneration. Considerable evidence points to CP's ability to remove Aβ from CSF -73. ThisThe diminished ability of CP to form fluid in advanced ageing and AD begs the question of how epithelial ion transport proteins are altered by distorted neurochemistry in senescence. In very old laboratory mammals the Na-K-ATPase activity of CP and the CSF generated by it are cut in half ,76. AnotThe cation-Cl superfamily of cotransporters includes NKCC1 and consists of 7 isoforms that actively transport Na and/or K electroneutrally with Cl . NKCC1 iThe NKCC1 helps cells and organs adjust to disrupted fluid balance. One thus expects homeostatic upregulation of this CP cotransporter in AD with its altered CSF dynamics. For delineating NKCC1 expression we used the T4 antibody to immunostain CP at various stages of AD dementia. Robust staining of the lateral ventricle plexus (even at Braak stage V/VI) occurred in the apical membrane and cytoplasm , then it might be feasible to prevent the early manifestations of AD (Braak stages I/II) from intensifying into the debilitating pathology of V/VI.One class of agents with considerable potential for AD therapeutics is the growth factor group. A useful paradigm for CSF growth factors and neuroprotection has evolved from experiments on transient forebrain ischemia in rats ,60. The Several other growth factors synthesized by CP and secreted into CSF should be explored in translational research dealing with interacting ischemia, hydrocephalus and AD ,103,104.In searching for agents to modify CP epithelial protein expression, the route of delivery of the active drug is of primary importance. Unlike the brain with its impermeable microvessels, the CP readily takes up water-soluble drugs from the plasma due to the highly-permeable choroidal capillaries. Consequently water-soluble agents freely diffuse to receptors or binding sites at the basolateral surface of the epithelium. Access of blood-borne hydrophilic compounds to the CSF-side of CP however is problematic because the tight junctions and basolateral membrane impede diffusing molecules as small as mannitol . MoleculTo circumvent the blood-CSF barrier, therapeutic agents are delivered into CSF by lateral ventricle catheters in experimental animals  or hydroA compelling aspect of CSF translational research is to identify agents that effectively regulate fluid formation by CP. Whereas the difficulty in congenital hydrocephalus is to downregulate CSF formation, the challenge in AD is to enhance CSF turnover perhaps by accelerating fluid production as well as outflow. Augmented flow of CSF enhances 'sink action' ,109. ThiAnother CP pathology meriting pharmacological attention is the massive fibrosis that progressively envelops the interstitium in old age. This interstitial fibrosis is even more extensive in AD . FibrosiThe CP has the main responsibility for CSF homeostasis. Therefore the functional status of the blood-CSF barrier is of great consequence to the CNS. Maintaining the CSF at a stable, specialized composition is of the utmost importance to neurons. CSF is prominent in regulating brain interstitial fluid with which it exchanges nutrients and waste products. Diseases markedly affect these molecular exchanges. Maintaining healthy bidirectional transport across the CP epithelium (CSF-blood) and ependyma (CSF-brain) is thus integral to a sound brain fluid environment.CSF macrocirculation through the ventriculo-subarachnoid system together with CSF microcirculation in the perivascular Virchow-Robin spaces ,111 perfAs the main generator of CSF, the CP has a pivotal role in helping the brain cope with the twin stressors of ageing and disease. More attention should be focused on the blood-CSF interface for pharmacologic opportunities to stave off CP dysfunction. Our findings on human CP expression of HSP90, FGFr and NKCC1 demonstrate that this epithelium in AD reacts to metabolic insults by upregulating certain proteins. This suggests that even diseased CP could respond to therapeutic agents, thus opening new vistas for treating CSF dysfunction in age-related dementias.Investigation of CP in AD is an area that is opening up. Translational research can now intensely focus on molecular factors that disable the CP to the point of reducing its ability to preserve brain integrity. Systematic CSF analyses using mass spectrometry and other cutting-edge biotechnology should generate neurochemical data specific for disease stages. New imaging approaches are essential to provide much needed functional data for CP, CSF and periventricular regions in AD patients. This should expedite the modeling of CP-CSF malfunctions and their resolution. Deeper insight into the pathophysiology of the blood-CSF transport interface will help to realize the development of novel therapeutic regimens for the AD family of diseases.AD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, beta amyloid; AVP, arginine vasopressin; BBB, blood-brain barrier; CP, choroid plexus; FGF2, basic fibroblast growth factor 2; FGFr, receptor for fibroblast growth factor; GRP94, glucose regulatory protein 94; HSP90, heat shock protein 90; NGF, nerve growth factor; NKCC1, Na-K-2Cl cotransporter secretory isoform 1; NPH, normal pressure hydrocephalus; TGFβ, transforming growth factor beta; SVZ, subventricular zone; TFI, transient forebrain ischemia; VEGF, vascular endothelial growth factor; IGF-II, insulin-like growth factor II; HGF, hepatocyte growth factor;The author(s) declare that they have no competing interests.CJ had the primary responsibility of organizing and writing the review, and had NIH support (NS 27601) to do the NaK2Cl cotransporter experiments. PM carried out the NKCC1 immunostaining runs with the T4 antibody and provided interpretation of the regional stainings. RT conducted the experimental analyses of the heat shock proteins and generated figures. AS did the image processing analyses of the cotransporter expression and assisted with the literature analysis. JD contributed ideas for the altered CP-CSF dynamics in hydrocephalus and ventriculomegaly. GS has developed the model that CP-CSF malfunction exacerbates AD progression, and helped to revise the manuscript. ES was responsible for the FGFr experiments and interpreted the human CP data. All authors read and approved the final manuscript.


Chronic ethanol exposure has been shown to result in changes in neuronal cyto-architecture such as aberrant sprouting and alteration of neurite outgrowth. In PC12 cells, chronic ethanol treatment produces an increase in Nerve Growth Factor (NGF)-induced neurite outgrowth that appears to require the epsilon, but not delta, isoform of Protein Kinase C (PKC). Neurites contain a core of microtubules that are formed from polymerization of free-tubulin. Therefore, it would be expected that an increase in neurite outgrowth would correlate with an increase in microtubule content. We examined the effect of chronic ethanol exposure on microtubule content in PC12 cells and the role of PKC epsilon and delta in ethanol's effect on microtubule levels.Chronic ethanol exposure of wild-type and vector control PC12 cells resulted in a significant increase in microtubule content and a corresponding decrease in free tubulin. There was also a significant increase in microtubule content in PC12 cells expressing a dominate-negative inhibitor of epsilon PKC; cells which have previously been shown to have no ethanol-induced increase in neurite outgrowth. In contrast, ethanol had no effect on microtubule content in PC12 cells expressing a dominate-negative inhibitor of delta PKC.These results suggest that chronic ethanol exposure alters the relative ratio of free tubulin to microtubule-associated tubulin, an important component of the cytoskeleton. Further, the data from the PKC dominant-negative cell lines suggest that the effects of ethanol on microtubule content do not correlate with the effects of ethanol on neurite outgrowth. The delta isoform of PKC appears to be necessary for the ethanol-induced increase in microtubule content. These studies demonstrate an effect of chronic ethanol exposure which may contribute to previously documented alterations of neuronal cyto-architecture. Membranes were then incubated in primary antibody  overnight at 4°C. Following incubation in the primary antibody, membranes were washed with TBS/0.05% Tween-20 and incubated in secondary antibody . Membranes were again washed and tubulin bands were visualized by Enhanced Chemiluminescence (ECL) using standard luminol reagents captured using a BioRad Gel Doc 1000. The computer analysis software Molecular Analyst  was used to quantify tubulin concentrations . All blots were very clean and no filtering was needed.Tubulin concentrations in both the free-tubulin and microtubule fractions were expressed as a percent control relative density and presented as mean (+/-SEM). The data represent multiple replicates of the same experiment . Student's t-tests were used to make comparisons between control and ethanol-treated PC12 cells.Each individual band analyzed was an independent extract from the same experiment, with the experiment being replicated 5 times. Each replication of the experiment was composed of 2 separate wells of cells for each treatment condition. For each of the 5 replications, the 2 control bands were averaged and each sample, including the controls, from that replication normalized to the average. The data was combined after all 5 replications.CKR-F conceived and performed the experiments and participated in writing the manuscript. DPD participated in the design of the study and writing of the manuscript. Both authors read and approved the final manuscript.


All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used.We developed a novel configuration of a cooled charge-coupled device (CCD) for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures.The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves. This is in contrast to the more widely known bioluminescence from animals that visibly glow, such as certain species of jellyfish, fireflies and beetles, which employ fluorescent proteins and/or luciferase enzymes to catalyse reactions that result in chemiluminescence. Ultra-weak bioluminescence is generally considered to result from oxidative chemistry occurring within cells  , which consists of a cooled ultra-sensitive area photo-detector within a stainless steel vacuum vessel and associated image acquisition electronics. The input image is coupled to the detector with optical fibres . The detector contains 222,336 pixels, and in the experiments presented here, ran at an operating temperature of -58°C. The combination of the proprietary low read-out noise electronics, cooling of the detector and the optical fibre coupling results in a sensitive imaging system capable of recording the faintest luminescences at high resolution, (40 μm to 200 μm depending on geometry of sample and integration time). The spectral absorption response of the SPAD ranges from the near UV to IR, thus including the entire visible spectrum up to the band-gap energy barrier of Silicon (1.1 eV) , though readers should note that the transmittance spectra provided with the filters covers the range 300–800 nm, rather than the more limited 400–700 nm spectra shown on the web site. Filters were placed between the leaf and the sample stage prior to imaging.Coloured filters were obtained from Lee Filters . We used lighting effect filters LEE 182 – "light red," LEE 118 – "light blue," LEE 183 – "moonlight blue" and LEE 735 – "velvet green." Spectral characteristics of the filters can be viewed at MF-H designed the PiXx*ell 1A luminescence imager. MF-H and MRR conceived of the current study, participated in its design and coordination, carried out experiments and wrote the manuscript. MF-H performed data analysis. TCM performed the norflurazon experiment, and both TCM and GLdB participated in some experimental design and execution. All authors read and approved the final manuscript.Arabidopsis leaves.Animated gif file showing images from five sequential 5-minute exposures of one control leaf (bottom) and four wounded Click here for fileArabidopsis leaves. Images were initially captured from samples on a cold imaging stage, but the heater in the lid of the sample stage was switched on when indicated.Animated gif file showing images from seven sequential 5-minute exposures of two control leaves (bottom) and four wounded Click here for file


Adolescents born very preterm have an increased risk for anxiety, social difficulties and inattentiveness, i.e. the ‘preterm behavioural phenotype’. The extreme end of these traits comprises the core diagnostic features of attention and hyperactivity disorders and autism spectrum disorder, which have been reported to show aberrant dynamic resting-state functional network connectivity. This study aimed to compare this dynamism between adolescents born very preterm and controls. A resting-state functional magnetic resonance imaging was performed on 24 adolescents born very preterm  and 32 controls born full term (≥37 weeks of gestation) at 13 years of age. Group-wise comparisons of dynamic connectivity between the resting-state networks were performed using both hard clustering and meta-state analysis of functional network connectivity. The very preterm group yielded a higher fraction of time spent in the least active connectivity state in hard clustering state functional network connectivity, even though no group differences in pairwise connectivity patterns were discovered. The meta-state analysis showed a decreased fluidity and dynamic range in the very preterm group compared with controls. Our results suggest that the 13-year-old adolescents born very preterm differ from controls in the temporal characteristics of functional connectivity. The findings may reflect the long-lasting effects of prematurity and the clinically acknowledged ‘preterm behavioural phenotype’. et al. report diminished dynamics in brain functional connectivity studied with the functional MRI in resting state as a possible sign of long-lasting consequences of very premature birth on brain connectivity in 13-year-old adolescents compared to the term-born peers.Lahti   These traits have wide-ranging effects on wellbeing, social relations and quality of life and they persist in adulthood.1-4Very preterm (VPT) birth  increases the risks for developmental difficulties in areas such as learning, language and motor skills.5 The static FNC method assumes that the connectivity is constant over the scanning time, while the dynamic approaches take into account fluctuations of the brain connectivity, which might be more informative.6Resting-state functional MRI (rsfMRI) is an imaging technique that measures fluctuations in the blood oxygen level dependent (BOLD) signal across the brain in the absence of tasks. Independent component analysis (ICA) is capable of extracting distinct networks (independent components) represented by spatial maps and related time courses of functional activity from the functional MRI (fMRI) data. The spatial maps of the independent components may represent various resting-state networks (RSNs), while the activity time courses can be used to explore correlative structure between the RSNs termed as functional network connectivity (FNC). The FNC can be analysed both in static and dynamic ways.et al.6 The second approach avoids such data reduction. Instead, it expresses each unique windowed connectivity situation as a weighted sum of the states where the weight vector constitutes a meta-state from a vast variety of other possible meta-states, thus allowing a high-dimensional analysis of dynamic FNC.7 While the hard clustering method can reveal a group difference in connectivity strength between each pair of RSNs and state changes in time, the meta-state method provides no data about pair correlations, but produces some metrics about fluidity and range of the meta-states ‘visited’ by subjects.9Therefore, in this study, we applied ICA to extract RSNs and analysed dynamic FNC between different RSNs using two different approaches. The first one interprets connectivity for each short period (time window) as the representation of one out of a few stable connectivity states thus reducing the data to a sequence of these reoccurring states over scan time. This approach is termed here as the hard clustering dynamic FNC method; it has been detailed in Allen 10-15 When compared to controls, preterm-born children did not differ in terms of static FNC at the age of eight years. However, the same participants underwent a new scanning at the age of 16 and at that age, it was found that preterm-born teenagers displayed greater relative connectivity compared to controls.14 The patterns between network and within-network connectivity have been shown to be altered differently. For example, in adolescents born VPT, the connectivity has been shown to be increased between the central executive network, salience network and sensorimotor network and decreased within the central executive network. Especially the central executive network and salience network are of high importance regarding keeping and shifting attention.15 In addition, altered connectivity of the amygdala has been shown to be associated with altered behavioural profiles in parent reports in VPT-born young adults.16The previous rsfMRI studies utilizing the static FNC method have shown controversial results when comparing VPT-born adolescents and controls.17 We aimed to compare the temporal FNC measures between 13-year-old adolescents born VPT and controls. We hypothesized that the adolescents born VPT would show an altered brain functionality due to prematurity and that the alterations might resemble those seen in adolescents with attention deficit hyperactivity disorder or autism spectrum disorder.19 To our knowledge, there are no dynamic FNC studies in adolescents born VPT.In a previous study, it has been shown, that preterm birth might alter the FNC between RSNs and that these alterations could be associated with the executive function deficits seen in attention deficit hyperactivity disorder or autism spectrum disorder.21 For this study, we invited the adolescents born VPT in 4/2004–2006. The flow chart is shown in 23 as a part of the longitudinal follow-up study.This study is part of the multidisciplinary PIPARI study (The Development and Functioning of Very Low Birth Weight Infants from Infancy to School Age) of VPT infants  born in Turku University Hospital in 2001–2006 and controls born full term  in the same hospital 2003–2004.24 A total of 97 controls were invited, 38 of whom agreed to attend. The reasons for dropping out were either refusal of the adolescent to participate in the imaging study or the parents withdrawing from the study in an earlier phase of this longitudinal follow-up. The parental education level did not differ between adolescents born VPT and controls.The recruitment protocol, including the inclusion and exclusion criteria of the healthy controls, has previously been described in detail.The Ethics Review Committee of the Hospital District of Southwest Finland approved the study protocol in 2012. The adolescents and their parents provided separate informed consents. Together with the informed consent, the families willing to participate were asked to report any diagnoses given to and medications affecting the central nervous system used by the adolescent.3 voxel) T1-weighted image  was acquired.MRI data were acquired with a 3.0 T Philips Ingenuity scanner  equipped with a 32-channel head coil. The BOLD series  were acquired in ascending order in a single session. For each participant, 234 volumes were collected during a 7.8 min fMRI sequence. In addition, a structural high-resolution  and the classification of the possible brain pathology is described in detail in Lind 26 a sample-specific brain template was created using the Computational Anatomy Toolbox, CAT12 (http://www.neuro.uni-jena.de/cat12-html). First, each structural volume was segmented and normalized to Montreal Neurological Institute brain space using affine transformation and age-specific tissue probability maps obtained with the Template-O-Matic TOM8 software.27 The affinely normalized images were checked for sample homogeneity, resulting in the selection of 30 subjects per group. The selected data were used to create a customized sample-specific template using a diffeomorphic algorithm, DARTEL.28 Then, the original individual structural images were registered again to the space of the sample-specific template with DARTEL to record individual deformation fields.The preprocessing was performed with free software packages implemented in MATLAB . In order to account for potential systematic structural differences between adolescent and adult brains,http://www.fil.ion.ucl.ac.uk/spm). Six starting volumes were discarded to allow T1 equilibration and subject state stabilization, resulting in 228 volumes for data processing. The fMRI data of each subject were corrected for slice-timing difference using the middle slice as a reference and corrected for head motion by co-registering scans to the realigned mean. Subjects with mean between-scan relative motion exceeding 0.2 mm in translation or 0.2° in rotation were rejected, after which 24 adolescents were born VPT and 32 controls remained. The resulting groups differed neither in translation (P = 0.95) nor rotation (P = 0.35) according to the Mann–Whitney U-test. In addition, the framewise displacement was calculated to be used later as a confound in the connectivity analyses. Its median values and interquartile intervals were 0.184 and 0.11 for the controls and 0.217 and 0.13 for the preterm-born group with no group difference (P > 0.05) according to the Mann–Whitney U-test. Functional images were co-registered to a scalp-stripped structural image for each subject using rigid-body transformation and then spatially transformed to the space of the sample-specific structural template using corresponding deformation fields, resliced to a 3 mm cubic voxel and smoothed with a 6 mm isotropic Gaussian kernel.The fMRI data preprocessing was performed with Statistical Parametric Mapping ; regressor for outlier scans , mean white matter and cerebrospinal fluid signals and their 16 principal components.31 Finally, the time series were band-pass (0.01–0.1 Hz) filtered.For additional cleaning of artefacts, the voxel time series were despikedhttp://trendscenter.org/software)8 after removing mean intensity over time for each voxel within the brain mask. We opted to use a low-order model to obtain easily recognizable networks and to avoid excessive splitting of the networks into smaller subnetworks. Given that 20–30 components appear to provide a representative characterization of large-scale networks, 25 independent components were extracted.32 Data dimensionality was reduced to 35 principal components, followed by the decomposition of the group-reduced matrix into 25 spatially independent components by the Infomax ICA algorithm, repeated 20 times in Icasso to ensure stability.33 Individual independent components  were back-reconstructed using the group ICA. The main maxima for each spatial map were defined using the results of one sample t-tests performed with SPM12. Components with maxima in the grey matter, frequency maxima within the 0.01–0.1 Hz range and spatial maps having only negligible overlap with non-grey matter areas were considered as RSNs.34 The remaining components were rejected from further analysis as related to noise factors.Spatial ICA was performed on the preprocessed and denoised fMRI data with the group ICA for the fMRI toolbox  rectangular window with a length of 60 s in steps of one repetition time (2 s), yielding 198 windowed FNCs for each of 153 pair correlations per subject. The calculation of correlation estimates included five runs of L1 regularization within the graphical lasso framework.35 The windowed FNC matrices were fed into two dynamic FNC analyses as illustrated by The dynamic connectivity was examined in two ways, using both standard (hard clustering) and meta-state approaches, both incorporated in the Temporal dynamic FNC toolbox in GIFT.6 each windowed FNCs was replaced with one of the prototype connectivity states that the windowed FNC resembled most closely. This made it possible to describe the connectivity dynamics as a series of transitions between these few reoccurring states. Before clustering, all the windowed FNC were concatenated along the subject × time dimension. The k-means clustering algorithm (iterated a maximum of 200 times), with the city distance measure, was used to define four stable FNC states (cluster centroids) based on a subset of subject windows corresponding to local maxima in the standard deviation. The number of the FNC states was equal to four as determined by the elbow criterion. The cluster centroids were used as starting points for clustering all of the data (98 subjects * 198 windows = 19 404 instances), resulting in the assignment of each windowed FNC to one of the four FNC states. Each state had to be present in a minimum of 10 windows for each subject.In the analysis,t-tests on group medians of correlation were performed for each pair correlation within each state. In addition, the percentage of scanning time spent in each state, i.e. the fraction rate, a mean time a subject was at a specific state, i.e. dwell time and the number of transitions between states was computed and compared between groups. To check the stability of the results, we repeated the hard clustering state FNC analysis for window widths of 50 and 44 s  structure in each of the 198-time windows.In a meta-state analysis,7 we additionally checked the stability of our results by repeating the analysis for four and six connectivity patterns while keeping the window width equal to 60 s  the number of distinct meta-states the subject occupied during the scan time; (ii) the number of times the subjects switched from one meta-state to another; (iii) the largest L1 distance (span) between the occupied meta-states and (iv) the overall distance travelled by the subject through meta-state space . The first two metrics characterize fluidity, while the second two describe the dynamic range. Higher values for each of the metrics are associated with higher dynamism. Though we used five connectivity patterns as providing reliable characterization of the meta-state connectivity60 s see .t-test was used. In the hard clustering state analysis, two-tailed two-sample t-tests on group medians of pair correlations between RSNs were performed within each state with the threshold of P < 0.05 after false discovery rate (FDR) correction. The fraction rate, dwell time and number of transitions were compared between groups with a two-tailed two-sample t-test and significance threshold of P < 0.05, with FDR correction. The results of the meta-state analysis were compared between the groups within each metric using a two-tailed two-sample t-test, with a significance threshold of P < 0.05 and FDR correction.The group differences in head motion parameters during fMRI the Mann–Whitney U-test. When finding local maxima for spatial map of each RSN see , one samP = 0.4, two-sample t-test) or gender proportions (P = 0.42 in Fisher’s exact two-tail test). The mean age of all participants at the time of the resting-state fMRI scan was 12.9 (12.3 and 13.8). The perinatal characteristics of adolescents born VPT are shown in The initial sample consisted of 35 adolescents born VPT and 35 controls, who were eligible after excluding cases with large susceptibility artefacts and anatomical abnormalities in the visual inspection of comprehensive MRI. As a result of the subject selection steps (see the Methods section for details), resting-state fMRI data from 24 adolescents born VPT (14 boys and 10 girls) and 32 controls (14 boys and 18 girls) were used in ICA. The groups did not differ in mean age  in comprehensive MRI. None of the adolescents born VPT fulfilled the criteria of significant cognitive impairment, even though two boys had FSIQ <70. Neither of these two had visible lesions in comprehensive MRI. One of the adolescents was born VPT and one of the controls had an attention deficit hyperactivity disorder diagnosis and medication. All the findings remained statistically significant after excluding adolescents with a FSIQ <70 or with attention deficit hyperactivity disorder.P = 0.02, FDR-corrected) in the fraction rate in the least connected state, State 1. The adolescents born VPT had a higher fraction rate  than controls  as shown in Correlation matrices representing four connectivity states for both groups are displayed in P < 0.05, FDR-corrected) reduction in the VPT group when compared to controls. The numeric results of the meta-state FNC analysis are shown in All of the meta-state FNC metrics—i.e. the number of states, changes between the states, state span and total distance—demonstrated significant , showed similar spatial orientation, but a functional hyperconnectivity in the default mode network, central executive network and salience network; and an increased anti-correlation within the central executive network at the age of 9–13 compared to controls. The study populations in their studies consisted of a combined sample of children and adolescents born late preterm who were also low to moderate-income twins. Both the population and the methodology make these studies not directly comparable with our study.This is, to our knowledge, the first study assessing dynamic FNC in adolescents born VPT, though static FNC analysis has been used by others for the prematurely born. Two static FNC studies by Degnan et al.14 found that at 8 years of age (13 preterm and 12 term-born children), the resting-state static FNC was similar between the groups. There was a difference between these groups at the age of 16, and the rsfMRI connectivity was markedly increased in the preterm group in several areas including occipito-temporal cortex, inferior frontal gyri and orbitofrontal and anterior prefrontal cortices. It remains unclear whether the present cohort would acquire similar changes in the static FNC connectivity patterns later in adolescence. The study of Rowlands et al.14 also included late preterm-born infants, which makes the study not directly comparable with our study.In a longitudinal static FNC study Rowlands 15 Differences were found especially in the networks involved in higher-order cognitive functions. The adolescents born VPT had a similar mean age compared to ours, but the age range was 10–16. This complicates the comparison with our study, as well as a seed-based analysis technique used by the authors. Also, a study by Wilke et al.11 found a group difference between adolescents born VPT and controls in language-related networks in static FNC. Their VPT and control groups differed from each other in terms of the age at scan, which might explain the between-group differences they found.One more static FNC study revealed both increased and decreased connectivity when comparing adolescents born VPT to the controls.36 found differences in the timing of hard clustering state FNC and meta-state FNC despite the lack of differences in the correlation matrices of hard clustering state FNC. They found an age-related decrease in the dwell time and transition frequency in a population with an age range of 9–32 years (32 children and 19 adults). This highlights the possibility of differences in temporal dimensions without differences in the group connectivity matrices.In this study, we did not consider the static FNC because of the contemporary conclusion of the dynamic fluctuation of brain connectivity at rest. The differences between adolescents born VPT and controls were found in the timing of hard clustering state FNC and meta-state FNC. These methods are more fine-grained and sensitive than static FNC analysis. We also used ICA, which allowed us to perform the analysis without a priori hypothesis. Previously, Hutchinson and MortonRegarding our meta-state analysis, we found that children born VPT occupied fewer states during the scan, made fewer switches between meta-states and travelled a shorter distance in the meta-state space. This means that the fluidity and range of RSN connectivity in the preterm-born group are decreased.18 The adolescents born VPT usually have traits belonging to the inattentive subtype.37Previous studies have shown a combined type of attention deficit hyperactivity disorder to associate with increased meta-state span in children and adolescents which was also found in our study.38It is also notable that the association between prematurity and temporal differences in resting-state FNC discovered in this study persisted when excluding adolescents with attention deficit hyperactivity disorder. This might suggest that our results were not driven by specifically attention deficit hyperactivity disorder-related alterations in FNC, but by the prematurity itself. On the other hand, the slow wave power in EEG at rest has previously been shown to be similarly increased in both adults with attention deficit hyperactivity disorder born full term and adults born VPT without attention deficit hyperactivity disorder. This supports the hypothesis of similarities in neural functioning between these groups.39 Rashid et al.39 found a longer dwell time in the least connected state in children with an autism spectrum disorder. De Lacy et al.40 found a significant decrease in the number of meta-state changes in adults with an autism spectrum disorder and Fu et al.19 in all meta-state metrics in adolescents with autism spectrum disorder. We have previously reported a trend of poorer social competence in adolescents born VPT.25Changes in hard clustering state dynamic FNC and meta-state dynamic FNC metrics have previously been observed in autism spectrum disorder, but also children with milder adversities in social competence.The lack of group differences in pair-wise RSN comparisons obtained with hard clustering method could suggest that there is only a negligible difference in connectivity patterns between the groups. Additionally, the moderate sample size could influence the detectability of such differences. Moreover, most of the participants excluded due to head motion were in the VPT group, which could diminish the differences.41 We were interested in examining large-scale connectivity, so we used a relatively low-order ICA model and analysed only the connectivity between, not within RSNs. Finally, the differences in preprocessing and certain ICA and FNC parameters may lead to somewhat different results between studies.Our series of 228 time points (7.6 min) exceeds 6 min duration that should provide a stable connectivity signal.Our study continues the discussion on functional disruptions in the neural networks of adolescents born VPT. Understanding the disruptions in brain functioning caused by VPT birth is important to ensure the proper growth environment and adequate support in daily life. We highlight the possible adverse effects in adaptive brain functions, in terms of dynamic fluidity, related to VPT birth.The long-term outcomes of VPT infants have improved over the last decades, but adolescents born preterm still suffer from anxiety, social problems and inattentiveness. In this study, we found aberrations in several characteristics of dynamic functional connectivity in adolescents born preterm. These findings resemble those made in previous studies on combined-subtype attention deficit hyperactivity disorder and autism spectrum disorder, which are the extreme versions of the features typical of the preterm behavioural phenotype. These findings could capture the brain mechanisms underlying the clinically observed behavioural features and the effects of prematurity on brain function.fcad009_Supplementary_DataClick here for additional data file.


T-lymphocytes immune response (CTL) in HIV-reservoir seems to be one of the reasons for shock-and-kill strategy failure. Ten non-controller patients on successful cART (TX) and seven elite controllers (EC) were included. HIV-Gag gene from purified resting memory CD4+ T-cells was sequenced by Next-Generation-Sequencing. HLA class-I alleles were typed to predict optimal HIV-Gag CTL epitopes. For each subject, the frequency of mutated epitopes in the HIV-Gag gene, the proportion of them considered as CTL-escape variants as well as their effect on antigen recognition by HLA were assessed. The proportion (%) of mutated HIV-Gag CTL epitopes in the reservoir was high and similar in EC and TX . Many of them were predicted to negatively impact antigen recognition. Moreover, the proportion of mutated epitopes considered to be CTL-escape variants was also similar in TX and EC . Thus, the most relevant finding of our study was the high and similar proportions of HIV-Gag CTL-escape mutations in the reservoir of both HIV-noncontroller patients with cART (TX) and patients with spontaneous HIV-control (EC). Our findings suggest that escape mutations of CTL-response may be another obstacle to eliminate the HIV reservoir and constitute a proof of concept that challenges HIV cure strategies focused on the reactivation of reservoirs. Due to the small sample size that could impact the robustness of the study, further studies with larger cohorts of elite controller patients are needed to confirm these results.Accumulation of mutations in epitopes of cytolytic- Combination antiretroviral therapy (cART) successfully curtails HIV replication and diminishes the circulating virus to undetectable levels , improviin vitro stimulation ) was 95%[93–97].Fresh peripheral blood mononuclear cells (PBMCs) were first obtained from 50 mL EDTA-anticoagulated blood samples by density gradient centrifugation with Ficoll-Hypaque . The population of resting memory CD4 T (Trm) cells defined by the expression of CD45RO marker (CD45RO+) and lack of expression of activation markers CD69, CD25, and HLADR (CD69-CD25-HLADR-) were purified from totally fresh PBMCs employing immunomagnetic microbeads technology . Briefly, memory CD4+ Total DNA was extracted employing a QIAamp DNA-MiniKit . HLA class I  genotyping was performed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) using LIFECODES HLA-SSO kit  and subsequent detection with SSO probes of the PCR product by bead array technology , with high level of resolution. The acquisition and analysis of the samples was carried out using the Luminex flow analyzer .Total DNA was obtained from Trm cells employing a QIAamp DNA-MiniKit . Proviral DNA was amplified by nested PCR using the Q5 Hot Start High-Fidelity DNA Polymerase PCR master mix . Due to the high genetic variability of HIV-Gag genes, we employed different sets of outer/inner patient-specific primers to successfully amplify the whole gene sequence in all patients (Suplementary Table S1). AMPure XP Kit  and QuantiFluor dsDNA System  were used to purify and quantify PCR amplicons respectively.Approximately 50 ng of DNA fragments of around 400 bp HIV-Gag genes in the autologous virus reservoir of each patient were used to construct DNA libraries with MiSeq Reagent Kit v3 , following the manufacturer’s instructions. DNA libraries were pair-end sequenced in a 2 × 300 format using the Illumina MiSeq sequencer . The pre-processed reads were subsequently mapped to the H×B2 reference genome (GenBank accession K03455.1) with Bowtie2 . The resulting alignment files were analyzed with VarScan2 software  for variant detection. No large internal deletions were found in the Gag gene of any of the studied subjects. Only variants with at least 20% of frequency along all reads per base were validated. All these programs were accessed using the integrated GPRO suite .www.hiv.lanl.gov/content/immunology/tables/optimal_ctl_summary.html). According to this list, an epitope is considered a best-defined HIV-1 CTL/CD8+ epitope if it meets all of the following criteria: (a) to induce a specific CTL response upon HIV infection in vivo; (b) to have the size of an optimal epitope to be recognized at the lowest epitope concentration; and (c) to have identified its HLA class I restricting molecules. To be included in this list, all the best-defined HIV-1 CTL/CD8+ epitopes were tested experimentally and validated mainly by intracellular staining of cytokines (ICS) assay, CD8 Elispot, and/or Chromium-release assays. Autologous sequences of HIV-Gag CTL epitopes of each patient enrolled in the study were checked against the list of the optimal best-defined HIV-1 CTL/CD8+ epitopes from Los Alamos database sequences. The proportion of mutated epitopes, as well as the ratio of mutated amino acids (aas) per epitope , was calculated for each patient.For each subject, optimal HIV-Gag CTL epitopes were determined depending on their HLA class I alleles , using “the list of the best-defined HIV-1 CTL/CD8+ epitopes” published in the Los Alamos Immunology Database available in September 2019 (TL/CD8+ Epitope Variants and Escape Mutations HIV database” (https://www.hiv.lanl.gov/content/immunology/variants/ctl_variant.html) was used. Sequence variants and mutations found in HIV CTL epitopes that have been tested for immunological response are reported in this database. A mutated epitope is considered a CTL escape variant only if it belongs to one of these types of variants: calculated escape; escape documented; inferred escape; literature escape; non-susceptible form; altered epitope processing; or TCR-related mutation.To calculate the proportion of autologous mutated epitopes that could be considered as CTL escape variants the “Cin-silico method to predict the binding strength of HLA molecules with viral peptides by means of an artificial neural network. The next default NetMHC threshold (% rank) values were used to consider the strength of binding of a viral peptide to an HLA allele: strong binding if % rank was ≤0.5, weak binding if % rank was >0.5 to ≤2%), and non-binding if % rank was >2%.The effect of mutated epitopes on recognition by HLA was analyzed employing the latest version of NetMHCpan Server (4.1 version) , which is the most current and accurate version of the best p-values <0.05 were considered as significant. Due to the relatively small sample size, median [interquartile range] was used to describe the main characteristics of the study population. Non-parametric tests were employed to check for inter-group differences .The SPSS software version 15  was employed for the statistical analysis of the data. All tests were two-tailed and only Clinical characteristics of patients recruited for this study are reported in We analyzed the proportion of mutations in HIV-Gag CTL epitopes obtained from the viruses archived in the HIV reservoir. For each patient, we first identified (according to the HLA class I haplotype) the best-defined CTL epitopes described in the Los Alamos Immunology Database. The number of optimal HIV-Gag CTL epitopes predicted was similar in EC and TX patients  . Next, wSince the proportion of mutated HIV-Gag CTL epitopes could be influenced by differences in the HLA-I alleles carried by EC and TX patients, we next analyzed the proportion of mutated epitopes on an HLA-specific basis. For this purpose, we selected EC and TX patients sharing a particular HLA-I allele and calculated the proportion of mutated CTL epitopes associated to that allele. Thirteen different HLA-I alleles were shared between EC and TX patients, and the proportion of mutated epitopes associated to these alleles was similar in EC and TX patients .Figure CTL/CD8+ Epitope Variants and Escape Mutations” HIV database to assess the proportion of autologous mutated epitopes in EC and TX patients that had been previously documented as CTL escape variants (www.hiv.lanl.gov/content/immunology/variants/ctl_variant.html). This analysis showed that the frequency of mutated epitopes considered as CTL escape variants were also similar in TX and EC patients   .Figure Additionally, we calculated the potential impact of mutated epitopes on HLA recognition. Interestingly, we found differences between EC and TX patients in the binding affinity to HLA of autologous mutated epitopes  .To the best of our knowledge, ours is the first study analyzing the CTL escape mutation landscape in HIV-Gag CTL epitopes present in one of the most relevant cell populations (Trm cells) in HIV reservoir, and comparing two groups of HIV patients classified depending on the mechanism of viral replication inhibition: spontaneous (EC group) or cART-mediated (TX group). The results of our study showed that: (a) the proportion of mutated HIV-Gag CTL epitopes present in the viral reservoir was high and similar in EC and in TX patients; (b) a large number of these mutations could potentially affect in a detrimental way the ability of CD8 T cells to recognize the mutated epitopes; c) the proportion of mutated CTL epitopes considered as CTL escape variants was similar in EC and TX patients.Contrary to expected, our results reveal that EC patients harbor a high frequency of mutations in HIV-Gag CTL epitopes present in the viral reservoir even greater than that found in patients with viral replication suppressed by cART. This high frequency observed in EC was not biased by differences in HLA alleles distribution between groups since the analysis of specific HLA alleles shared between the two groups of patients yielded the same results. Our results agree with previous studies in which immune-escape variants of the CTL response have been observed in EC patients  even at Viral replication control in elite controllers is reached soon after acute infection , so the CTL/CD8+ Epitope Variants and Escape Mutations” database, suggesting that these mutated variants could escape CTL recognition. In agreement with this, a previous study has shown a high prevalence of escape mutants to the CTL response in the reservoir of non-controller patients on cART [The most relevant finding of our study was a similar proportion of HIV-Gag CTL escape mutations in the viral reservoir of EC and TX patients. The majority of mutated variants in non-controller patients on cART had reduced affinity for HLA molecules and were included as escape variants in the “ on cART . Surpris on cART . In agre on cART ,22.in silico studies, and thus, it is of utmost importance to corroborate them with studies employing functional assays to measure CTL response to viral variants in these patients.Some limitations of our study deserve to be commented on. First, the sample size analyzed limits the robustness of the conclusions and thus further studies with large cohorts of patients are needed to support our findings. Second, we employed a set of optimally-defined dominant CTL epitopes and thus we lacked information about subdominant epitopes that may be relevant in the CTL-mediated control of HIV replication as suggested by Frahm et al., 2006 . FinallyTaken together, our results reveal a high frequency of CTL mutations both in the HIV reservoir of non-controller subjects with successful cART-mediated control and in the HIV reservoir of subjects that maintain spontaneous HIV-control since an early stage of infection. In fact, a large number of these mutations could potentially affect in a detrimental way the process of antigen recognition by CD8 T cells and have been included as CTL escape variants. Our findings suggest that escape mutations of CTL response may be another obstacle to eliminate the HIV reservoir and constitute a proof of concept that challenges HIV cure strategies focused on the reactivation of viral reservoirs.Click here for additional data file.


The tumor immune microenvironment (TIME) played an important role in immunotherapy prognosis and treatment response. Immune cells constitute a large part of the tumor microenvironment and regulate tumor progression. Our research is dedicated to studying the infiltrating immune cell in lung adenocarcinoma (LUAD) and seeking potential targets.  The scRNA-seq data were collected from our FDZSH and two public datasets. The code for cell-type mapping algorithms was downloaded from the CIBERSORTx portal. The bioinformatics data of LUAD patients could be approached from The Cancer Genome Atlas (TCGA) portal. Weighted gene coexpression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) analyses were performed to construct a risk model. TIMER2 and TIDE helped with the immune infiltration estimation, while PROGENy helped the cancer-related pathways' enrichment analysis. GSE31210 dataset and IMVigor ICB therapy cohort validated our findings as the external validation datasets.  We clustered the scRNA-seq dataset (integrating our FDZSH datasets and other public datasets) into 23 subpopulations. After curated cell annotation, we implemented Cibersort and WGCNA analysis to anchor the brown module and natural killer cell cluster1 due to the most relationship with tumor trait. The overlap of the brown module gene, natural killer cell signature, and DEGs of tumor and adjacent normal samples was screened by LASSO Cox regression. The obtained 5-gene risk model showed an excellent prognostic performance in the validation dataset. Furthermore, there was a correlation between risk score and tumor-infiltrating immune cells and tumor genomics abnormity. Patients with higher risk scores had a significantly lower immunotherapy response rate.  Our observations implied that immune cells played a pivotal role in TIME and established a 5-gene signature  on the basement of nature killer markers targeted by WGCNA analysis. The significance of clinical outcome and immunotherapy response prediction was validated robustly. Lung cancer is the most common cancer and the most prevalent cause of tumor-related death in the world . Lung adWith the development of sequencing technology over the past decade, molecular prognostic markers of tumors based on RNAseq technology emerged in endlessly. As a hot new technology in transcriptional analysis, scRNA-seq technology enables single-cell sequencing technology to reveal cellular gene expression that cannot be detected by bulk RNA sequencing . Single-This study integrated scRNA-seq data from our hospital and two external public databases with curated cell identity annotations. Furthermore, weighted gene coexpression network analysis (WGCNA) was implanted on the normal and tumor samples of the TCGA-LUAD cohort to explore the key module and cluster associated with tumor status. Consequently, the hub gene was selected to construct a five-gene risk model by the least absolute shrinkage and selection operator (LASSO) Cox regression algorithm and AUC validation. Our downstream analysis proclaimed the prospect and functionality of the signature in immune infiltration, mutational status, oncogenic pathways, and clinical prognosis; our results provide new sight into the immune cells in tumor heterogeneity and biomarker mining.Fourteen primary LUAD patients who had received surgical resection in the Department of Thoracic Surgery in Zhongshan Hospital (FDZSH) were included for scRNA sequencing . The diaPreparation for single-cell transcriptomic sequencing followed the protocol for the 10x Genomics Chromium Single-Cell platform. Our previous published literature described the detailed tissue processing, and the single-cell suspension was described in our previous published literature .http://biocc.hrbmu.edu.cn/CellMarker/) with the assistance of SingleR [We followed the Seurat v3 guidelines for the routine procedure. Cells expressing less than 200 genes or greater than 7000 genes or more than 20% mitochondrial genes were removed in the cell QC procedure. After normalization and PCA dimension reduction, the harmony  R packag SingleR  and scTy SingleR . The marhttps://gdc.xenahubs.net). The differential gene analysis between the 56 paired tumor and normal sample of TCGA-LUAD was performed using the R package limma [The bulk RNA sequencing (RNA-seq), genomics data, and clinical features of The Cancer Genome Atlas (TCGA)–LUAD tumor and adjacent normal samples were collected from the UCSC Xena (ge limma  with thehttps://cibersortx.stanford.edu). WGCNA was performed using the R package WGCNA [The cell type abundance of TCGA samples from our scRNA-seq annotation was calculated by CIBERSORTx  using ouge WGCNA  (versionhttps://cran.r-project.org/package=caret) was applied to build the classification of the TCGA-LUAD cohort and assess machine learning classifiers for the classification task.LASSO is a widely used regression method appropriate for analyzing data with high dimensions and strong relationships like high-throughput data. We used the R package glmnet to perform the LASSO Cox regression analysis as previously described , includihttp://tide.musc.edu/) and TIMER2 (http://timer.cistrome.org/). The MAF file was used to illustrate the distribution of mutation frequency and status by the R package maftools [The immune infiltration and cell composition estimation of TCGA patients were based on the TIDE .The statistical analysis was conducted using R4.2.0. The Log-rank survival analysis and univariate and multivariate Cox proportional hazards regression by the stepwise method were performed using “survival” and “survminer.” The nomogram construction, validation, and calibration were performed and plotted using “rms” and “Hmisc” packages. The statistical analysis was performed via unpaired Student's FindAllMarkers function (Threshold: LogFC:1), we defined the signature genes of each cluster. Each cluster's label was firstly annotated by the SingleR and scType automatically. The final curated annotation was completed via the CellMarker database. The detailed information on cell cluster identification was demonstrated in Table First, we performed the routine quality control and normalization before integrating three datasets' scRNA-seq data by harmony algorithm. Figures During the cell annotation, universal marker genes classified the main cell type shown in the dotplot in Figure Under the UMAP dimension reduction and cell-type annotation of our datasets, we formulated the CIBERSOT progress to calculate the cell abundance based on the transcriptome profile of our scRNA-seq. Considering the heterogeneity of each cluster, we included all the clusters rather than cell types to establish the signatures. The cell abundance of tumor and normal samples in the TCGA-LUAD database showed significant differences among 21 cell subpopulations  except fWGCNA analysis was performed to determine the correlation between gene expression module and cell abundance of specific cell subpopulations. All 585 TCGA-LUAD samples mingled with 59 adjacent and 526 tumor samples. The hierarchical clustering results and deleted outlier sample are shown in p adjust value < 0.05 and logFC > 1, we obtained 1972 differential expressed genes (DEGs), of which 1048 genes were upregulated and 1048 were downregulated  and higher tumor infiltration . Additionally, the mRNAsi stemness score in high group was higher than low group (p < 0.001). The correlation analysis between the immune cell abundance and a risk score is shown in Figures The immune cells in the tumor microenvironment play an essential role in tumor prognosis. The association between the risk score and infiltration of immune cells was explored. The ESTIMATE formula computed the ImmuneScore and TumorPurity. Corresponding to the above result, the high risk score tumors had statistically significantly lower levels of immune infiltration  (ImmuneSow group  (0.350 vp < 0.001). Consistent with the analysis above, the patients in the high-risk group hold an elevated tumor mutation burden . The above results suggested that this model revealed that immune cell infiltration in the TIME affects the tumor's genomics status and mutation load.The investigation of the genomic features was conducted to reveal the tumor characteristics in TIME. The distribution of variants and somatic interactions of the low- and high-risk groups was shown in p < 0.0001). Due to the baseline imbalance and confounding factors. We identified that the risk score group was a crucial independent prognostic factor by univariate and multivariate Cox regression analysis )2(IDH2), adrenoceptor beta 2(ADRB2), surfactant protein C (SFTPC), coiled-coil domain containing 69 (CCDC69), and cyclin D2 (CCND2). IDH2 is often considered to have a similar prognostic effect to IDH1 in glioma . Li et ao et al.  reportedo et al. . In summThis study provides a new perspective on understanding the immune cells in TIME and sets a novel risk model; limitations to our research highlight the need for further work to optimize our work. Firstly, the internal validation of the model showed a good performance, and further external real-world validation is needed. Downstream and functional experiments underlying the mechanism of immune cells and model genes could help discover potential therapeutic targets. We plan to pursue applying the risk model to the diagnosis of early-rate LUAD.Our study utilized the scRNA-seq data to identify the heterogeneous cell population in LUAD, applied the CIBERSORTx algorithm to map the cell type into the bulk RNAseq, revealed the key role of immune cells, especially natural killer cells, in TIME, and constructed the 5-gene model with the robust prognostic prediction and potential to evaluate immunotherapy response.


The rapid spread of the coronavirus disease COVID-19 has imposed clinical and financial burdens on hospitals and governments attempting to provide patients with medical care and implement disease-controlling policies. The transmissibility of the disease was shown to be correlated with the patient’s viral load, which can be measured during testing using the cycle threshold (Ct). Previous models have utilized Ct to forecast the trajectory of the spread, which can provide valuable information to better allocate resources and change policies. However, these models combined other variables specific to medical institutions or came in the form of compartmental models that rely on epidemiological assumptions, all of which could impose prediction uncertainties. In this study, we overcome these limitations using data-driven modeling that utilizes Ct and previous number of cases, two institution-independent variables. We collected three groups of patients  from different time periods to train, validate, and independently validate the models. We used three machine learning algorithms and three deep learning algorithms that can model the temporal dynamic behavior of the number of cases. The endpoint was 7-week forward number of cases, and the prediction was evaluated using mean square error (MSE). The sequence-to-sequence model showed the best prediction during validation (MSE = 0.025), while polynomial regression (OLS) and support vector machine regression (SVR) had better performance during independent validation , which exhibited better generalizability of the latter. The OLS and SVR models were used on a dataset from an external institution and showed promise in predicting COVID-19 incidences across institutions. These models may support clinical and logistic decision-making after prospective validation. Coronavirus disease COVID-19) was declared a pandemic by the World Health Organization (WHO) on March 2020 following the global spread of the underlying severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 9 was dec. SARS-CoRT-qPCR is a serological test that remains the gold standard for COVID-19 diagnosis . It measReverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a serological test that remains the gold standard for COVID-19 diagnosis . It measWe believe that the use of Ct for now-casting has its merits since it is a commonly available parameter irrespective of demographics and is highly correlated with transmissibility and incidence rates ,23. A poWe demonstrate the merits of this approach using a robust framework that leverages observed viral load measurements for time series now-casting of new COVID-19 cases for an upcoming 7-day time frame. The models are developed using a large cohort from a single cross-sectional virologic test center in Lebanon with a hold-out cohort for independent testing after the model is finalized. The Lebanese patient cohort used in this study is the largest and most consistent one in terms of serological assessment. This fact made the retrieved Ct values representative and reflective of the whole country.Now-casting the pandemic trajectory can facilitate its containment and improves healthcare providers’ preparedness against new SARS-CoV-2 variants and the surge in new cases caused by them. Furthermore, now-casting the pandemic trajectory can support policymakers during the decline phase of the pandemic  to suggest the best time frame for relaxing current PHSMs without the risk of the pandemic relapsing.We retrospectively collected de-identified data for all COVID-19 patients diagnosed at Rafik Hariri University Hospital (RHUH) in Lebanon between 1 March 2020, and 31 March 2021. Rafik Hariri University Hospital (RHUH) is the country’s leading institution for COVID-19 testing and treatment, and the collected cohort represents the nation’s COVID-19 trajectory well . Ct valuWe created 3 cohorts  using a longitudinal data split. The discovery group (Group 1) was used for training and cross-validation  to tune We first identify the relevant input features needed by the models to predict epidemic trajectories in Lebanon using Spearman’s correlation test. We analyzed the association between the patients’ Ct values  and age t values . The daiIn addition to the previously mentioned features, the epidemic trajectory also depends on the past number of COVID-19 confirmed cases and is therefore aggregated with the input features during now-casting ,7. The pThe epidemic trajectory is given by a sequence of predicted case counts in the upcoming ectories . We deven-th layer LSTM cell is represented by its hidden The first two models are built around recurrent neural networks (RNNs), which accommodate time-series data that are often temporarily correlated  assumption does not hold for time series data). This type of neural network can capture the temporal relationship between a decrease in Ct value and a subsequent (possibly delayed) rise in the number of cases. The RNN unit used in the models is the long short-term memory (LSTM) cell, which can capture long-term temporal effects and trends encoded by a long sequence of inputs and avoid the problem of vanishing gradients during backpropagation . The LST neurons . We usedformance . TemporaThe first model is given by a sequence-to-sequence (S2S) model commonly used in natural language processing (NLP) translation tasks. The model consists of an encoder RNN that accepts an input sequence of features of length Pteacher . The arcWe also developed a second RNN model based on the stacked LSTM cells alone  . This moThe average number of predicted and actual number of cases for the next We then developed a third model based on deep learning using feedforward neural networks. The feedforward neural network (DNN) model has several hidden layers (function . All deeze bsize . Early satience) .We developed three additional models that are not based on deep learning, namely a support vector machine regression (SVR) model , a gradiThe hyperparameters of each model (listed in and described in The outer loop split (Group 1) into five groups and sent four into the inner loop for training the models and subsequent hyperparameter optimization with respect to the average k-fold cross-validation error . The croEquation .(3)MSE=Several models used in this paper  involve random variables associated with the training algorithm (backpropagation and gradient boosting), which are often ignored in the literature of applied machine learning. Examples of these random variables include the initial value of learnable parameters , dropout, and gradient descent step sizes. Fixing the random seed of these random variables could result in model bias.We address this issue by randomly sampling different training runs during hyperparameter optimization and optimizing the mean cross-validation errors of all the sampled runs. We apply this approach to a grid search on the hyperparameter space to discern the sensitivity of the cross-validation error to the hyperparameters. We then use a stochastic derivative-free optimization (DFO) algorithm (stochastic mesh adaptive direct search (StoMADS)) to fine-tune the hyperparameters . StoMADSAfter obtaining the optimal model in the internal loop, we scored it using the outer loop data. We then performed a random draw to obtain 30 models using the tuned hyperparameters. These models were binned by training error and the top-performing model was stored and used to make predictions for the test group (Group 2).We note that binning and sampling of the cross-validation error are unnecessary for the OLS and SVR models since their training is deterministic and does not involve random variables.The entire dataset included 23,185 patients with a median age of 37 years. We aggregated the individuals’ Ct into a sequence of daily mean Ct values. Group 1 contained 6296 patients admitted to RHUH between 2 March 2020 and 17 October 2020; Group 2 contained 3228 patients from 18 October 2020 to 30 November 2020, and the unseen group contained 12,097 patients from 1 December 2020 to 16 March 2021. All three groups have comparable median ages . Group 1 was further split into five groups during model development for cross-validation: four training and one validation interchangeably.We observed a temporal delay between the incidence rate and the observed Ct values. For example, the trough in mean Ct values on 8 October 2020 (Trough 3 in p < 0.001), quantified by the Spearman correlation test . The optimal hyperparameters for each model are listed in Group 2) . The modGroup 2)  footnoteThe RNN models (S2S and SEQ) performed well on Group 2 , followed by the DNN model with an MSE that is two-fold larger (0.042). The OLS and SVR had an MSE that is four-fold larger than that of the RNN models . The GBM was heavily biased and did not generalize well on Group 2 (MSE of 0.326). The training error for the RNN models was higher than that of the parametric models (OLS and SVR) due to the regularization performed by the early-stopping criterion to avoid overfitting. Movie S1 shows an example training run of the S2S model with arbitrary hyperparameters, where early stopping helped avoid overfitting.The MSE error of the SVR, OLS, and DNN models was comparable on the unseen data group . The SEQ and S2S performed worse on the unseen group, implying that simpler models perform better on the unseen group due to the limited number of data points available for training and hyperparameter tuning. Deep learning models generally excel when a large dataset is available for model development and has been reported by several studies in the literature ,42.To verify this, the RNN models were re-developed using both Groups 1 and 2 for training, hyperparameter tuning and validation. The generalization performance improved significantly, bringing the MSE error down from 0.571 to 0.106 for the S2S model . This imHost viral load and the resultant Ct values have been widely proposed to evaluate the progression of SARS-CoV-2 infection and address patients’ contagiousness . MathemaHay et al.  used BayIn comparison, the presented data-driven approach of inferring the epidemic trajectory using past case counts and Ct values makes very little assumptions about the pandemic trajectory and viral kinetics models that gave rise to the observed Ct values. This has the benefit of potentially generalizing to a wide range of scenarios. To prove this, we used all the models developed in this paper using Group 1  to inferThe inferred trajectories for the state of Massachusetts (15 April 2020–15 December 2020) and Lebanon (1 December 2020–31 March 2021) show that simple machine learning models (such as OLS and SVR) perform well with limited training data (when developing the models using data from Group 1 only). Deep learning models begin to outperform such models when including more data in the development set (Groups 1 and 2) to infer the trajectory in Lebanon . AlthougThe dataset used in this study contained fluctuations that allowed us to extract the Ct temporal effect on the trajectory of the pandemic. Since the data came from a single institution, the fluctuations are likely to be signals in the data rather than noise. The significant changes in the Ct values mirrored the well-recognized political, economic, and social turning points that happened in Lebanon during the pandemic. These incidences impacted the population’s behavior towards COVID-19 in a consistent and well-defined manner, allowing us to track and correlate these changes with the variation in the mean Ct values and subsequently the disease spread. The early reported high mean Ct values and the low number of COVID-19 cases in Lebanon between March 2020 and June 2020 co-occurred with a strictly imposed lockdown and a harsh awareness campaign executed by local media platforms . In compThe detected inverse relationship between Ct values and the number of national COVID-19 positive cases reflects population dynamics of transmission and demonstrates the temporal significance of Ct values. The results emphasized the importance of early testing when the patient’s viral load and infectivity are low to prompt isolation practices and thus, suppress the national spread of the virus. The models were able to predict the upcoming one-week expected number of national COVID-19 cases based on a commonly available diagnostic measurement, the Ct value. This shows that viral load measurements are a rigid input that can enhance the outcomes of disease forecasting models. Interestingly, this model is still valuable among vaccinated patients as these patients were shown to have a similar viral load pattern as unvaccinated patients and thus, can efficiently transmit the disease in the same manner upon infection . UltimatThis study is limited to a single-institution cohort. Although the cohort represents the national number of cases, and the model’s variable (Ct) is country-independent, a prospective validation on multi-institutional data is needed before translation. To facilitate this process, we have hosted the models on a web interface to be used in future studies that compare the predicted and observed number of cases . AnotherThis study is a first attempt at combining serologic assays from a representative cross-sectional patient cohort with epidemiological indicators such as incidence rates and infection size to now-cast the number of nationwide positive COVID-19 cases in a specific region . This wa