Skilled nursing facilities (SNFs) are high-risk settings for SARS-CoV-2 transmission. Infection rates among employees are infrequently described.To describe SARS-CoV-2 rates among SNF employees and residents during a non-outbreak time period, we measured cross-sectional SARS-CoV-2 prevalence across multiple sites in the Seattle area.SARS-CoV-2 testing was performed for SNF employees and residents using quantitative real-time reverse transcription polymerase chain reaction. A subset of employees completed a sociodemographic and symptom questionnaire.Between March 29 and May 13, 2020, we tested 1583 employees and 1208 residents at 16 SNFs for SARS-CoV-2.SARS-CoV-2 testing results and symptom report among employees and residents.p = 0.62, McNemar’s test).Eleven of the 16 SNFs had one or more resident or employee test positive. Overall, 46 (2.9%) employees had positive or inconclusive testing for SARS-CoV-2, and among those who completed surveys, most were asymptomatic and involved in direct patient care. The majority of employees tested were female , and most employees were Asian , Black , or white . Among the 1208 residents tested, 110 (9.1%) had positive or inconclusive results. There was no association between the presence of positive residents and positive employees within a SNF  contains supplementary material, which is available to authorized users. The COVID-19 pandemic has accentuated realities of the SNF care environment in which resources and personnel are often inadequate to meet the demands of an infectious disease outbreak, and SNFs have also reported employee and supply shortages since the COVID-19 pandemic began.16 Despite these precautions, cases of SARS-CoV-2 continue to be observed in SNFs. To address this, testing for SARS-CoV-2 among SNF employees regardless of symptoms is needed in order to develop strategies for decreasing transmission in SNFs and the larger community. In this study, we describe the results of cross-sectional resident and employee SARS-CoV-2 testing, and infection control and personnel policies associated with 16 Seattle area SNFs.In Washington State, a government call to action on March 10, 2020, led to SNFs barring visitors and engaging in employee symptom-based screening.Through two testing strategies, a total of 16 SNFs offered testing to either residents, employees, or both. The first testing strategy was directed by Public Health of Seattle & King County (PHSKC) and focused on SNF resident testing with employee testing offered at select sites. The second testing strategy was facilitated by the Seattle Flu Study (SFS) and directed at testing only employees. PHSKC testing was conducted by providers from University of Washington, between March 29, 2020, and May 8, 2020, at 13 SNFs and one assisted living facility, of which six offered both resident and employee testing and eight had only resident testing. Employee testing by SFS was designed to coincide with resident testing done by PHSKC when possible. SFS testing was conducted between April 14, 2020, and May 13, 2020, at 13 SNFs. At three SNFs, both the PHSKC and Seattle Flu Study teams tested SNF employees.PHSKC identified SNFs in need of SARS-CoV-2 testing, including sites with known COVID-19 cases, facilities with no known cases, or where COVID-19 testing of residents had not occurred. For testing through PHSKC, teams of healthcare workers collected nasopharyngeal (NP) swabs from all residents in a SNF during a single visit. For testing through Seattle Flu Study, facilities identified by PHSKC were contacted by the study team for employee testing. Facilities agreeing to participate messaged all employees before the visit to inform them of the upcoming testing event and distributed a copy of the informed consent form for previewing. Employees were eligible to participate if they worked at the facility and were over 18 years old. All testing was voluntary and not required by the employer, and employees were advised that results would not be reported directly to employers. Employees who reported prior testing for SARS-CoV-2 through other mechanisms were eligible for enrollment. Study staff consented individuals in English or in the participant’s language of preference using an interpreter. After informed consent was obtained, individuals completed an electronic tablet–based questionnaire  and self-collected a mid-nasal swab under observation by trained study staff.17 No samples tested through PHSKC were resulted as indeterminant.For testing through PHSKC, NP swabs from SNF residents were placed in universal viral transport media  and transported to the University of Washington Virology Laboratory for testing via a one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assay following the SARS-CoV-2 CDC assay protocol, as previously described.18 Briefly, SARS-CoV-2 detection was performed using real-time RT-PCR with a probe set targeting Orf1b and S with FAM fluor (Life Technologies 4332079 assays # APGZJKF and APXGVC4APX) multiplexed with an RNase P probe set with VIC or HEX fluor (Life Technologies A30064 or Integrated Data Technologies custom made) each in duplicate on a QuantStudio 6 instrument (Applied Biosystems). Three or four replicates for RNase P and SARS-CoV-2 were required to have a detection cycle threshold less than 40 for a sample to be considered positive for this laboratory-developed test, or both replicates must be positive in the research assay. Samples resulting with two replicates of positive SARS-CoV-2 detection were defined as inconclusive. Because tests determined to be inconclusive had SARS-CoV-2 detected in multiple replicates, these results were grouped with positive results for reporting purposes.For testing through Seattle Flu Study, self-collected mid-nasal nylon-flocked swabs were placed in universal viral transport media  and transported to the Brotman Baty Institute for Precision Medicine and the Northwest Genomics Center for testing using a laboratory-developed test for SARS-CoV-2, as previously described.For individuals tested through PHSKC, data available included name, date of birth, date of testing, and whether the individual was a resident or an employee. For employees tested through the Seattle Flu Study, data included participant date of birth, date of testing, race and ethnicity, location and nature of work, new symptoms experienced during the last 7 days, and history of SARS-CoV-2 testing . Frequencies were tabulated for social and demographic data. To test the association between residents and employees who tested positive for SARS-CoV-2, a two-tailed McNemar’s test was used. For employees, positive or inconclusive SARS-CoV-2 test results were reported directly to participants by phone within 48 h and to the Washington State Department of Health. Resident results were reported to the ordering physician at the SNF.The Seattle Flu Study was approved by the University of Washington Institutional Review Board. Other testing of residents and employees was conducted as a public health surveillance activity under the direction of PHSKC.Overall, 1583 employees at 16 SNFs were tested, with 287 (18%) tested through PHSKC and 1296 (82%) through SFS. A total of 46 (2.9%) employees had positive or inconclusive testing for SARS-CoV-2  of employees on-site on the day of testing participated  tested positive for SARS-CoV-2 .Of the 14 SNFs with resident and employee testing  in the Seattle area during the spring 2020 peak of the COVID-19 pandemic. To our knowledge, this is the first study to report occupational SARS-CoV-2 for SNF workers outside of an outbreak investigation and the largest study to date evaluating prevalence of SARS-CoV-2 in SNF workers.4 Similar to other congregate environments like cruise ships, correctional institutions, and long-term care facilities,22 SNFs are an environment where introduction of one case may lead to rapid transmission. We did not assess for routes of disease transmission during the study period, and transmission of SARS-CoV-2 to SNF residents could have occurred from contact between residents, infected employees, or other outside contacts, such as exposure to dialysis centers outside of the SNF.We detected SARS-CoV-2 in both residents and employees at multiple sites. The majority of employee testing positive for SARS-CoV-2 were asymptomatic and involved in direct patient care. In this study, all surveillance was performed starting at least 2.5 weeks after the implementation of a strict no visitor policy at SNFs throughout the region. Given that many infected SNF employees were asymptomatic, transmission events have the potential to go undetected without broad-based testing of all employees. This is critical, because asymptomatic and presymptomatic infections have led to significant transmission events in other high-density congregate living facilities with high-risk residents.25 We found that SNF employees in our study were disproportionately more non-white and non-Hispanic individuals, including a higher proportion of Asian and Black participants, than are represented in the population of the Seattle area.26 While all facilities reported some form of paid sick leave program, many had limits on eligibility for part-time and contracted employees. Lacking paid sick leave is a financial disincentive to report symptoms or positive tests. These factors highlight the vulnerability of SNF employees as an often overlooked group in the transmission of SARS-CoV-2.Minority communities are over-represented in low-wage healthcare clinics and include populations known to be disproportionately impacted by COVID-19.This study was conducted during an ongoing pandemic in Seattle. In the conduct of this study, we encountered obstacles including shortages of PPE, viral transport media, and nasal swabs. Participation was voluntary across sites, and an average of 70% on-site employees participated. The administration at each SNF presented the opportunity for testing with different levels of enthusiasm and support, and this may have impacted employee participation. Furthermore, when approached with the offer of employee testing, the administrative leadership of some of the SNFs in the Seattle area declined to participate. Common concerns about mass employee testing from SNF administration were that testing would result in increased fear, employee absenteeism, and/or consequent staffing shortages.Strengths of this study include broad testing of both residents and employees in a group of SNFs at the spring 2020 peak of the COVID-19 pandemic in a major metropolitan area. We collected sociodemographic and symptom data on the majority of the employees, and information on infection control policies across sites. Limitations of this study included that sites were included only if administrative leadership agreed to participate, and the sites that did not participate may have differed in infection rates and PPE practices compared with those that agreed. Testing at sites did not include all employees; only employees volunteering for testing participated. Collection of SARS-CoV-2 samples used different collection methods between residents and employees, and the timing of testing for resident and employees was not simultaneous. However, both methods of collection have proven to be concordant (Citation of a manuscript under review will be inserted here) and the mean difference between employee and resident testing was small (mean of 1.6 days). Symptom data was self-reported and may be limited by a social desirability bias and/or by recall bias. To mitigate bias, employees filled out the questionnaire using an electronic tablet while at a six foot distance from other participants, which afforded some privacy. Additionally, we do not have longitudinal data on participants and do not know how many asymptomatic individuals were presymptomatic.As SARS-CoV-2 infections continue to cause disproportionate numbers of deaths in facilities for older adults throughout the country, strategies to prevent mortality in this fragile population are critical. We found that infections in both employees and residents persisted even with no visitor policies, and facilities had heterogenous paid leave policies. Based on our findings, implementation of periodic point prevalence testing of both residents and employees, coupled with rigorous infection control precautions and universal paid sick leave for employees, may provide an improved strategy to reduce mortality in this highly vulnerable population. Future research should focus on trials of strategies, such as routine employee testing, to understand their effectiveness in SARS-CoV-2 high-risk occupational settings.ESM 1(DOCX 22 kb)ESM 2(DOCX 20.3 kb)


Hepatocellular carcinoma (HCC) is a serious cancer which ranked the fourth in cancer-related death worldwide. Hence, more accurate diagnostic models are urgently needed to aid the early HCC diagnosis under clinical scenarios and thus improve HCC treatment and survival. Several conventional methods have been used for discriminating HCC from cirrhosis tissues in patients without HCC (CwoHCC). However, the recognition successful rates are still far from satisfactory. In this study, we applied a computational approach that based on machine learning method to a set of microarray data generated from 1091 HCC samples and 242 CwoHCC samples. The within-sample relative expression orderings (REOs) method was used to extract numerical descriptors from gene expression profiles datasets. After removing the unrelated features by using maximum redundancy minimum relevance (mRMR) with incremental feature selection, we achieved “11-gene-pair” which could produce outstanding results. We further investigated the discriminate capability of the “11-gene-pair” for HCC recognition on several independent datasets. The wonderful results were obtained, demonstrating that the selected gene pairs can be signature for HCC. The proposed computational model can discriminate HCC and adjacent non-cancerous tissues from CwoHCC even for minimum biopsy specimens and inaccurately sampled specimens, which can be practical and effective for aiding the early HCC diagnosis at individual level. Liver cancer is the fourth leading cause of death in patients with malignant cancerous . HepatocThe existed diagnostic signatures are mainly on the basis of risk scores obtained from signature genes’ expression , which aMachine learning method is a good choice to uncover underlying patterns . It has 1 . The details have been listed in The gene expression profiles datasets were freely gained from GEO  and TCGAFor the raw data (.CEL files) detected by the Affymetrix platform, the RMA (Robust Multi-array Average) algorithm was used for background adjustment. If a gene was matched to multiple probes, the arithmetic mean expression value was used as the gene expression level. For the data sets detected by the Illumina platforms, we directly used the processed expression data.a and b) is expressed as Ea > Eb (or Ea < Eb) if gene a has higher (or lower) expression level than gene b. The REOs pattern of a gene pair is regarded as stable if the REOs kept in at least 95% of the samples. A reversal gene pair is a gene pair with stable REOs in both cirrhosis tissues in patients without HCC (CwoHCC) samples and HCC samples, but the REOs patterns are reversed in the second group (Ea < Eb or Ea > Eb in CwoHCC samples but Ea > Eb or Ea < Eb in HCC samples). Here, the reversal gene pairs are selected as the candidate REOs signature for the identification of HCC. Then we obtained the common genes between training datasets and validation datasets and its corresponding gene expression profile. Subsequently, based on the gene expression profiles and reversal gene pairs, we generate a new profile by using 1, 0, and −1 to represent Ea > Eb, Ea < Eb, and other cases (Ea or Eb do not exist), respectively.Within a sample, the REOs of two genes   was applgi is a gene pair from the 857 gene pairs and T is the disease type. The mutual information (I) can be formulated as:Here, Ω represents all 857 gene pairs, The mRMR function:I is mutual information between the gi gene pair and disease type T, I is mutual information between gi and gj. Then we used incremental feature selection (IFS) (where on (IFS)  method ton (IFS) .C and γ for SVM are determined by the use of grid search with fivefold cross-validation. In present work, the optimal values are C = 0.125 and γ = 0.5, respectively.Support Vector Machine (SVM) is a powerful classification method which has been used extensively in the fields of biological data mining . Here, tThe sensitivity, specificity and accuracy ,b was apwhere TP, FN, TN, and FP denotes the number of true positives, false negatives, true negatives, and false positives, respectively. Additionally, the ROC curve and AUC are commonly used to test the balance between true positive rate and false positive rate.The flow diagram for identifying and validating the diagnostic signature is shown in Subsequently, we used biopsy and surgically resected samples to estimate the performance of the 11-gene-pair see . For 73 For biopsy samples, all of 80 cirrhosis tissues in patients with HCC (CwHCC) samples in GSE54236 and all of 97 NwHCC biopsy tissues from 2 datasets (GSE64041 and GSE121248) were correctly classified to HCC. The results proved again that, the 11-gene-pair still displayed good performance that most of HCC adjacent non-cancerous patients (CwHCC and NwHCC) can be correctly recognized, even for the inaccurate samples from biopsy specimens. For surgically resected samples, 93.7% of the 254 CwHCC samples and 100% of the 644 NwHCC samples can be accurately identified see in . All aboTo further demonstrate the performance of our proposed signatures, we compared our method with 19-gene-pair-based models and recorded results in For biopsy samples, our proposed model could correctly identify the 70 HCC samples in GSE121248 and the 97 NwHCC biopsy tissues from 2 datasets (GSE64041 and GSE121248) with the accuracy of 100%. Moreover, all 80 CwHCC samples in GSE54236 can be predicted as HCC. Compared with the accuracy (77.5%) of 19-gene-pair based model, the accuracy of 11-gene-pare model could increase to 100%.For surgically resected samples, based on the predictor of 11-gene-pair, 79.8% of the 475 HCC samples from 3 datasets  and 93.7% of the 254 CwHCC samples from 5 datasets  can be corrected as HCC. Moreover, the model can accurately predict the 644 NwHCC biopsy tissues integrated from 7 datasets . Also, the sensitivity of HCC samples increases to 79.8% (19-gene-pair: 79.3%) and the accuracy of NwHCC samples to HCC increases to 100% (19-gene-pair: 96.6%). It can be seen from The above results showed that the proposed 11-gene-pair-based model is powerful on both training datasets and independent datasets. This achievement can be attribute to using within-sample REOs and SVM.Clinical practice has demonstrated that diagnosing the tumors in early stages is key to improve the survival of patient. Although pathology is used as a gold standard for HCC diagnosis, the histological analysis of the HCC biopsy specimen is influenced by the sampling location and tissue amount. In present work, a set of diagnostic signature including 11-gene-pair consisting of 18 genes was identified, which can be used to discriminate HCC and adjacent non-cancerous tissues (CwHCC and NwHCC) from CwoHCC individuals for the early HCC diagnosis.Ten genes in the signature set, including LAMC1, UBE4B, HSPH1, HNF1A, SF3B1, APC2, CHST4, HGF, MTHFD2, and AGO3, might have a vital role during the hepatocarcinogenesis and are key genes for cancer. For instance, LAMC1 mRNA can promote the development of HCC by competing with miR-124 and supporting the excretion of CD151 . UBE4B c2  were used for functional enrichment analysis by using Metascape2  on the KIn current study, we showed that 11 gen pairs can be applied to accurately diagnose the tumors found in the liver. Further, we shall try to establish a user-friendly web-server for the proposed “11-gene-pair” model. In the future, we will apply other feature selection techniques and algorithms to further improve the diagnosis of cancers.https://www.ncbi.nlm.nih.gov/geo/) and TCGA  repository.The datasets used in this study can be freely download from the GEO (HL designed the study and revised the manuscript. Z-MZ carried out all the data collection and drafted the manuscript. Z-MZ, J-XT, FW, F-YD, and Z-YZ performed the data analysis. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pre-injection of probenecid, furosemide, mannitol or colchicine had no influence on activity uptake in kidneys compared to the control group. Mice pre-injected with maleate and fructose had 33% and 51% reduction in the kidney-associated activity, respectively, compared to the control group. Autoradiography images showed that the accumulation of activity after [99mTc]Tc-ZHER2:2395 injection was in the renal cortex and that both maleate and fructose could significantly reduce it. Results from this study demonstrate that pharmacological intervention with maleate and fructose was effective in reducing the kidney uptake of affibody molecules. A presumable mechanism is the disruption of ATP-mediated cellular uptake and endocytosis processes of affibody molecules by tubular cells.Affibody molecules are the most studied class of engineered scaffold proteins (ESPs) in radionuclide molecular imaging. Attempts to use affibody molecules directly labelled with radiometals for targeted radionuclide therapy were hampered by the high uptake and retention of radioactivity in kidneys. Several promising strategies have been implemented to circumvent this problem. Here, we investigated whether a pharmacological approach targeting different components of the reabsorption system could be used to lower the uptake of [ Advances in protein engineering such as molecular display techniques made it possible to generate alternatives to monoclonal antibodies (mAbs). A promising type of binding proteins is the non-immunoglobulin engineered scaffold-based affinity proteins (ESPs) ,2. Of thA common issue for radiometal-labelled affibody molecules is the high radioactivity retention in kidneys. The size of affibody molecules (7 kDa) is below the kidney filtration barrier (60 kDa), making them readily filtered through the glomerulus ,11. AfteHER2:V2 labelled with the cytotoxic radionuclide rhenium-188 that could deliver high absorbed doses to tumours without exceeding kidney and bone marrow toxicity limits Tc was performed in 96.5 ± 1.5% chemical yield and 100% radiochemical purity. Maleate, colchicine, probenecid, fructose, furosemide and mannitol act on various parts of the reabsorption system in kidneys and have been previously shown to interfere with the reabsorption of protein-based agents. To investigate their effect on reabsorption of affibody molecules in the kidneys, they were administered to healthy female NMRI mice prior to the injection of [99mTc]Tc-ZHER2:2395  lower than in the control group (151.3 ± 21.3% ID/g) (p > 0.05) in activity uptake in other organs or tissues. Fructose reduced the kidney uptake of [99mTc]Tc-ZHER2:2395 by 2-fold (74.1 ± 6.4% ID/g) compared to the control group. However, an increased activity uptake was observed in the blood and other normal tissues (Radiolabelling of ZER2:2395 . The con3% ID/g) . A one-w tissues . No diff tissues .Autoradiograms of kidney sections of mice from the control and treated groups showed that the activity was mainly localized in the renal cortex for all studied groups . The lev99mTc]Tc-ZHER2:2395 affibody molecule could be reduced by administration of other drugs and compounds that are known to act on different parts of the renal excretion pathway.ESPs and protein-based targeting agents below 60 kDa are readily reabsorbed in the renal tubular cells after glomerular filtration. Following reabsorption, lysosomal degradation of radiometal-labelled affibody molecules in the tubular cells generates “non-leaky”, residualizing radiocatabolites that are retained inside cells. This renders the radiosensitive kidney more prone to harmful radiation in targeted radionuclide therapy. Therefore, the use of affibody molecules for targeted radionuclide therapy is hampered by this elevated renal uptake of radioactivity. Direct pharmacological intervention using megalin blockers, Gelofusine and lysine, had no influence on the kidney uptake of affibody molecules . Alterna111In]In-DTPA-octreotide, in rat kidneys in a dose-dependent manner [Colchicine is an anti-gout drug that interferes with the process of endocytosis mainly by inhibiting the microtubules polymerization and hence disrupting intracellular trafficking of organelles between different cell compartments . Disruptt manner . No effet manner .111In]In-DTPA-octreotide by 85% compared to the control group. An important finding was the less impressive reduction of renal uptake when maleate was given hours prior to the administration of the radiolabelled peptide, thus indicating a transient effect of maleate. Based on that, and in order to maximize the effect of maleate, we have chosen to administer maleate shortly before the injection of [99mTc]Tc-ZHER2:2395 affibody molecule. Interestingly, we have observed a significant 33% decrease in the kidney activity uptake compared with the control group. It is worth mentioning that the convoluted segments of the proximal tubules, where reabsorption mainly takes place, are confined entirely to the renal cortex. Autoradiograms of kidney sections from the maleate group have demonstrated a reduction in the accumulated radioactivity in the cortex , thus, it is of interest to see if lower doses or different administration schedules could provide a similar effect.Fructose is another compound that decreases intracellular ATP in liver and kidneys mainly through the sequestration of phosphates into metabolic intermediates . Fructostreotide . Mice in maleate . We spec99mTc]Tc-ZHER2:2395. Earlier, Stahl et al. reported that probenecid administration reduced kidney uptake of [111In]In-DOTATOC by 30% Tc-ZHER2:2395 injection. These results indicate that the capacity of reabsorption of small proteins, specifically affibody molecules, is neither dependent on the filter volume or load, nor on its rate of flow.Furosemide, a loop diuretic, may affect the urinary and plasma concentrations of some co-administered drugs by either increasing the glomerular filter load or inducing an increased renal excretion in the tubules . MannitoAlthough interesting, the effect of fructose and maleate on the kidney uptake of radiolabelled affibody molecules is suboptimal and hence cannot justify the use of these drugs in radionuclide therapy protocols involving radiolabelled affibody molecules. We believe that findings from this study may help in understanding the underlying mechanism of kidney uptake of affibody molecules, as well as be of help for researchers working with other classes of targeting agents with elevated kidney uptake. A similar approach may provide an indication for researchers whether they should optimize therapy protocols by co-administering blocking agents or adopt more efficient strategies, for example, pretargeting or plasma half-life extension. We, therefore, recommend not to generalize the findings from this study to other classes of targeting agents and that the kidney uptake lowering strategies should be evaluated for each class on a case-by-case basis.99mTcO4− was provided by elution of 99Mo/99mTc generator  with sterile saline solution. Sodium maleate, D-fructose, colchicine, probenecid and L-lysine were purchased from Sigma . Furosemid , mannitol , and Gelofusine  were purchased as solutions for injections. Radioactivity was measured by an automated γ-spectrometer with a NaI(Tl) detector .Technetium pertechnetate HER2:2395 were produced and purified as described by Ahlgren et al. Tc-ZHER2:2395 affibody molecule was injected intravenously into the tail vein (100 μL). In the treatment groups, mice were pre-injected with probenecid , furosemide , mannitol , colchicine , maleate  and fructose  prior to the injection of [99mTc]Tc-ZHER2:2395 affibody molecule. Control groups were injected with [99mTc]Tc-ZHER2:2395 only. Animals were euthanized and dissected 4 h after injection of [99mTc]Tc-ZHER2:2395 and organs were collected and measured for radioactivity using an automated gamma counter.All experiments had been approved by the Ethics Committee for Animal Research in Uppsala County (Permit Number: C4/ 2016) and were performed according to the Swedish laws on laboratory animal welfare. Thirty-two female NMRI mice, divided into eight groups of four mice, were used in the experiments. The average mouse weight was 27 ± 3 g, and the average kidney weight was 272 ± 35 mg at the start of the experiment. Mice and kidney weights did not differ significantly .The study was done in two days. Day 1 included treatment groups and a control group, shown in GraphPad Prism version 8.00 for Windows  was used for statistical analysis. Biodistribution data were analysed using descriptive and dispersion statistics to measure; the frequency (percentage), distribution or central tendency (mean), dispersion or variation (standard deviation and one-way ANOVA test). The one-way ANOVA was evaluated with Bonferroni correction for multiple comparisons.


The novel coronavirus (COVID-19) pandemic continues to be a significant public health threat worldwide, particularly in densely populated countries such as Bangladesh with inadequate health care facilities. While early detection and isolation were identified as important non-pharmaceutical intervention (NPI) measures for containing the disease spread, this may not have been pragmatically implementable in developing countries due to social and economic reasons . Hence, to elucidate COVID-19 transmission dynamics with respect to the NPI status—e.g., social distancing—this study conducted spatio-temporal analysis using the prospective scanning statistic at district and sub-district levels in Bangladesh and its capital, Dhaka city, respectively. Dhaka megacity has remained the highest-risk “active” cluster since early April. Lately, the central and south eastern regions in Bangladesh have been exhibiting a high risk of COVID-19 transmission. The detected space-time progression of COVID-19 infection suggests that Bangladesh has experienced a community-level transmission at the early phase , primarily introduced by Bangladeshi citizens returning from coronavirus epicenters in Europe and the Middle East. Potential linkages exist between the violation of NPIs and the emergence of new higher-risk clusters over the post-incubation periods around Bangladesh. Novel insights into the COVID-19 transmission dynamics derived in this study on Bangladesh provide important policy guidelines for early preparations and pragmatic NPI measures to effectively deal with infectious diseases in resource-scarce countries worldwide. The novel coronavirus COVID-19) infection, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 9 infecti. As of 1While public and private sectors in Bangladesh keep fighting to cope with the ramifications of this deadly pandemic, the scientific communities are contributing toward improving existing understanding on various aspects of this disease ,10,11,12Spatial, temporal, and spatio-temporal statistics are essential approaches for identifying transmission dynamics of COVID-19, which can enable evaluating ongoing efforts as well as inform new, innovative solutions for containing the disease ,17. ExisTimely detection and evaluation of active space-time clusters of COVID-19 infections that currently pose threat to public health;Track space-time propagation of previously existed clusters for an understanding of ‘where’ and ‘when’ infections spread;Identify linkages between the retrospective emerging patterns of COVID-19 spread and time-lagged NPI scenarios.Specifically, this study has following contributions to the understanding of COVID-19 transmission dynamics relative to NPI efforts in the example of Bangladesh:https://www.iedcr.gov.bd/) that contain cumulative counts of confirmed cases over the past 24 h nationwide. Note that, our study could not incorporate data on COVID-19 cases beyond 15 June because such data at the administrative levels  were not publicly available. At the time of this study , daily confirmed cases were on the rise in Bangladesh, particularly in Dhaka megacity. Although district-level reported cases were made available since 4 March by the IEDCR, currently location-based  daily counts are only available from 4 April Bangladesh and 7 April for Dhaka. We used freely available COVID-19 daily reports from the IEDCR website  at different geographic locations—i.e., districts and thanas. Therefore, we subtracted daily cumulative counts from the previous day’s count to obtain the present day’s new cases for the entire available timeline. Our analysis required the specification of population size over space in each spatial unit. For that, we used the LandScan™ Population dataset with a ~1 km spatial resolution ,27. The We detected currently emerging space-time clusters of COVID-19 cases in Bangladesh using the prospective space-time scan statistic ,29. For 0 stipulates that the model represents a diverse Poisson process of contracting COVID-19 with an intensity μ , whereas the alternative hypothesis HA supports that the number of observed cases is greater than the number of expected cases. The following equation calculates an expected number of COVID-19 cases (μ) [The prospective space-time scan statistic detects active clusters of COVID-19 infections that are still being found at the end of the study period. ases (μ) ,31:(1)μ=0 is the likelihood function for H0; nZ is the number of COVID-19 cases in a cylinder; μ(Z) is the number of expected cases in Cylinder Z; N is the total number of observed cases for the entire study areas—Bangladesh or Dhaka citys—across all time periods; and μ(T) is the total number of expected cases in the study area across all time periods. Risk elevates for the cylinder when it has a likelihood ratio greater than 1.For each cylinder, the number of infections inside and outside the cylinder, as well as the expected number of cases reflecting the population at risk and relevant covariates, are used to calculate the likelihood for each cylinder. The most likely cluster has a cylinder with the maximum likelihood and more than the expected number of cases. Equation (2) was used for calculating maximum likelihood ratio that identified scanning windows with elevated risk for COVID-19 .(2)L(Z)Multiple geographic units can belong to a significant space-time cluster, which assumes that the relative risk (RR) of COVID-19 is homogeneous across a cluster. To avoid that assumption, we derived relative risk for each geographic unit—district and thana—that lies within a cluster. The RR is calculated using Equation (3) ,32:(3)RRIn the following section, we present active and emerging clusters of COVID-19 cases in the Dhaka city and all 64 districts of Bangladesh up to 15 June. To elucidate the space-time transmission dynamics of COVID-19 cases in Bangladesh relative to NPI scenarios, first, we divided current timeline of COVID-19 cases into two groups: 4 April to 30 April and 4 April to 15 June. 30 April was used as a cut-off date as it signifies one of the major breaks in social distancing over the past two weeks , which mBeing the capital and largest industrial and economical hub of the country, Dhaka experienced the inception of the COVID-19 outbreak in Bangladesh. Although the Bangladesh government-initiated intervention measures in mid-March see , it was We found eleven statistically significant emerging space-time clusters of COVID-19 at the thana level in Dhaka megacity from 7 April to 15 June, 2020. As mentioned previously, travel to and from Dhaka city, before and during the nationwide lockdown, may have propagated a community-level transmission in other parts of the country. Hence, in the next sections, we show results from district-level analyses for two distinct periods that characterize large-scale NPI violations and increase in reported COVID-19 cases.Ten statistically significant emerging space-time clusters were detected in Bangladesh between 4 April and 30 April. Cluster 1—with the highest RR of 20.07, contains, collectively, the Dhaka metropolitan area and suburban Dhaka. The second relatively higher risk cluster (Cluster 13) contained the neighboring Faridpur (RR = 4.54) and Rajbari district (RR = 1.22). The third relatively higher risk cluster (Cluster 3) with an RR of 2.97 contained five districts that are located near Dhaka: Narayanganj (RR = 3.26), Munshiganj (RR = 6.42), Madaripur (RR = 2.4), Shariatpur (1.24), and Chandpur (RR = 1.14). The fourth higher-risk cluster (Cluster 2) with an RR of 2.72 included three south-eastern districts: Chittagong (RR = 2.66), Cox’s Bazar (RR = 2.94), and Bandarban (RR = 0.91). The fifth higher-risk cluster (Cluster 6) with an RR of 2.13 contained Comilla (RR = 2.06), Noakhali (RR = 2.05), and Feni (RR = 2.3). Thirteen statistically significant emerging space-time clusters were detected in Bangladesh between 4 April and 15 June. Cluster 11 was the longest (6 May–15 June) active cluster over the period of this analysis (4 April–15 June). This cluster contained two relatively lower-risk COVID-19 districts in the north-eastern Bangladesh: Sylhet and Maulvibazar. Cluster 4, 5, 7, 8, 9, and 12 are the second-longest clusters with lower RR districts. Cluster 1 and 2 were the third-longest  active high-risk cluster that are primarily characterized by industrial  and tourist areas . Cluster 13 was the most recently formed high-risk cluster that included two neighboring districts: Faridpur and Rajbari. Cluster 10 was also a recently formed clusters with relatively lower-risk districts in southern Bangladesh that included areas of the Sundarbans mangrove forest.The suburban region of Dhaka, which is characterized as a high-density working-class population, emerged as a new high-risk cluster with an RR of 16.0 during 1 May–15 June, likely responding to a large-scale violation of social distancing in late April. Although Dhaka metropolitan area experienced a reduced level of risk , it remained the epicenter of outbreak with the highest risk level (RR = 16.54) over the same period. Similarly, the relative risk in Narayanganj and Kishoreganj districts decreased as well, although Narayanganj with RR = 3.26 remained one of the high-risk districts. The areas that manifested >3.00 increase in RR over this period were: Faridpur and Munshiganj. Districts with >2.00 increase in RR were: Madaripur, Comilla, Feni, Chittagong, and Cox’s Bazar, followed by Chandpur, Shariatpur, and Noakhali, where RR increased by 1. The relative risk of COVID-19 increased all over the country after 30 April, as seen in With an understating of the weekly progression of emerging clusters, we can begin to link these emergences with intervention scenarios in accordance with the incubation period, which can be up to 2 weeks. This study leveraged prospective space-time scan statistics for identifying currently active or emerging clusters of COVID-19 in Bangladesh. We conducted this analysis at the district level for Bangladesh and the thana level for the Dhaka metropolitan area—one of the most densely populated megacities in the world. This study, to the best of our knowledge, is a first attempt to showcase the space-time progression of COVID-19 risk in Bangladesh. It is also the first work that utilized prospective scanning statistics to detect emerging clusters of COVID-19 in the country. As the COVID-19 cases were still on the rise at the time (mid-June) of this study, with more real-time data becoming available, the prospective approach demonstrated here can be utilized for rapidly monitoring evolving space-time patterns of COVID-19  risk in Bangladesh and elsewhere. This will enable government and health officials to take appropriate and time-sensitive intervention measures by considering disease’s space-time diffusion pathways and potentially prepare for future outbreaks of a highly contagious disease.We investigated emerging space-time cluster patterns of COVID-19 at the thana level in Dhaka. Utilizing available 7 April–15 June, 2020 daily COVID-19 reported cases, we have identified 11 emerging clusters with 23 relatively higher magnitudes of risky thanas . MultiplWhile exploring the space-time progression of COVID-19 cases at the district level, we found that there were only two high-risk clusters in four districts in the central Bangladesh before May. This was most likely introduced by travelers coming from abroad, including China, Italy, the UK, Saudi Arabia, Kuwait, Bahrain, and India. However, although that is the most likely case, as suggested by the chronicle of government press releases and newspaper reports, without rigorous contact tracing of COVID-19 patients in the early phase , any such assumption could not be scientifically validated. The Bangladesh government-initiated measures on safe evacuations of Bangladeshi citizens from Wuhan, China, travel restrictions, and social distancing measures may have been effective in restricting further outbreak until early March. However, very insufficient and limited testing facilities may have been contributed to that perceived scenario as well in the country. In addition, large-scale violations of the social distancing effort a few times, particularly between 27 April and 30 April, contributed to the transmission outside of Dhaka. Our analysis identified a substantial increase in the number of emerging clusters in the second phase after 30 April. The onset and duration of the emerging clusters  suggest The prospective space-time scanning results presented here suggest that Bangladesh may well have experienced a community-level exposure to the deadly coronavirus pandemic in March. The primary transmission agents were people returning from abroad in February and March, especially from highly infected countries in Europe  and the Middle East . Although the government started taking initiatives in March, it was belated to contain the outbreak primarily in Dhaka city. For that, early detection and effective isolation measures were necessary. In addition, the space-time progression from early April that highlights community-level spread in districts was potentially facilitated by the large-scale violations of social distancing orders. Despite mass gatherings due to poor education as well as behavioral, and dogmatic religious practices , the govThe timeliness and strength of this study lie in its contribution to rapid surveillance measures as manifested by the prospective space-time scan statistics approach. The prospective version of SaTScan is an excellent exploratory statistic approach for closely monitoring disease outbreaks . HoweverThis study provides novel insights into the COVID-19 transmission dynamics and impacts of NPI scenarios—e.g., social distancing in Bangladesh. We found that the central and south eastern regions in Bangladesh are currently exhibiting a high risk of COVID-19 transmission. Dhaka megacity has remained the highest-risk “active” cluster since early April. The space-time progression of COVID-19 infection was validated against the chronicle of government press releases and newspaper reports, which suggests that Bangladesh has experienced a community-level transmission at the early phase  primarily introduced by Bangladeshi citizens returning from coronavirus-affected countries in Europe and the Middle East. We also identified potential linkages between the violation of NPIs and the emergence of new higher-risk clusters over the post-incubation periods around Bangladesh.Overall, the space-time emerging patterns and change in the relative risks of the COVID-19 outbreak identified here can be further compared and validated with local-level epidemiological and travel network datasets. Thus, our study should be taken as a benchmark to understand and unraveling complex pattern and process of COVID-19 transmission in Bangladesh and elsewhere as more heterogeneous data become available and rigorous cross-disciplinary research is conducted in the future. Meanwhile, the knowledge derived in this study on Bangladesh highlights three specific guidelines for reducing disease transmission and minimizing impacts, particularly in the developing countries: (i) in the face of a highly likely disease outbreak, act early to prevent or minimize infection cases by putting necessary travel restrictions, (ii) coordinate with local civic and religious leaders to control human mobility—e.g., cultural or religious gatherings, sporting events, etc.—and (iii) take early preparations on the NPI measures as there are generally inadequate hospital and health care facilities in resource-scarce countries.


The latter enabled full-depth eradication of tumor tissue, demonstrated by the absence of S100 and Ki67 immunostaining. These studies are the first to demonstrate complete melanoma response to PDT in an immunocompromised model in vivo, with quantitative assessment of tumor volume and thickness, confirmed by (immuno) histological analyses, and with non-pigmented melanomas used as controls to clarify the critical role of melanin in the PDT response. The results indicate the potential of OCA-enhanced PDT for the treatment of pigmented lesions, including melanoma.Treatment using light-activated photosensitizers  has shown limited efficacy in pigmented melanoma, mainly due to the poor penetration of light in this tissue. Here, an optical clearing agent (OCA) was applied topically to a cutaneous melanoma model in mice shortly before PDT to increase the effective treatment depth by reducing the light scattering. This was used together with cellular and vascular-PDT, or a combination of both. The effect on tumor growth was measured by longitudinal ultrasound/photoacoustic imaging in vivo and by immunohistology after sacrifice. In a separate dorsal window chamber tumor model, angiographic optical coherence tomography (OCT) generated 3D tissue microvascular images, enabling direct in vivo assessment of treatment response. The optical clearing had minimal therapeutic effect on the in control, non-pigmented cutaneous melanomas but a statistically significant effect ( Melanoma arises from the melanocytes and is highly invasive and metastatic. According to the American Cancer Society, melanoma rates have been rising for the last 30 years and are expected to increase by 2% in 2020 , with abMost melanomas ≈96%) are highly pigmented , which n% are higBaldea et al.  and Huan−1 in the 670–690 nm wavelength range. The latter was significantly higher than in any other lesions, ascribed as being due to the atypically large nuclei [In the optical diffusion regime, the effective penetration depth, i.e., the depth, borators  measurede nuclei . Thus, oe nuclei  and by ie nuclei . We havee nuclei . The clee nuclei . Hydrophe nuclei . These ce nuclei . Two hypotheses were tested here: (1) that using a combination of cellular-PDT with Photodithazine (PDZ) and vascular-PDT with Visudyne (VIS) improves the treatment response in melanoma compared to single-agent PDT, and (2) that topical application of OCA immediately before treatment improves the PDT efficacy sufficiently to enable complete tumor destruction up to about 1 mm thickness, which corresponds to Clark level II melanoma . TreatmeAll animal procedures were approved by institutional Animal Care Committees .PEG-400 and 1,2 propanediol were purchased from Sigma-Aldrich (USA). A 19:1 mix was prepared using PEG-400 and the transdermal permeation enhancer 1,2-propanediol, as described by Wen et al. (2012) .2. Cutaneous melanoma was induced by intradermal injection of 105 cells in one flank of adult female nude mice  using a 30ga needle under inhaled anesthesia (isoflurane 5% for induction and 2% for maintenance). Animals were kept in micro-isolator cages with access to food and water ad libitum. B16F10 pigmented murine melanoma cells were purchased from American Type Culture Collection . B78H14 non-pigmented murine melanoma cells were kindly donated by Dr. Pier-Luigi Lollini, University of Bologna, Italy. Both cell lines were cultured in Dulbecco Modified Eagle’s Medium supplemented with 10% fetal bovine serum and antibiotics at 37 °C and 5% CO−2 for 1000 s, giving an energy fluence of 100 Jcm−2. A 690 nm diode laser was similarly used to activate Visudyne at 80 mWcm−2 and 80 Jcm−2. With the combined photosensitizers, the energy fluences were reduced to 60 and 40 Jcm−2, respectively, and Photodithazine-PDT was given first, followed immediately by Visudyne-PDT. The fluences were reduced compared to the single treatment to minimize the off-target effects, such as inflammation, erythema and pain. This sequence was chosen to avoid reducing the tumor oxygenation due to the vascular shutdown following VIS-PDT that could compromise the direct tumor-cell cytotoxicity of PDZ-PDT. Buprenorphine (0.1 mg/kg) was administered for analgesia before and then every 8 h for 72 h after treatment. Two different photosensitizers were used, either singly or in combination: Photodithazine  to target primarily the tumor cells and Visudyne  for vascular-mediated treatment. The treatment groups are summarized in The tumor volume was measured using 3D ultrasound scanning on a Vevo 2100 system  operating at 32–56 MHz, with the images analyzed using ImageJ software . This instrument also incorporated a tunable pulsed laser  for photoacoustic imaging at 700 nm that served primarily to confirm the presence and absence of melanin in the pigmented and non-pigmented tumors, respectively, and to complement the ultrasound measurements. The tumor volume was measured immediately before treatment and then every 48 h until the sacrifice by cervical dislocation under general inhaled anesthesia, either at 10 d after treatment or earlier if humane endpoints  occurred or if the tumors grew to >10 mm diameter. Sacrifice at 10 d post-treatment was chosen to allow histological analyses and quantification of S100 and Ki67 biomarkers. Moreover, we used immunocompromised animals specifically to avoid any immune-regulation component of the PDT response and, since the tumors were imaged every 48 h to track the change in volume accurately and longitudinally over time, this required procedures outside the biosafety environment, with the corresponding risk of infection for these immunocompromised animals. Studies with longer follow up in immunocompetent mice are in progress. The specific growth rate (SGR) and doubling time (DT) were calculated as in Mehrara et al. , using t®; Leica, Canada) and the images were processed using commercial software  to measure the tumor area and total tumor cell count and to quantify the specific protein-expression levels. Immunostaining was considered negative when <10% of tumor cells showed positivity. The S100-stained sections were used to measure the largest tumor thickness, with three measurements being performed in each section. Upon sacrifice, the tumors were excised and processed for (immuno) histology. Tissue sections (5 μm thick) cut perpendicular to the surface and showing all layers of skin and tumor (top to bottom) were stained with H&E and immunostained for S100  and Ki67  expression as markers of the presence of melanoma cells , respectp < 0.05 was considered statistically significant.The immunohistological and tumor-depth results are presented as mean values ± standard deviation, and statistical comparisons were performed to test differences between the treatment groups and untreated controls, as well as between treatment groups using one-way analysis-of-variance (ANOVA) with post-hoc comparisons with the Tukey test : 6 cells were injected intradermally into the dorsal skin of nine mice (n = 3 per group) and a window chamber was installed 3 days later to allow direct visualization of the tumor. The tumor vasculature was assessed 2 days later by speckle-variance optical coherence tomography (svOCT) to visualize subsurface tissues with sensitivity to capillary-level microvasculature and micron-scale resolution. PDT was performed through the chamber window using the same photosensitizer and light doses as in the intradermal tumor model. The microvascular response was assessed at 2, 24, and 48 h and 7 d post-treatment. In this model, only the vascular response to PDT without optical clearing was investigated, since adding OCA risked compromising the window preparation. Moreover, the effects on melanoma of OCA alone has been previously reported by us [Additional experiments were performed to image directly the effect of PDT on the tumor vasculature using a dorsal-window-chamber model, as described previously . Brieflyed by us . The OCT system was based on a quadrature Mach-Zehnder interferometer with optical amplification , incorpoThe svOCT images demonstrated the rich vascular network around and within the tumors before treatment. As seen in PDT with Visudyne at a short drug-light interval of 15 min  was morThe use of both pigmented tumors and non-pigmented melanoma controls allowed factors such as photosensitizer uptake and intrinsic photodynamic effect, both of which are clinically important parameters that are independent of the presence of melanin, to be distinguished from melanin-dependent antioxidant and light absorption/scattering factors. An example of a pigmented tumor and corresponding ultrasound/photoacoustic images is shown in The tumor volume assessment in the non-pigmented tumors showed that PDZ-PDT halted tumor growth for the first 6 days but regrowth was observed subsequently. VIS-PDT showed similar growth arrest, but this was maintained through day 10: By contrast, in the pigmented tumors the single-agent treatments produced only minimal to mild responses, with Visudyne being slightly more effective than Photodithazine: The volumetric specific growth rate (SGR) and tumor doubling time (DT) profiles are shown in The DT values showed the same general behavior. For the non-pigmented tumors, single-agent treatments increased the DT values by 2- to 8-fold over the untreated controls, corresponding to altered tumor growth and progression within the 10 d evaluation period. The doubling times for the dual-agent photosensitizer, with and without optical clearing, were negative, indicating tumor remission. In the pigmented tumors, no difference in doubling time was observed between single-agent PDT and untreated controls, but dual-agent PDT increased the DT value by ≈10-fold. With the addition of optical clearing, all treatment groups showed negative values of the doubling time.While the in vivo imaging was able to monitor the tumor volume (ultrasound/photoacoustic) and microvasculature (svOCT), it does not give information on the cellular response. Hence, this was assessed by quantitative histology in H&E, S100, and Ki67 staining of tissue sections. , indicating that targeting the vasculature was more effective than targeting the tumor cells directly; this is consistent with the fact that melanomas have a rich vascular network [In the non-pigmented tumors, the treatment decreased the overall number of tumor cells in all groups. However, the PDZ-PDT group still showed 53% and 64% of the cells expressing Ki-67 with and without optical clearing, respectively. (The fact that these levels were lower in the non-treatment control group is an artifact due to the negatively-staining necrotic core that develops as the tumors grow). By contrast, none of the groups treated with VIS-PDT had detectable S100 or Ki-67 staining in the non-pigmented tumors . In these lesions, a reduction in tumor thickness of ≈65% was observed for PDZ-PDT, with no significant change by the addition of OCA. Correspondingly, no residual tumor was seen in either the VIS-PDT or dual-agent PDT groups, with or without optical clearing. By contrast, for pigmented melanoma VIS-PDT reduced the tumor thickness by ≈30% and ≈20% compared to the untreated controls and PDZ-PDT, respectively. Dual-photosensitizer PDT reduced the tumor thickness reduction by ≈75%, ≈70% and ≈50% compared to the control, PDZ-PDT and VIS-PDT groups, respectively. The addition of OCA gave greater enhancement with VIS-PDT than PDZ-PDT (p < 0.0001) and, when used with dual-agent PDT, resulted in complete tumor eradication within the 10 days observation period.A summary of the key results in the intradermal model is presented in Melanoma is an aggressive and highly lethal cancer that survives in a hypoxic environment and invades surrounding tissues ,40. If nPDZ-mediated cellular PDT showed poor responses in both cutaneous models, with only a slight reduction in tumor volume even in the non-pigmented tumors. OCT-angiography assessment of pigmented tumors in the window chamber model showed minimal vascular response immediately after PDZ-PDT and recovery of blood microvasculature within 2 h. Interestingly, the larger feeder vessels were completely destroyed at day 7 and replaced by a rich microvasculature network, preventing full tumor eradication. Vascular-PDT should, in principle, be effective for treating well-vascularized tumors such as melanoma, providing that the poor light penetration can be addressed. Here, VIS-PDT alone, which is predominantly vascular-mediated due to the short drug-time interval used, resulted in a strong response in the non-pigmented tumor. Not surprisingly, however, only partial response was observed in pigmented lesions, with regrowth in the tumor periphery. As also expected, the vascular response was more evident in the pigmented window chamber model for VIS-PDT than for PDZ-PDT, with progressive vascular damage and partial shutdown, together with blood leakage at 2–48 h after PDT. Nevertheless, the larger feeder vessels were re-established at day 7 and the tumor was not eradicated, despite there being growth delay. PDT treatment protocols, including in melanoma, are usually based on a single photosensitizer. Acedo et al. proposed combining two photosensitizers, zinc(II)-phthalocyanine (ZnPc) and a cationic porphyrin meso-tetrakis(4-N-methylpyridyl)porphine (TMPyP), to improve the PDT effect. A synergistic effect was seen in vitro in the three tumor cell lines studied  and tumor growth delay was also seen in vivo . In the As predicted, but not previously reported, the effect of optical clearing on the efficacy of PDT was more marked in the pigmented than non-pigmented tumors. Indeed, minimal improvement in treatment response was seen in the latter, where both vascular- and dual-agent PDT were already effective in these ≈1 mm thick lesions. The relatively poor responses for non-pigmented tumors treated with cellular-PDT, with or without OCA, suggests that other factors such as photosensitizer efflux and/or defects in the apoptotic pathways may be involved, as identified by Baldea et al.  and HuanThe positive effect of the optical clearing was substantial in the pigmented tumors, both for single- and, particularly, dual-agent PDT for which no tumor was detectable in vivo at 10 days post-treatment. This was supported by the negative S100 protein and Ki67 expression data. To our knowledge, this is the first demonstration of effective PDT treatment of pigmented melanoma in an immunocompromised mouse model. The fact that a dual-photosensitizer approach supplemented with OCA was needed to achieve complete response underscores the refractory nature of this tumor, but also suggests that its successful management is possible, albeit procedurally challenging. The high efficacy may translate into improved survival and this is currently under investigation. Additionally, we are evaluating two complementary strategies to enable effective treatment of even thicker lesions: (1) combining OCA with whitening agents that fragment the melanin granules, thereby further reducing the light attenuation  and (2) In addition to the destruction of the primary tumor, PDT has immune-modulation/immunoregulatory effects, including stimulation of tumor-specific cytotoxic T-cells to destroy distant untreated tumor cells, thereby inhibiting tumor metastasis, and induction anti-tumor memory to alter tumor progression and prevent recurrence . These iPDT using conventional photosensitizers has not resulted in clinically useful responses in cutaneous melanomas, which are usually heavily pigmented. The preclinical in vivo experiments reported here support the hypotheses that using combined cellular- and vascular-PDT improves the therapeutic response compared with single-agent PDT and that optical clearing results in better tumor responses for both single- and dual-agent PDT. Complete tumor eradication in a cutaneous pigmented melanoma model by combining optical clearing with dual-agent PDT has been demonstrated for the first time. The next steps will be to optimize the combination PDT treatment and the OCA delivery and to assess the resulting impact on local control, survival and metastatic spread over longer follow-up periods. The translational potential of this novel strategy is that it could make PDT as effective in pigmented melanoma as in other skin tumors, which, given the refractory nature of this disease and its lack of effective treatments, would represent a significant advance.


Furthermore, aMMP-8 levels were less correlated with plaque levels than bleeding on probing (BOP) ; respectively). Thus, aMMP-8 was more robust to the confounding effects of oral hygiene than traditional periodontal parameter bleeding on probing. The aMMP-8 point-of-care mouthrinse test can be utilized as an adjunctive and preventive diagnostic tool to identify periodontal disease, classified by stage and grade, and ongoing periodontal breakdown chairside in clinical practice in only 5 min. Overall, integrating aMMP-8 into the new periodontitis classification system seems beneficial.The aim of this study was to investigate the utility of incorporating active matrix metalloproteinase-8 (aMMP-8) as a biomarker into the new periodontitis classification system (stage/grade) presented in 2018. This study included 150 Greek adults aged 25–78, of whom 74 were men and 76 women. Participants were tested with an aMMP-8 point-of-care mouthrinse test, after which a full-mouth clinical examination was performed to assess their periodontal and oral health. The aMMP-8 levels in mouthrinse were significantly lower among healthy patients compared with patients in more severe periodontitis stages and grades (Kruskal–Wallis test and Dunn–Bonferroni test for pairwise post-hoc comparisons;  A new revised periodontitis case definition system was introduced in 2018 to define periodontitis more appropriately by its stage , and grade (the future risk of progression) [As Tonetti et al.  note, thThe new periodontitis classification system includes grading parameters to assess the future risk of periodontitis progression. Although they are associated with periodontitis and increase the likelihood of future periodontal breakdown, they cannot be considered to be reliable enough to indicate when periodontitis is in its active phase. This applies to even the grade modifiers smoking and diabetes that are well-known risk factors for periodontitis ,4,5. TheOral fluids have been studied intensively to find suitable biomarkers from saliva and gingival crevicular fluid (GCF) for the diagnostics of periodontitis. One of the most studied biomarkers is matrix metalloproteinase-8 (MMP-8). Several studies ; p < 0.001). The association between dental plaque levels and BOP levels and their potential confounding is well understood in the literature [Firstly, aMMP-8 levels in mouthrinse were significantly lower among healthy patients compared with patients in more severe periodontitis stages and grades . All in all, aMMP-8 was the preferable method for differentiating periodontal health and disease, because the risk of false positives seemed generally much lower for aMMP-8 than BOP or VPI . Regarding periodontitis patients, the same applies to BOP and VPI levels among Grade A patients when compared to Grade B patients . This ki VPI see . Our findings indicate that the aMMP-8 mouthrinse PoC/chairside test ,24,25,26The utility of mouthrinse measurements is based on a simple technique to collect and sample gingival crevicular fluid (GCF) ,21,53, iBased on this evidence, therefore, we strongly support the call of Tonetti et al.  for a paTimo Sorsa and Dirk-Rolf Gieselmann are inventors of US-patent 10 488 415 B2 and a Japanese patent 2016-554676.


Randomized controlled trials have demonstrated increased all-cause mortality in elderly patients with dementia treated with newer antipsychotics. It is unknown whether this risk generalizes to non-elderly adults using newer antipsychotics as augmentation treatment for depression.This study examined all-cause mortality risk of newer antipsychotic augmentation for adult depression.Population-based new-user/active comparator cohort study.National healthcare claims data from the US Medicaid program from 2001–2010 linked to the National Death Index.Non-elderly adults (25–64 years) diagnosed with depression who after ≥3 months of antidepressant monotherapy initiated either augmentation with a newer antipsychotic or with a second antidepressant. Patients with alternative indications for antipsychotic medications, such as schizophrenia, psychotic depression, or bipolar disorder, were excluded.Augmentation treatment for depression with a newer antipsychotic or with a second antidepressant.All-cause mortality during study follow-up ascertained from the National Death Index.The analytic cohort included 39,582 patients  who initiated augmentation with a newer antipsychotic  or with a second antidepressant . The median chlorpromazine equivalent starting dose for all newer antipsychotics was 68mg/d, increasing to 100 mg/d during follow-up. Altogether, 153 patients died during 13,328 person-years of follow-up . An adjusted hazard ratio of 1.45  indicated increased all-cause mortality risk for newer antipsychotic augmentation compared to antidepressant augmentation  per 10,000 person-years). Results were robust across several sensitivity analyses.Augmentation with newer antipsychotics in non-elderly patients with depression was associated with increased mortality risk compared with adding a second antidepressant. Though these findings require replication and cannot prove causality, physicians managing adults with depression should be aware of this potential for increased mortality associated with newer antipsychotic augmentation. Antidep support –8. Augme support ,10, but  support , 12.Further, newer antipsychotics have well characterized serious adverse effects, including rapid weight gain, diabetes, and tardive dyskinesia , 14. NewThe safety of newer antipsychotic augmentation for depression should be viewed in the context of limited therapeutic benefits , 18 and The uncertain safety profile of augmentation with newer antipsychotics for depression complicates clinical decisions concerning their appropriate role in treatment for patients who do not have an adequate response to antidepressant monotherapy. This study aimed to estimate the real-world mortality risk of newer antipsychotics in non-elderly adults with depression and an incomplete response to antidepressant monotherapy.The study cohort was assembled from the Medicaid Analytic eXtract (MAX) for 45 states from 2001–2010 . Data frThe study compared all-cause and select cause-specific mortality between non-elderly adult (25–64 years) patients with depression who, after a 90-day period of stable antidepressant treatment, either initiated augmentation with a newer antipsychotic or with a second antidepressant . InitiatPatients with alternative indications for newer antipsychotics including psychotic depression, schizophrenia, bipolar disorder, autism-spectrum disorders, or dementia were excluded. Patients were also excluded if they had any antipsychotic medication use (including older antipsychotics) during the baseline period, used more than one antidepressant medication during the 60 days preceding their index date, initiated a second antidepressant and an newer antipsychotic on their index date, or were diagnosed with a life threatening disorder .All newer antipsychotics available in the U.S. during the study period, except clozapine, were included in the analysis . Although only some of these newer antipsychotics are FDA-approved for the adjunctive treatment of depression, all except for clozapine are used clinically for this indication , 21. AntThe primary outcome was death that occurred during study follow-up. In addition to all-cause mortality, we separately examined natural and unnatural deaths, as well as non-cancer deaths  and sex. In addition, we performed individual comparisons between antidepressant augmentation and the most commonly used newer antipsychotics and explored dose-response based on the initial dose of the newer antipsychotic.AD*(aHR−1) with aHR as the estimated adjusted hazard ratio and IAD as the unadjusted incidence rate for patients in the antidepressant augmentation cohort with corresponding 95% CIs [Lastly, we approximated the absolute difference in the incidence of death between patients in both groups. The adjusted rate difference was approximated as I 95% CIs .post-hoc analysis stratified across the distribution of the propensity score. To provide perspective regarding the robustness of the study results to unmeasured confounding, we estimated the strength of the residual confounding required to fully explain the observed associations for all-cause mortality if in fact no association existed [We performed a series of sensitivity analyses to examine the robustness of our results to changes in design or assumptions:  Alternat existed .P-values were 2-sided with a P value < .05 indicating statistical significance. The study used de-identified data and was approved with a waiver of informed consent by the Rutgers Institutional Review Board.All analyses were performed with SAS version 9.4 . All The initial study cohort included 44,301 patients: 25,172 initiators of antipsychotic augmentation and 19,129 initiators of augmentation with a second antidepressant . BaselinPatients in the initial cohort were on average 44 years old, predominantly female (78%), non-Hispanic white (70%), and eligible for Medicaid due to disability (64%). The most prevalent comorbid baseline diagnoses were anxiety (25%), hypertension (23%), hyperlipidemia (15%), and diabetes (14%) . Over thIn the analytic cohort, initiators of newer antipsychotics had 105 deaths during 7,601 person-years of follow-up  . InitiatEstimates were consistent when the endpoint was restricted to natural death  or non-cancer death . Risk for unnatural death showed a modest numerical increase but confidence intervals were consistent with both harmful and protective effects .Analyses stratified by age group and sex and for individual newer antipsychotics are shown in When augmentation with individual newer antipsychotics was compared to augmentation with a second antidepressant, olanzapine showed the greatest increase in risk , followed by risperidone , quetiapine , and aripiprazole . Due to sample size limitations, stratified analyses and analyses of individual newer antipsychotics were considered exploratory and no formal interaction tests were performed.Sensitivity analyses related to follow-up period, study period, and propensity score implementation, yielded consistent findings . Adding Analyses adjusted for age, sex, race/ethnicity, and index year were generally consistent with inverse probability of treatment-weighted results . The magnitude of the observed relative increase in mortality risk is broadly similar to the findings of a meta-analysis of placebo-controlled randomized controlled trials for newer antipsychotics in elderly dementia patients (54%) , a findiAlthough the absolute mortality risk in patients with depression is markedly lower than in elderly patients with dementia, the magnitude of excess risk in the non-elderly depression group is substantial and warrants careful clinical consideration. A mortality rate difference of 37.7 per 10,000 years of follow-up corresponds to a number-needed-to-harm of approximately 265 per year. For higher risk subgroups the number-needed-to-harm decreases substantially, e.g., to 112 for patients between 55 and 64 years of age. Because of the small-to-moderate-size benefits of newer antipsychotic augmentation  and lack of demonstrated benefit with regards to quality of life or functional impairment , the addStratified analyses suggest potential clinically relevant heterogeneity by age, sex and choice of newer antipsychotic, with particularly high absolute risk differences in patients 55 to 64 years of age, women, and patients treated with olanzapine or risperidone. Importantly, the study’s power to examine these subgroups was limited and stratified results should be considered as hypothesis generating until refuted or confirmed by future research. Newer antipsychotic augmentation showed an association with increased risk for mortality in women but not in men. Although this finding may reflect chance, prior reports have suggested several adverse effects of antipsychotic medications, including weight gain, diabetes, and cardiovascular death may disproportionately affect women . FurtherWhen we restricted the analyses to mortality from natural causes or to non-cancer mortality, the point estimates increased and remained statistically significant. The estimate for unnatural death was numerically small and not statistically significant. Sample size limitations prevented examination of specific causes of death. In contrast to some prior studies in older adults , 40, we We selected augmentation treatment with a second antidepressant as the comparator group for this study as a means of controlling by study design for confounding by indication . This apOur study had some limitations. First, as a nonrandomized study, residual confounding by factors associated with both newer antipsychotic use and increased risk of death, such as unmeasured psychotic symptoms, cannot be completely excluded. However, quantitative sensitivity analysis demonstrates that a strong and prevalent unmeasured confounder would be needed to fully explain the observed association. Second, study results were sensitive to the protocol-specified implementation of propensity score trimming, which excluded a small proportion of the study population in the tails of the propensity score distribution due to concerns for unmeasured confounding. However, this approach was supported by the strong heterogeneity of newer antipsychotic-associated mortality risk in the extremes of the propensity score distribution . eTable 2: ICD-10 Codes for Selected Causes of Death. eTable 3: Baseline Characteristics for New Initiators of Study Augmentation Regimens. eTable 4: Newer Antipsychotic Dose by Generic. eTable 5: Dose-Response for All-Cause Mortality. eTable 6: All-cause Mortality Stratified by Percentiles of the Propensity Score. eTable 7: Mortality According to Underlying Cause of Death . eTable 8: All-cause Mortality by Age Group, Sex, and Individual Newer Antipsychotic Medication . eTable 9: Sensitivity Analyses . eTable 10: Mortality According to Underlying Cause of Death .(DOCX)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.


Our understanding of the impact of severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) on pregnancies and perinatal outcomes is limited.The clinical course of neonates born to women who acquired coronavirusdisease 2019 (COVID-19) during their pregnancy has been previouslydescribed. However, the course of neonates born with complex congenitalmalformations during the COVID-19 pandemic is not known.We report a case series of seven neonates with congenital heart and lungmalformations born to women who tested positive for SARS-CoV-2 during theirpregnancy at a single academic medical center in New York City.Six infants had congenital heart disease and one was diagnosed withcongenital diaphragmatic hernia. In all seven infants, the clinical coursewas as expected for the congenital lesion. None of the seven exhibitedsymptoms generally associated with COVID-19. None of the infants in our caseseries tested positive by nasopharyngeal test for SARS-CoV-2 at 24 hours oflife and at multiple points during their hospital course.In this case series, maternal infection with SARS-CoV-2 during pregnancy didnot result in adverse outcomes in neonates with complex heart or lungmalformations. Neither vertical nor horizontal transmission of SARS-CoV-2was noted. Neonatalinfections with SARS-CoV-2 have been described although robust data on verticaltransmission are lacking. In most instances where neonatal infection has beenreported, close contact with infected mother or caregiver is postulated to have occurred.621 Miscarriage, intrauterine fetal demise, and premature birth have beenreported with COVID-19 while most neonates who acquire SARS-CoV-2 infections haveeither been asymptomatic or experienced a mild course.621 There are currently no reports on the impact of COVID-19 on pregnanciescomplicated by complex malformations in the fetus nor the clinical and hospitalcourse of these neonates after birth. In this case series, we report on ourexperience at a single large medical center in NYC, with newborn infants diagnosedwith complex congenital malformations born to mothers who acquired COVID-19 duringpregnancy.Pregnant women infected with the Severe Acute Respiratory Syndrome Coronavirus 1(SARS-Cov-1) and Middle East Respiratory Syndrome (MERS) coronaviruses were atgreater risk of severe infection and adverse perinatal outcomes compared to womenwho were not pregnant at the time of infection.This study was conducted at New York Presbyterian’s Morgan Stanley Children’sHospital and Columbia University Irving Medical Center, a quaternary carehospital that predominantly serves northern Manhattan and neighboring boroughsincluding Westchester county and New Jersey. Patients were admitted to theInfant Cardiac Unit, a 17-bed intensive care unit dedicated to the care ofneonates and young infants with congenital heart disease. The InstitutionalReview Board of Columbia University has approved this study under expeditedreview with waiver of informed consent.Eligible patients were identified from the Infant Cardiac Unit’s administrativedatabase. Data were obtained by manual chart review and included neonates bornto mothers who were diagnosed with COVID-19 between March 16, 2020, and May 7,2020. Data collected included maternal and infant demographic information,maternal and infant SARS-CoV-2 test results, maternal symptoms, timing ofmaternal clinical presentation, mode of delivery, maternal disposition, Apgarscores, resuscitation at birth, cardiac and other diagnoses, hospital course,and disposition of neonates.This case series included neonates with prenatal diagnosis of complex congenitalmalformations of the heart or lung born to women who tested positive bynasopharyngeal swab for SARS-CoV-2 between March 16, 2020, and May 7, 2020.Infants born to women with symptoms suggestive of COVID-19, but not confirmed bylaboratory test, were not included.A dedicated multidisciplinary COVID-19 team from the Infection Prevention andControl (IP&C) department provided guidance on infection prevention andcontrol measures, breast milk and breastfeeding practice, as well as guidance ontiming and frequency of testing and visitation to our unit and hospital.SARS-CoV-2 testing: All neonates underwent testing for SARS-CoV-2 bynasopharyngeal swab at 24 hours, at 14 days, and in-between or after if symptomsemerged or if there was a scheduled procedure when a repeat test was performed24 hours prior to the procedure as per institutional guidelines. Cobas (Roche)or Xpert Xpress (Cepheid) Reverse transcription polymerase chain reaction(RT-PCR) assay were used to test for neonates for SARS-CoV-2.Based on Centers for Disease Control and Prevention and local IP&C interimguidance, neonates born to COVID-19 positive mothers were considered personsunder investigation (PUI). While a PUI, newborns were placed on droplet andcontact isolation in negative pressure rooms until infection in the neonate wasexcluded at the end of the incubation period, that is, 14 days. Airborneprecautions were implemented if aerosol-generating procedures such as continuouspositive airway pressure (CPAP), oral suctioning, or intubation wereperformed.For neonates born at our hospital, attendance at delivery was organized tominimize exposure of staff and preserve personal protective equipment (PPE)while maintaining adequate numbers of personnel to provide effectiveresuscitation. Hospital policies were followed to ensure staff protection withappropriate PPE usage. After initial stabilization in the delivery room,transport of these neonates was accomplished by placing the infants in anenclosed incubator with an additional team outside of the delivery room orobstetrical operating room. The transport team wore appropriate PPE, wasaccompanied by support staff to facilitate movement through hospital, andtransported patients immediately to a negative pressure room in the InfantCardiac Unit at our hospital.During the pandemic, visitation has been restricted to the Infant Cardiac Unit.For all infants, only one designated, asymptomatic parent or guardian waspermitted, all of whom wore a gown, gloves, and a surgical face mask. Motherswho tested positive for SARS-CoV-2 were prohibited from entering the InfantCardiac Unit until the local IP&C team cleared her for visitation. Clearanceparameters generally included an afebrile period of three consecutive dayswithout antipyretics and at least 14 days (seven days if asymptomatic) hadpassed since the onset of symptoms and other symptoms had markedly improved.22 Asymptomatic mothers who were cleared for visitation were allowed tobreastfeed or provide pumped milk to their child while wearing a mask and havingperformed good hand and breast hygiene.Symptomatic mothers who were not yet cleared for visitation were encouraged toexpress breast milk at home, store appropriately, and deliver to the InfantCardiac Unit. Per hospital policies and CDC recommendations, safe expression ofbreast milk in symptomatic mothers could be performed while mothers wore a mask,performed good breast and hand hygiene, and cleaned the pump between each session.Descriptive statistics and data collection were performed using Microsoft Excel2016 MSO (16.0.4266.1001). Parametric results were expressed as mean andstandard deviation (SD). Nonparametric results were expressed as median andinterquartile range (IQR).Between March 16 and May 7, 2020, seven neonates with complex congenital heart andlung malformations born to women who tested positive for SARS-CoV-2 were admitted tothe Infant Cardiac Unit at our institution. Notably, of the seven, three women wereasymptomatic during their hospital course and denied prior history of COVID-19symptoms. Three women experienced mild to moderate symptoms of COVID-19 prior todelivery and tested positive between five and eleven days prior to delivery. Onewoman who was asymptomatic at admission to the labor and delivery ward developedsymptoms of COVID-19 immediately after delivery and tested positive for SARS-CoV-2.During this period, approximately 15% of women who delivered at our hospital testedpositive for SARS-COV-2.d-transposition of the great arteries with intactventricular septum and a restrictive atrial communication. The patient’smother developed respiratory symptoms and fever two-and-a-half weeks priorto delivery and was diagnosed with COVID-19 11 days prior to delivery. Afterbirth and immediate stabilization, balloon atrial septostomy was performedin a negative pressure room in the Infant Cardiac Unit. Arterial switchoperation was performed on day of life (DOL) 4. His postoperative course wasunremarkable, and he was discharged home on DOL 13. SARS-CoV-2 tests on thisinfant on DOL 1, 2, 4, and 8 were negative.This is a full-term male neonate born at our institution with a prenataldiagnosis of This is a late preterm male neonate born at our institution with prenataldiagnosis of left congenital diaphragmatic hernia (CDH). Prenatal magneticresonance imaging confirmed left-sided defect containing stomach, bowel andliver, a lung area-to-head circumference ratio (LHR) of 0.7, andobserved-to-expected LHR ratio of 0.25. Based on these reports, the infantwas at higher risk for needing extracorporeal membrane oxygenation (ECMO) inthe postnatal period. Although his mother developed mild symptoms and testedpositive one week prior to delivery, she presented on the day of admissionwith worsening respiratory distress and a nonreassuring fetal heart tracingprompting delivery. On DOL 2, the neonate required escalation of support forhypoxic respiratory failure and pulmonary hypertension and was placed onveno-arterial ECMO for three days. The ECMO course was complicated bycoagulopathy prompting decannulation on DOL 4. Gortex patch repair of theCDH defect was accomplished on DOL 5 without complications. SARS-CoV-2 testson DOL 1, 4, and 14 were negative. At the time of this writing, this neonateis recovering well, weaning off noninvasive respiratory support, andestablishing feeding skills.d-transposition of the greatarteries, pulmonary atresia, ventricular septal defect, Dandy Walkermalformation, micro/retrognathia, glossoptosis, and intrauterine growthrestriction. A postnatal diagnosis of trisomy 13 was established. A stent inthe ductus arteriosus was placed on DOL 10. This patient is currently in theInfant Cardiac Unit on nasal CPAP, awaiting tracheostomy for upper airwayobstruction. SARS-CoV-2 tests on DOL 1, 7, 9, and 14 were negative.This is a full-term female neonate born at our institution with complexcongenital anomalies including This is a term male neonate born at an outside institution with pulmonaryatresia and intact ventricular septum who underwent radiofrequencyperforation and balloon dilatation of the pulmonary valve on DOL 10. Due tothe inability to wean off from Prostaglandin E1 (PGE-1) infusion, thispatient underwent ductal stent placement on DOL 19. SARS-CoV-2 tests on DOL1, 5, 9, 15, and 18 were negative. This neonate was discharged home on DOL29.This is a term female neonate born at our institution with prenatal diagnosisof critical pulmonary stenosis who underwent balloon dilatation of thepulmonary valve on DOL 2. SARS-CoV-2 testing on DOL 1 was negative. Thisneonate was discharged home on DOL 7 without additional SARS-CoV-2testing.This is a term male neonate born at our institution with prenatal diagnosisof pulmonary atresia with intact ventricular septum and postnatal diagnosisof critical pulmonary stenosis. This patient underwent balloon dilatation ofthe pulmonary valve on DOL 4. Currently, this patient remains in theintensive care unit and is weaning from CPAP and PGE-1. SARS-CoV-2 testsperformed on DOL 1 and 3 were negative.l-transposed greatarteries, large ventricular septal defect, mild pulmonary stenosis, and 2:1atrioventricular block. This infant was placed on CPAP and weaned to roomair at term gestation. No cardiac intervention was performed. She wastransferred back to the referring institution to improve feeding skillsprior to discharge. All SARS-CoV-2 tests were negative on DOL 2, 5, 14, and23.This is a preterm female infant born at an outside institution at 34 4/7weeks’ gestation due to placenta previa, vaginal bleeding, and nonreassuringfetal heart tracings. Cardiac diagnoses included 621 In this series, except for two, all neonates were born at term gestation. Incase 2, the infant was born early primarily due to symptoms in the mother leading tofetal intolerance of labor. In case 7, early delivery may have been related tomaternal COVID-19 diagnosis. All seven neonates in our case series followed ahospital course that appears comparable to patients with similar diagnoses cared forat our institution in the pre-pandemic era.The clinical course of neonates with complex cardiac or lung malformations born towomen with COVID-19 has not been described previously. Adverse pregnancy outcomesincluding preterm births have been reported with COVID-19.23 In the same study, while the risk of preterm birth (<37 weeks’ gestation)was increased, spontaneous preterm birth was not. We do not yet know the impact ofsevere COVID-19 on the fetus if delivery occurs remote from infection. During the2009 H1N1 influenza pandemic, 25% of neonates who were delivered after the motherhad a hospital discharge from severe or critical illness were born small forgestational age and of lower birth weight than the general population.24 During the SARS epidemic, neonates delivered several weeks after maternalinfection experienced intrauterine growth restriction.3In all seven neonates, infection in the mother occurred in the late third trimester.In six cases, delivery occurred 5 to 11 days after testing positive for COVID-19with one case of postnatal maternal diagnosis. We are only now beginning tounderstand the impact of SARS-CoV-2 infection on pregnancy outcomes if the infectionoccurred earlier in pregnancy, including in the first or second trimesters. Rates ofspontaneous abortion and fetal growth restriction in early trimester maternalinfection do not appear increased in a recent report.25 In our series, nasopharyngeal swab test for SARS-CoV-2 was negative at 24hours and at multiple points thereafter including on DOL 14 when neonatal infectionwas finally excluded in all seven cases. The usual routes of transmission ofSARS-CoV-2 are by respiratory droplets, by close contact with infected individuals,or through fomite transmission from contaminated surfaces or objects.26 The mode(s) of transmission of SARS-CoV-2 to the newborn has not been clearlydefined, and the potential contributions of antepartum and intrapartum infection areunclear. Although placental invasion of SARS-CoV-2 has been reported, robust datathat support in utero transmission are lacking.2729 Studies have detected immunoglobulin M in newborns suggestive of intrauterineinfection, although the sensitivity and specificity of these tests are unknown.17 Newborns can also potentially acquire infection through horizontaltransmission from infected contacts.1113 Thus far, to our knowledge, there are published reports of 28 neonates whohave tested positive for SARS-CoV-2.621 While the modes of transmission to the neonate have not been clearly outlinedin these reports, close contact with the infected mother or other caregiver is presumed.1113 In our case series, all of the seven infants were separated from the motherfor the entire duration of the incubation period, that is, 14 days and have testednegative on multiple tests. This is a specific practice we employed in our Neonatalintensive care unit and Infant Cardiac Unit and not our standard well baby nursery.Our hospital IP&C guidelines restricting maternal visitation to NeonatalIntensive Care Unit and Infant Cardiac Unit infants has likely prevented horizontaltransmission to these infants.In general, vertical transmission can occur from mother to newborn antepartum acrossthe placenta, intrapartum during the birthing process, or after birth through theingestion of breast milk.7In our case series, four of the seven women did not exhibit any symptoms of COVID-19at the time of arrival to the labor and delivery ward, and three continued to beasymptomatic during the entire hospital course. Based upon the high asymptomaticcarriage rate and the highly efficient manner in which this virus is transmitted, wethink it is prudent to test all pregnant women who present for delivery forSARS-CoV-2 infection based on local prevalence and resources.This study has several limitations. It is a small case series of seven patients froman academic University hospital at the epicenter of the global pandemic in theUnited States. Although our results are in accordance with the majority of reportsof vertical transmission, large data sets and data registries are needed to confirmthe low probability of vertical transmission of SARS-CoV-2. Additionally, this studydoes not provide data on pregnant women with fetuses with complex cardiac or lungmalformations who were positive for SARS-CoV-2 and experienced either miscarriage orintrauterine fetal death.In conclusion, neonates with complex cardiac or lung malformation born to womendiagnosed with COVID-19 during pregnancy followed an unremarkable course. We did notidentify vertical transmission in our series. Additionally, we did not experienceany horizontal transmission from mothers with COVID-19.