Cervical cancer (CC) is the leading cause of morbidity and mortality from cancer in Nepalese women. Nearly all cases of CC are caused by infection with certain genotypes of human papillomavirus (HPV). Data on HPV genotype distribution in Nepalese CC patients is sparse. We aimed to determine the distribution of HPV genotypes in biopsies of CC tissue from Nepalese women.This study examined 248 archived paraffin-embedded tissue specimens from CC cases from patients of B.P. Koirala Memorial Cancer Hospital, Bharatpur, Chitwan, Nepal. DNA was extracted from the biopsies and HPV detection performed by PCR. HPV genotyping was then carried out by a reverse line hybridization technique capable of identifying 36 distinct HPV genotypes.Most of the samples were from tumors that had been designated by hospital pathologists as squamous cell carcinoma (77.6%). 165 of the 248 samples contained DNA of sufficient quality for rigorous PCR testing. All the analyzable specimens were positive for HPV. The most common HPV genotypes, in decreasing order of frequency were 16, 18, 45, 33, 52, 56 and 31; most were found as single infections (94.5%). Together, HPV types 16, 18, and 45 were found in 92% of the tumor samples.This study strengthens the knowledge-base of HPV genotype distribution in CC cases in Nepal. Hopefully, this information will be useful to the medical community and public health policy-makers in generating improved HPV-surveillance, −prevention and -treatment strategies in Nepal. In 1983, Harald zur Hausen and colleagues isolated human papillomavirus (HPV)-16 and 18 from human cervical cancer (CC) tissues and helped define the central role of HPV in the development of CC .Papillomaviridae, more than 200 HPV genotypes have been characterized [Alphapapillomavirus genus, can infect the female and male anogenital region. Infection with some of the genotypes can cause benign genital warts while others cause precursor cervical lesions, cervical intraepithelial neoplasia (CIN) and CC [Currently, within the family cterized . Over 40) and CC . HPV gen) and CC . Of the ) and CC .Worldwide, CC is the fourth most common type of cancer among women, and causes over 40% mortality . CurrentIndia, southern China, Myanmar, Tibet and other areas of Central Asia, making Nepal a multiethnic and multicultural country. Nepal’s population has been steadily rising in recent decades, currently reaching approximately 26 million (census 2011). As in most developing countries, a spectrum of sanitary and public health conditions are present, due at least in part to transportation limitations and reduced access to medical services in rural compared to urban areas.Nepal is located in Southern Asia, adjacent to both India and China, in the Himalayan region. Along with indigenous peoples, many of Nepal’s inhabitants are descendants of migrants from Several studies have addressed the prevalence and genotypes of HPV circulating in Nepalese females having non-cancerous cytology, as well as in those having preneoplastic and neoplastic cervical lesions . HoweverAlthough HPV-16 and 18 are the predominant HPV genotypes associated with CC worldwide, at least 25% of CC cases are associated with other HPV genotypes . DiffereThree prophylactic HPV vaccines are currently available worldwide: a quadrivalent vaccine was first licensed in 2006, a bivalent vaccine in 2007 and a nonavalent vaccine in 2014. All protect against infection with HPV-16 and 18; while the most recent also protects against five additional HR-HPV types . Current evidence suggests the three vaccines offer comparable efficacy in prevention of CC. The choice of HPV-vaccine is typically based on data regarding a number of factors, including the type and scale of local HPV-associated pathologies, the target-populations for which the vaccine has been approved and product characteristics  [The goal of this study was to determine the distribution of HPV-genotypes found in CC cases of Nepalese women. We are hopeful that this information will be useful to public-health officials for designing improved strategies for the prevention of HPV-infection and its related diseases.A retrospective cross-sectional study was designed and coordinated by the BP Koirala Memorial Cancer Hospital (BPKMCH), Bharatpur, Chitwan, Nepal, in collaboration with the Central Department of Biotechnology, Tribhuvan University, Kritipur, Nepal, Kathmandu Research Institute for Biological Sciences, Lalitpur, Nepal, and the National Institute of Infectious Diseases (INEI) -ANLIS “Dr. Malbrán”, Buenos Aires, Argentina. The study was approved by the Nepal Health Research Council.Archived formalin-fixed paraffin-embedded sections of tumor tissue biopsies that had been excised from CC patients (30–99 year old women) during 2011 to 2014 at BPKMCH were examined. A random sampling method was used for tissue specimen selection and de-identification was done prior to specimen handling, to avoid any sampling-bias. Information regarding the age at collection, year of collection, and histological findings generated from the samples was obtained.All cervical tumor tissue blocks were processed at INEI under stringent laboratory conditions to avoid contamination of or between samples. For each ten case-block of samples, a paraffin blank-block was also sectioned and included, as a control for contamination. One to five 10 μm sections of tissue from each sample were transferred to a 1.5 mL screw-cap tubes using a fresh tooth pick for each section, for use in downstream techniques described below. Fresh gloves were used for each case. After the sectioning of each case the scalpel used was cleaned with R-WAX . A blank-block was sectioned after each ten case-block.DNA was extracted from the sectioned blocks using a method developed at the United States Centers for Disease Control (US CDC) using the QIAamp DNA Blood MiniKit (QIAGEN) . Briefly. Genotyping was carried out by a reverse hybridization line which identifies 36 HPV- genotypes . Briefly, the denaturated biotynilated amplicons, obtained from amplification of sample DNA with BSGP5+/GP6+ primers, were hybridized with genotype-specific oligonucleotide probes immobilized as parallel lines on membrane strips . The hybrids were treated with alkaline phosphatase-streptavidin conjugate and substrate (ECL Detection Reagents) resulting in a chemiluminescent product subsequently detected by exposure to autoradiography film. The β-globin gene was co-amplified and its relative abundance detected in each sample, to serve as an internal control [HPV detection was performed using PCR with biotinylated Broad-Spectrum General Primers (BSGP) 5+/6+ designed to amplify a highly conserved 140 bp fragment of the HPV-L1 gene . Genotyp control . The INEData analysis was performed using Epi Info version 3.5.3 (US CDC). In determining the frequency of HPV genotypes, each sample was scored for the genotype detected and, if more than a single genotype was detected, for the combination of genotypes detected.Of the 248 paraffin embedded CC tissue blocks originally selected, the β-globin gene was amplifiable in only 165, thus our further analysis was restricted to this subset. Histological analysis of these samples revealed that 81 of the 165 blocks contained squamous cell carcinoma  were infected with a single genotype and 9 (5.5%) were infected with multiple genotypes. Almost all cases were positive for at least one HR-HPV genotype, and one sample harbored HPV-6 . One or more of these three genotypes were detected in 152/165 (92%) of the tumor tissue samples.Samples testing positive for HPV-18 or 45 were from women of a higher age than those infected with HPV-16 Table . The meaThis study defines the HPV-genotype distribution in the largest series of CC tissue samples from Nepal addressed to date. We analyzed archived biopsy specimens from the B.P. Koirala Memorial Cancer Hospital, one of the few hospitals in Nepal specializing in cancer diagnosis and treatment. Patients from throughout Nepal are referred to this hospital, thus the samples analyzed can be considered as representative of the Nepalese population overall.In our study, HPV detection was performed by PCR using the BSGP5+/6 + multiplexed with-globin system, which is technically superior to the original GP5+/6+ PCR system , and suiIn agreement with previously published studies on CC in local, regional and worldwide populations, we found that squamous cell carcinoma was the dominant CC histo-type (77.6%) ., but a little smaller than those rates from Eastern India that exceeds 100%, considering multiple infections  [Our results are consistent with previous studies documenting HPV-16 as the most common and HPV-18 as the second-most common genotypes associated with CC worldwide. We found HPV-16 and 18 to be associated with 87% of the total CC tissue samples in our study, consistent with a previous study of 54 Nepalese cases detecting HPV-16/18 in 90% of samples  . Our st 21.08%) .HPV45 was the next most common genotype found in our study (4.8%), in agreement with previous publications on local and regional populations , 20. TogThe obtained results confirmed the inverse correlation between HPV genotype diversity and progressive disease . Also inMost of the samples examined in this study were infected with only one HPV genotype, which is consistent with the previously observed inverse correlation between HPV diversity and neoplastic lesion . MoreoveOur detection of HPV-6 in one CC biopsy containing no other HPV-genotypes is a rare and noteworthy event. According to the IARC-WHO Working Group Reports, the carcinogenic potential of HPV-6 and 11 is considered “not classifiable”, a category which includes agents that are considered to have low carcinogenic potential, based on the available epidemiological and experimental data . The ratThe results from this and previous studies documenting the presence of HPV-16 and 18 in a high percentage of CC tissue samples represent further evidence that implementation of an HPV vaccine program designed to address at least these two HPV genotypes could significantly lower the incidence of CC in Nepal. Fortunately, all three HPV vaccines provide considerable cross-protection against numerous disease-associated HPV types not specifically included as antigenic targets in the respective vaccine formulations. Based on evidence from clinical trials and post-introduction impact evaluations, the bivalent and quadrivalent HPV vaccines provide protection against HR-HPV genotypes other than HPV-16 and 18, such as HPV-31, 33 and 45, all of which have been implicated to cause preneoplastic lesions and subsequent cancer .Numerous previous studies have made similar optimistic predictions regarding the potential of HPV-vaccination to greatly reduce the overall number of cervical abnormalities , 31. PreThis study increases our knowledge of HPV genotype distribution in cervical cancer cases from Nepal. In anticipation that Nepal will soon implement more effective widespread public health measures to prevent HPV infections and their sequelas, particularly CC, it is important to have epidemiologic data regarding the prevalence of HPV genotypes. Considering the high prevalence of HPV-16 and 18 (83%) found in the CC from Nepalese women in this study, we expect that the widespread introduction of any of the approved HPV-vaccines will sharply decrease the incidence of CC in Nepal. Considering the cross-protection generated against most or all of the disease-associated HR-HPV genotypes by the currently-marketed vaccines, including the relatively inexpensive bivalent vaccine , 33, wid


Staphylococcus aureus, but the dearth of evidence from non-outbreak situations means that routine health-care worker screening and S aureus eradication are controversial. We aimed to determine how often S aureus is transmitted from health-care workers or the environment to patients in an intensive care unit (ICU) and a high-dependency unit (HDU) where standard infection control measures were in place.Health-care workers have been implicated in nosocomial outbreaks of In this longitudinal cohort study, we systematically sampled health-care workers, the environment, and patients over 14 months at the ICU and HDU of the Royal Sussex County Hospital, Brighton, England. Nasal swabs were taken from health-care workers every 4 weeks, bed spaces were sampled monthly, and screening swabs were obtained from patients at admission to the ICU or HDU, weekly thereafter, and at discharge. Isolates were cultured and their whole genome sequenced, and we used the threshold of 40 single-nucleotide variants (SNVs) or fewer to define subtypes and infer recent transmission.S aureus in health-care workers at 4-weekly timepoints was 36·9% (IQR 35·7–37·3), and 115 (58%) health-care workers had S aureus detected at least once during the study. S aureus was identified in 8–50% of environmental samples. 605 genetically distinct subtypes were identified  at a rate of 38 (IQR 34–42) per 4-weekly cycle. Only 25 instances of transmission to patients  were detected.Between Oct 31, 2011, and Dec 23, 2012, we sampled 198 health-care workers, 40 environmental locations, and 1854 patients; 1819 isolates were sequenced. Median nasal carriage rate of S aureus epidemiology in the ICU and HDU is characterised by continuous ingress of distinct subtypes rather than transmission of genetically related strains.In the presence of standard infection control measures, health-care workers were infrequently sources of transmission to patients. UK Medical Research Council, Wellcome Trust, Biotechnology and Biological Sciences Research Council, UK National Institute for Health Research, and Public Health England. Staphylococcus aureus is a common commensal bacterium but also a leading cause of health-care-associated infection. Colonisation usually precedes infection, and the risk of invasive disease is greatest immediately after acquisition of a new strain.S aureus is transmissible between patients, particularly in high-dependency settings.S aureus eradication as routine control measures is controversial.S aureus is transmitted from health-care workers or the environment to patients in a non-outbreak situation, using whole-genome sequencing to establish genetic relatedness and infer recent transmission.In this study, we aimed to determine how often In this longitudinal cohort study, we sampled health-care workers, the environment, and patients at the adult intensive care unit (ICU) and high-dependency unit (HDU) of the Royal Sussex County Hospital, a large acute hospital in Brighton, England. Since the rate of transmission between groups was unknown, we conducted the study for 14 months  and did not prespecify fixed sample sizes. The ICU has one five-bedded area, one four-bedded area, three double side-rooms, and one single side-room. The HDU, two floors below the ICU, has two four-bedded areas, one two-bedded area , and two single side-rooms. During the study, infection control practice followed UK National Health Service Guidelines .12Evidence before this studyStaphylococcus aureus”, “MRSA”, “healthcare worker or staff”, “transmission”, “whole-genome sequencing”, and “molecular epidemiology”. We found two published reports documenting the use of whole-genome sequencing and describing the involvement of health-care workers in S aureus outbreaks. The first report implicated a health-care worker in a persistent meticillin-resistant S aureus (MRSA) outbreak in a neonatal intensive care unit (ICU). In the second study, the investigators characterised two outbreaks of meticillin-susceptible S aureus (MSSA) in separate neonatal ICUs and identified a health-care worker who was colonised with highly related strains during each outbreak. Most published reports of S aureus transmission in health-care settings focus on MRSA and the investigation of outbreaks. To our knowledge, no prospective, whole-genome sequencing-based studies of nosocomial carriage and transmission of S aureus in the endemic setting have been done.We searched PubMed from April 1, 2001, to Sept 1, 2016, for studies published in any language using the terms “Added value of this studyS aureus, thus providing a comprehensive description of colonisation and transmission. Quite unexpectedly, we found continuous ingress of genetically diverse S aureus strains with little onward transmission, despite high rates of carriage by health-care workers and patients, and in the environment.In this study, we systematically sampled health-care workers, the environment, and patients in a critical care unit over 14 months and used whole-genome sequencing to characterise all available isolates of Implications of all the available evidenceS aureus transmission are unlikely to reduce infection rates. Many acquisitions might be attributable to recrudescence of cryptic carriage and should not be routinely ascribed to transmission and breached infection control. We present methods that are of value to those involved in the development and use of methods based on whole-genome sequencing to investigate S aureus colonisation and transmission. The possibility that patients experience recrudescence of cryptic S aureus carriage requires further investigation to understand its mechanism and develop strategies to identify and protect patients from invasive disease.In settings where good infection control practice is in place, additional measures to prevent In this study, patient sampling and data collection without individual consent were approved by the Research Ethics Committee (10/H0505/83) and Health Research Authority (ECC 8-05 (e)/2010), and screening and data collection of health-care workers were approved by the Research Ethics Committee (11/LO/1451). All health-care workers provided written informed consent.S aureus carriage were collected through anonymised questionnaires completed with each sample . Demographic characteristics, comorbidities, and risk factors for We sampled each bed space monthly by swabbing three sites of frequent staff contact  and two less accessible sites (floor behind bed and underside of bed). The blood gas machine in a central utility room was swabbed monthly. Air samples were taken from ten sites monthly  with an intake of 100 L/min and an impact speed of less than 20 m/s , as recommended by the International Organization for Standardization.S aureus (MRSA) and meticillin-susceptible S aureus (MSSA). All patients admitted to the ICU or HDU were eligible and included if they were screened. Additionally, to optimise detection of transmission to patients, from March 1, 2012, sputum, respiratory, urine, wound, and blood culture samples taken for diagnostic purposes yielding S aureus were included. Anonymised data regarding the patient, hospital stay, and ICU or HDU bed stay were collected from patient records and ICU or HDU routine screen results from the laboratory database.As per routine clinical practice, nasal swabs were obtained from all patients at admission to the ICU or HDU , weekly thereafter, and at discharge. Perineal swabs were also taken for most patients. All available screening swabs were included in the study and cultured for meticillin-resistant S aureus comprises a small number of lineages within which closely related strains cannot be reliably distinguished by conventional molecular typing techniques,S aureus, swabs from health-care workers and environmental samples underwent broth enrichment.All isolates were cultured with SaSelect  and MRSAselect chromogenic agar plates . Since the nosocomial population structure of S aureus subtype, which was used to infer compatibility with involvement in recent direct or indirect transmission.S aureus acquisition was defined by a culture-negative screen followed by a culture-positive screen or diagnostic sample, or by a culture-positive sample followed by a culture-positive sample of a different subtype. Transmission from health-care worker to patient was defined as patient acquisition of a subtype cultured from a health-care worker either at the same time or at any previous timepoint in the study. Patient-to-patient transmission was defined as patient acquisition of a subtype cultured from a previous patient, irrespective of whether they shared time in the ICU or HDU. Transient health-care worker carriage was defined as culture-negative pre-shift nasal screen preceding culture-positive post-shift screen on day one, followed by culture-negative screen on day two.All analyses were done at the level of The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.S aureus nasal carriers at enrolment, of whom eight (4%) carried MRSA  and at 4-weekly sampling intervals  consented to participate in the study. 73 (37%) were ied MRSA . During Longitudinal data were available for 191 health-care workers who returned a median of ten (range 2–15) nasal swabs taken every 4 weeks. Three patterns of nasal carriage were observed . 82  of 122 nurses were culture positive by nasal swab, 61 (50%) were culture positive when throat swabs were included, and 64 (52%) were culture positive when all body sites were included. Hence, the sensitivity of nasal swabbing was 70·3% (95% CI 57·6–81·1).S aureus carriage transiently following patient contact, 103 nurses had nasal swabs before and after a shift on day one and before a shift on day two. Concordance of culture results between swabs was 98%. Transient nasal acquisition was not detected.To assess whether nursing staff acquire S aureus isolates were obtained from health-care workers during the study, and whole-genome sequencing was successfully done for 902 (96%) of these isolates  of 191 health-care workers. Acquisition was defined by change of colonisation status from culture negative to culture positive for 40 acquisitions and change of subtype for 29 acquisitions . The genetic diversity of environmental isolates across the study was bimodal. 107 (60%) of 178 isolates were within 40 SNVs of another environmental isolate (of which 81 [76%] of 107 were within one SNV), whereas 71 (40%) of 178 isolates were more than 100 SNVs (predominantly >1000 SNVs) apart  were taken from 40 locations  during the study. imepoint , with 8%s) apart .S aureus in the ICU or HDU  to the ICU and HDU were recorded, including 20 patients who were already in the ICU or HDU at study initiation, 1889 admitted and discharged, and 24 remaining in the ICU or HDU at study end. Median length of stay was 3·0 days (IQR 1·6–6·0), and median age was 65·5 years (IQR 48·8–76·6). 1164 (60%) admissions involved male patients. 1854 (96%) of 1933 admissions were screened at least once for U or HDU , and 178S aureus, of whom 39 (2%) carried MRSA  than were patients admitted to ICU or HDU more than 24 h after hospital admission . Most strains of S aureus carried by different patients were distinct  of 1854 screened admissions carried ied MRSA . 357  and 29 were new subtype acquisitions  acquired S aureus were identified during the study. A median of 38 (IQR 34–42) new subtypes was detected per 4-weekly cycle  of 169 subtypes identified in health-care workers were also identified in patients, only seven fulfilled criteria for transmission from health-care worker to patient—ie, the newly acquired patient subtype was identified in a health-care worker before or at the same time as the patient . These sAlthough 30 (34%) of 88 environmental subtypes were also found in patients, only two were found in the environment and then acquired by a patient . One subOf the 416 subtypes found in patients, 27 were identified in more than one patient. For 14 subtypes, there were 16 acquisitions where a subtype present or previously present in one patient was acquired by another patient, in addition to the transmissions from health-care workers and the environment . Eight iS aureus transmission in an ICU and an HDU where standard UK infection control measures were in place. We sampled health-care workers, the environment, and patients and used whole-genome sequencing to determine the genetic relatedness of isolates. Using a threshold of 40 SNVs  to define genetic subtypes, we detected 605 genetically distinct subtypes separated by a median of 273 SNVs, equating to roughly 34 years of evolution. Strikingly, although a third of health-care workers carried S aureus, only seven instances of transmission from health-care worker to patient were detected. S aureus was also widely present in the environment, but we only detected two instances of patient acquisition from the environment. Consistent with results from our previous study,We have exhaustively investigated S aureus as a reservoir or as vectors. Patient acquisition of S aureus has been associated with overcrowding, understaffing,S aureus, particularly in MRSA outbreaks.S aureus outbreaks. Harris and colleaguesS aureus colonisation and transmission in a well defined high-dependency clinical setting. We showed the presence of highly diverse strains and continuous ingress of new subtypes into the unit rather than ongoing transmission of strains between health-care workers, the environment, and patients.Health-care workers might contribute to nosocomial transmission of S aureus transmission,S aureus in the same month. However, since health-care workers were screened every 4 weeks, transmission from patient to health-care worker is an alternative interpretation. Because the sensitivity of nasal screening is imperfect, we might have underestimated colonisation rates in patients and health-care workers. Our 4-weekly sampling interval for health-care workers could have missed some transient carriage, although the carriage patterns we observed were strikingly consistent. We also staggered recruitment of different groups of health-care workers, starting with nurses in view of their high patient contact, followed by doctors and physiotherapists after successful study implementation. Nevertheless, our method represents the reality of clinical practice; even if some transmissions from health-care workers to patients were not detected, such additional carriage would have to account for a substantial and disproportionate number of transmissions to change our fundamental observation—namely, in the context of good infection control, patients are not at high risk of S aureus acquisition, despite the substantial burden of S aureus detected in health-care workers, the environment, and patients.Our study has limitations. It was done in a single hospital, and our findings might not be generalisable to all locations. However, our study setting is likely to be typical, in terms of risk factors for The high genetic diversity and low level of transmission we observed contrast strikingly with findings from studies in low-income settings with lower barriers to transmission, in which multiple transmissions of small numbers of strains have been documented.Despite intensive sampling, only 25 (26%) of 97 patient acquisitions in our study could be linked to putative donors. The lower carriage rates for patients compared with health-care workers suggest that a substantial proportion of suspected acquisitions might not actually be true acquisitions. Low levels of colonisation  might result in a false-negative admission screen. Some acquisitions might thus represent recrudescence from cryptic (eg, intracellularS aureus colonisation of patients, staff members, and the environment, but transmission to patients occurs infrequently. Therefore, use of additional measures to reduce S aureus colonisation of health-care workers and the environment might provide little extra protection to patients in a non-outbreak situation.To conclude, in the presence of robust infection control measures, the critical care setting is characterised by genetically diverse and continually changing patterns of


Most trials investigating new drugs around the world, including phase I trials, are conducted in outpatient clinics. However, in Japan, regulatory authority requirements and traditional domestic guidelines often require hospitalization of phase I study participants.Patients participating in single-agent phase I clinical trials at National Cancer Center Hospital between December 1996 and August 2014 were monitored. Toxicity requiring hospitalization is defined as toxicity that needs intensive treatment. Study designs were classified into three types: first-in-human (FIH) study, dose-escalation study  in Japanese patients), and dose-finding study (to assess safety and pharmacokinetic profiles up to the MTD previously determined in the West).n = 582, 62%), first-in-human , or dose-finding . A total of 76 study drugs were evaluated as part of this pool of phase I studies. Subdivided by mechanism of action, 20 (26%) were cytotoxic, 50 (66%) were molecularly targeted, and 6 (8%) were immune checkpoint inhibitor. Thirty-six patients (3.8%) had severe toxicities requiring hospitalization during the first cycle. The overall number of toxicities requiring hospitalization and/or grade 4 toxicities during any cycle was 5.0%.A total of 945 patients who participated in a variety of single-agent phase I clinical trials between December 1996 and August 2014 were included in this study. Patients participated in one of three study types: dose-escalation (The frequency of severe toxicity that needs to be hospitalized was unexpectedly low. The data did not demonstrate the need for hospitalization in the phase I trials, suggesting that phase I trials in Japan could be conducted in outpatient settings. Phase I clinical trials of oncologic drugs are conducted to determine the proper dosage of both single-agent and combination therapies. If the therapeutic index is very narrow, it is critical to evaluate the study drug toxicity in dose-escalation studies. Dose-limiting toxicity (DLT) is evaluated during the first cycle of each new dosage level for every trial participant to determine the maximum tolerated dose (MTD). Close monitoring for symptoms as well as physiological and laboratory data are required to determine if DLT is observed.Clinical trials in Japan and Western countries are carried out under the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines . These gBecause the assessment period for DLT is approximately 1 month in most phase I oncology clinical trials, in Japan study participants are required to be hospitalized for 1 month. Phase I clinical oncology trial participants are generally refractory to standard treatments and usually have very limited options. Even though these patients have a good performance status and generally good health, their life expectancy is limited, and consequently, the phase I hospitalization requirement is sometimes in conflict with ongoing end-of-life care. Additionally, the patient is usually charged with the cost of hospitalization, making a patient’s participation in the phase I trial burdensome with respect to time and cost. For all these reasons, it is very difficult to recruit patients to participate in phase I clinical trials. Furthermore, Japan’s hospitalization practice makes it an outlier from standard ICH guidelines, limiting the globalization of Japanese oncology drug development.Toxicity assessment is an essential objective of a phase I trial. Hyman et al. have developed a nomogram-based model to predict the risk of a patient developing a cycle-one serious drug-related toxicity (SDRT), but its practical use remains very limited . AdditioIf the frequency of severe toxicity during first cycle in phase I trials is low, it may be more practical to conduct trials in an outpatient setting or with only minimal hospitalization. Furthermore, minimizing or avoiding hospitalization altogether may facilitate recruitment of participants and accelerate clinical drug development. However, to date, there has been no evaluation of the incidence and risk of severe toxicity in hospitalized patients participating in phase I clinical oncology trials in Japan.In this study, we surveyed severe toxicity observed in the phase I single-agent clinical trials conducted in our institution and investigated the frequency of toxicity that did or did not require hospitalization.Patients who participated in single-agent phase I clinical trials at National Cancer Center Hospital between December 1996 and August 2014 were included in this study. Drug type, treatment course, and toxicity were retrieved from medical records and databases. Severity of toxicity was assessed by Common Terminology Criteria for Adverse Events v. 4.0 . Severe The present study involving human subjects was approved by the National Cancer Center Institutional Review Board (2014-148). A copy of the letter from the Institutional Review Board is available for review by the Editor of this journal.n = 207, 22%), molecularly targeted drugs , or immune checkpoint inhibitors . Patients participated in one of three study types: dose-escalation , first-in-human , or dose-finding . Tumor types were non-small cell lung cancer , colorectal cancer , sarcoma , esophageal cancer , pancreatic cancer , bile duct cancer , breast cancer , and gastric cancer  . Median patient age was 58 years ; 537 patients (57%) were men and 408 (43%) were women. Patients were assigned to receive cytotoxic drugs  were cytotoxic, 50 (66%) were molecularly targeted, and 6 (8%) were immune checkpoint inhibitor (Table Ninety-eight patients 10.4%) developed severe toxicities during the first cycle; a total of 126 patients 13.3%) developed severe toxicities during any cycle. Thirty-six patients (3.8%) had severe toxicities requiring hospitalization during the first cycle  in dose-escalation studies, 14 patients (10.9%) in FIH studies, and 11 patients (4.7%) in dose-finding studies developed severe toxicity, including DLT. Twenty-seven (4.6%) dose-escalation study participants, 4 (3.1%) FIH study participants, and 5 (2.1%) dose-finding study participants needed hospitalization for toxicity , neutropenia , thrombocytopenia , anorexia , and proteinuria . During the first cycle, toxicities requiring hospitalization included thrombocytopenia , febrile neutropenia , ileus/bowel obstruction , arrhythmia , and pneumonia   compared to the doses used in dose-escalation studies. Severe toxicity in dose-finding studies was low (4.7%), likely because the dose level in these studied may not reach the MTD. Consequently, in phase I oncology dose-escalation and dose-finding studies, hospitalization during the first cycle should not be mandatory. In contrast, in FIH studies conducted to determine MTD, careful observation is required because the drug’s toxicity profile is not well characterized, and hospitalization is one option to ensure patient safety. In FIH studies in Japan for drugs studied in earlier Western trials, it can be useful to use the Western clinical data to design phase I trials that reduce the risk of severe toxicity because it has been observed that the both the MTD and DLT frequency tends to be very similar between Western and Japanese patients [The frequency of toxicity by study types was similar in both the dose-escalation and FIH studies   or by dosage levels. Of particular note, there is no standardization regarding the type of toxicity requiring hospitalization in Japan compared to other countries. We employed the definition described in the Methods, although it should be noted that hematological toxicity can be managed in an outpatient setting by blood transfusion with close monitoring in some cases. An effort between clinical investigators, regulatory authority, and other key stakeholders to identify a consensus definition of “toxicity requiring hospitalization” is warranted.Our results indicate that there is a low rate of severe toxicity requiring hospitalization in phase I oncology trials in Japan. Consequently, the conventional approach requiring participants to be hospitalized during the first cycle of the phase I trial is not necessary. Rather, the outpatient setting should be considered for phase I clinical trials in Japan.


This event aims to raise awareness that overdose deaths are preventable, reduce stigma about substance use disorders and drug-related deaths, highlight community drug-related services, and support evidence-based policies and programs to prevent or reduce drug-related harms. Additional information is available at The opioid overdose epidemic resulted in the deaths of approximately 300,000 persons in the United States during 1999–2015, including 33,000 in 2015 (MMWR 1) highlight how increases in deaths and death rates related to heroin and synthetic opioids mirror data tracking illicit drugs and 2) describe the role of IMF and fentanyl analogs in 281 overdose deaths in 2 months in Ohio. Additional data and information on CDC’s state-level efforts to address drug-related deaths are available at https://www.cdc.gov/drugoverdose/index.html.Reports in this issue of


Herein, we review current experience of auto-HSCT for the treatment of CD as well as approaches that have been used to monitor immune reconstitution following auto-HSCT in patients with ADs, including CD. We also detail immune reconstitution studies that have been integrated into the randomized controlled Autologous Stem cell Transplantation In refractory CD—Low Intensity Therapy Evaluation trial, which is designed to test the hypothesis that auto-HSCT using reduced intensity mobilization and conditioning regimens will be a safe and effective means of inducing sustained control in refractory CD compared to standard of care. Immunological profiling will generate insight into the pathogenesis of the disease, restoration of responsiveness to anti-TNF therapy in patients with recurrence of endoscopic disease and immunological events that precede the onset of disease in patients that relapse after auto-HSCT.Patients with treatment refractory Crohn’s disease (CD) suffer debilitating symptoms, poor quality of life, and reduced work productivity. Surgery to resect inflamed and fibrotic intestine may mandate creation of a stoma and is often declined by patients. Such patients continue to be exposed to medical therapy that is ineffective, often expensive and still associated with a burden of adverse effects. Over the last two decades, autologous hematopoietic stem cell transplantation (auto-HSCT) has emerged as a promising treatment option for patients with severe autoimmune diseases (ADs). Mechanistic studies have provided proof of concept that auto-HSCT can restore immunological tolerance in chronic autoimmunity  NOD2) and autophagy-related protein 16-1 (ATG16L1) genes which suggest impairment of bacterial sensing and clearance. CD is associated with increased IL-12/IL-23 release from antigen-presenting cells and an imbalance in the differentiation of Th1/Th17 effector and regulatory lymphocytes  populations. Defects in the innate immune pathway in CD are implicated by pathogenic mutations in the nucleotide-binding oligomerization domain 2  has been identified as a promising therapeutic option for patients with severe autoimmune diseases (ADs). Mechanistic studies suggest that restoration of immunological tolerance in chronic autoimmunity occurs after auto-HSCT uto-HSCT , 15, conuto-HSCT , 17. Thiuto-HSCT , 17.2) and granulocyte colony-stimulating factor (G-CSF), after which they were randomized to immediate auto-HSCT or conventional care for 1 year  and conditioning (50 mg/kg for 4 days). The median duration of anemia and neutropenia was shorter after both mobilization and conditioning than that seen in previous reports using higher cyclophosphamide dosing, and few episodes of febrile neutropenia were reported. The lower intensity regimen still resulted in marked reduction in clinical disease activity with 13 patients achieving disease remission (CDAI < 150) at 30 days  for 3 months, with no medication for CD and no evidence of active disease on imaging and endoscopy at 1 year. Few patients randomized to immediate HSCT or who underwent mobilization and were then randomized to conventional care achieved the ambitious primary endpoint at 1 year. However, ASTIC did demonstrate benefits of auto-HSCT over conventional care in more traditional endpoints for therapeutic trials in this area , such as0 for 3 m 30 days .In addition, favorable long-term outcome after auto-HSCT in a single-center cohort of 29 patients with CD  has been described . This inThe long-term outcomes for all adult patients undergoing auto-HSCT for CD in Europe between 1997 and 2015 (outside the ASTIC study) have been evaluated using the EBMT registry. Clinical data were obtained for 82 patients from 19 centers in 7 countries, with clinical response being categorized as remission , significant improvement (improved pain and frequency), no change, or worsening of symptoms.Median follow-up was 41 months (range 6–174). At 100 days post-HSCT, 64% of patients were in CR and a further 28% had experienced significant improvement. At 1 year after transplantation, data from 75 patients indicated that 43% were in CR, 20% improved, 17% unchanged and 20% worsened. 37% patients required surgery after auto-HSCT, and 73% re-started medical therapy. Of those requiring further treatment, 57% responded to therapies to which they had previously been refractory. Treatment-free survival, defined as survival without major surgery or medical therapy, was 27 and 22% at 3 and 5 years respectively.In addition, the EBMT ADWP has produced a historical summary of the AD section of the registry, which has characterized outcomes following auto-HSCT in various indications. Compared with MS, relapse is more common in CD, which may reflect fundamental differences in disease processes .As of November 2017, there have been a total of 166 transplant registrations for auto-HSCT of CD within the EBMT registry, with the majority 91.5% being in adults over 18 and 24% being treated as part of the ASTIC trial. Patients are predominantly being treated in Spain, UK, Italy, Belgium, The Netherlands, and France   , 35 and T (Treg) . In addiT (Treg) . Functioter HSCT . Likewister HSCT . CollectCompared with MS and rheumatological diseases, there are relatively little data specifically relating to immune reconstitution following auto-HSCT in CD. The following sections will discuss how components of aberrant immunity may be investigated further in CD.The intestinal innate immune system not only protects against invading pathogens, it also regulates the interactions between the commensal intestinal microbiota and the host at multiple levels, many of which are altered in CD. A small study investigating the effect of auto-HSCT on the innate immune system in seven patients with moderate to severe CD has reported elevated numbers of peripheral blood TLR-4 expressing monocytes that also express TNF-alpha and IFN-gamma at baseline compared to healthy controls, which were normalized after transplantation . The imp+ Th17/Th1 cells toward bacterial-derived proteins that have been described in patients with CD suggest a potential mechanism for sustaining persistent disease  . Clinica+ memory T cells in the periphery, as well as a greatly enhanced CD123+ plasmacytoid DC infiltration of the lamina propria. Interestingly, IFN-γ production was higher in non-responders to G-CSF compared with responders. Although a randomized controlled placebo trial has not yet been performed, these studies suggest that G-CSF per se might offer some therapeutic benefit for patients with CD. Whether stem cell mobilization with G-CSF affects the functional orientation of immune cell populations in patients with CD and whether G-CSF-mobilized immune cell subsets, such as Treg cells and/or plasmacytoid DCs, favor the re-establishment of immune tolerance after auto-HSCT remains to be evaluated.A clinical trial in nine patients with CD at the USA National Institutes of Health confirmed the potential benefit of subcutaneous G-CSF given over a 4-week period . In thisIt is essential to determine whether there is a real “reset” of autoimmunity following auto-HSCT for CD, as has been observed for multiple sclerosis and systemic lupus erythematosus and which forms the basis for treatment-free remissions, or simply a temporary downregulation of innate and acquired immune responses. The relapse rate in CD after auto-HSCT is considerably higher compared to other ADs, which could be related to an insufficient eradication of the autoreactive immunologic memory by the conditioning regimens applied and/or a more prominent genetic contribution that favors disease recurrence. The latter fact is supported by epidemiologic studies demonstrating concordance rates among monozygotic twins of up to 50% . AlternaGuidelines and expert recommendations to develop and implement systematic approaches to monitor immune responses in patients with cancer have been recently published . LikewisFor the analysis of TCR repertoire, several different approaches and methods exist. The approach to be used is dependent on the experimental questions being asked and methodological bias can make it difficult to compare results across different studies. The latest advances, available tools, the choice of starting material, and the method for preparing samples have been reviewed in detail elsewhere . Most of® Beta Mark TCR V-beta Repertoire Kit, Beckman Coulter).Comprehensive protocols relating to multi-parameter flow cytometric analysis have been published previously , 49. In via a thymic-independent pathway, which involves the expansion of graft-derived mature donor T cells, or a thymic-dependent pathway, the consequence of which is a regeneration of T cells with a more diverse TCR repertoire from graft-derived precursor cells  has become an important discovery tool, and the preferred choice for unbiased biomarker discovery of transcriptional signatures associated with disease activity, treatment outcomes, and mechanism of action studies of therapeutic agents. However, data analysis and interpretation require advanced bioinformatics approaches. Quantitative PCR (qPCR) provides a more accurate insight into gene expression than microarrays and requires lower amounts of RNA. However, the analysis of the expression of multiple genes with classical qPCR and digital PCR, which can also be used for high-throughput high-precision analysis, is more difficult.Gene expression profiles reflect the immune NanoString-based molecular “bar coding” enables the high-throughput analysis of the expression of multiple genes using defined panels such as the nCounter™ Human Immunology, Human Inflammation, and Human Myeloid Innate Immunity Panels. The Human Immunology module could be particularly relevant to the post-HSCT immune monitoring of patients with CD, given that the ASTIC trial reported treatment-related infections to be the most frequent serious adverse events during the 100 days after conditioning and the subsequent follow-up . The Nanvia a soon-to-commence clinical trial (ASTIClite). This UK NIHR-funded national multicenter randomized controlled clinical trial aims to recruit 99 patients with a 2:1 randomization to compare HSCTlite with standard of care. For this, eligible outpatients with treatment refractory CD will be randomized to auto-HSCT with cyclophosphamide 1 g/m2 + G-CSF for mobilization followed by transplant conditioning with fludarabine 125 mg/m2, cyclophosphamide 120 mg/kg, and rabbit anti-thymocyte globulin 7.5 mg/kg  versus standard of care and followed for 48 weeks. The reduced doses of cyclophosphamide in the mobilization and conditioning regimens are based on the concerns raised following the single case of TRM in the original ASTIC trial, which may have been related to cumulative toxicity from higher mobilization dose of cyclophosphamide (4 g/m2) followed by the transplant conditioning  using multi-parameter flow cytometry. Representative panels that can be used for the monitoring of immune reconstitution in patients with CD following auto-HSCT are summarized in Table Reconstitution of immune potential is being monitored by determining the re-appearance of monocyte subsets, T cell subsets , B cell subsets, and NK cell subsets  before and after auto-HSCT and during the progression toward disease relapse.Recently, the EBMT and European Crohn’s and Colitis Organisation have published a review to encourage and guide inter-specialty collaboration in both clinical and scientific development of this auto-HSCT in CD . Thus, iAP drafted the initial version of the manuscript, to which all other authors made significant content and editorial contributions, with JS and TA leading the contribution from the EBMT Autoimmune Diseases Working Party. All authors are integrally involved in the clinical and/or scientific aspects of the ASTIClite trial .Although drug doses have been checked against primary sources, this review should not be used as a means of assuring prescription of chemotherapy and other drugs in clinical practice.JS has received honoraria for speaking from Sanofi and Jazz. JG has received honoraria for advisory boards from Abbvie, Celgene, Gilead, Janssen, Roche/Genentech, and Novartis. Otherwise, the authors confirm that there are no commercial or financial interests and relationships that could be construed as being a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors JS, and a shared affiliation with the reviewer MR.


It is still unclear whether the time interval from transplant to DSA occurrence influences graft damage. Utilizing sera collected longitudinally, we evaluated 114 consecutive primary pediatric kidney recipients grafted between 2002 and 2013 for dnDSA occurrence by Luminex platform. dnDSAs occurred in 39 patients at a median time of 24.6 months. In 15 patients, dnDSAs developed within 1 year (early-onset group), while the other 24 seroconverted after the first posttransplant year (late-onset group). The two groups were comparable when considering patient- and transplant-related factors, as well as DSA biological properties, including C1q and C3d complement-binding ability. Only recipient age at transplant significantly differed in the two cohorts, with younger patients showing earlier dnDSA development. Late AMR was diagnosed in 47% of the early group and in 58% of the late group. Graft loss occurred in 3/15 (20%) and 4/24 (17%) patients in early- and late-onset groups, respectively (p = ns). In our pediatric kidney recipients, dnDSAs predict AMR and graft loss irrespective of the time elapsed between transplantation and antibody occurrence. De novo posttransplant donor-specific HLA-antibody ( This observation has prompted recommendations on posttransplant HLA-antibody monitoring as a tool to identify patients at risk for antibody-mediated rejection and graft loss [Humoral alloimmunity leading to chronic antibody-mediated rejection (AMR) has been recognized as a major obstacle to long-term kidney graft (KTx) survival –5. In adaft loss –16. early-onset DSAs being associated with lower graft survival [It has also been shown that DSA development continues as an active process even many years after transplantation , 14–17, survival , 17. dnDSA appearance could influence AMR development and graft outcome.We conducted a longitudinal analysis on a pediatric cohort of pretransplant HLA-antibody-negative, first kidney recipients sequentially monitored for posttransplant DSA onset and alloantibody biological properties, in order to evaluate whether the timing ofBetween July 2002 and March 2013, 125 consecutive patients were referred to the Genoa Pediatric Kidney Transplant Program for first allografting. Pretransplant patient sera were screened periodically for the presence of panel reactive anti-HLA antibodies by complement dependent cytotoxicity technique and by a bead-based assay  or MDRD Graft biopsies were performed for clinical indication (graft function decline and/or proteinuria); since 2010, DSA positivity was also included among indications. Rejections were histologically graded following the Banff 97 criteria with updates. Banff 2009 and Banff 2013 criteria were employed for classifying C4d positive and negative AMR . All bioThis study was approved by the Institutional Review Board of the Fondazione Ca' Granda, Ospedale Maggiore Policlinico, Milano (867/2014).n = 11) were not included, resulting in a total of 114 nonsensitized first kidney allograft pediatric recipients monitored for dnDSA . p values < 0.05 were considered statistically significant. Event-free survival was estimated with the Kaplan-Meier method and was compared between risk groups with the log-rank test. For graft failure, censoring event was death with functioning graft. For AMR, censoring event was graft failure. Patients who did not experience graft failure or AMR were censored at the end of the follow-up. Stata 13  or the NCSS System  was used for computation.Data were described as the mean and standard deviation (SD) or median and range if continuous and as count and percent if categorical. To determine differences among patient groups, categorical variables were compared by chi-squared analysis, continuous variables with  dnDSAs at a median time of 24.6 months (range 3–115 months). The mean number of DSA specificities found per patient was 1.97 (±1.29).The cohort median follow-up was 6.7 years (range 2.0–12.6). Antibody identification was based on longitudinal analysis of collected sera in both retrospective and prospective sample series. Among the 114 patients analyzed, 39 patients (34%) developeddnDSA-positive KTx recipients were stratified in two groups, based on time to DSA appearance. We considered patients with antibody occurrence within the first 12 months (the period of greater immunosuppression reduction) as those more prone to mount an immune response to the graft  and patients with antibody occurrence beyond the first posttransplant year as the late-onset group (n = 24) ( dnDSA development ((n = 24) . The medelopment . dnDSA-positive patients recognized a total of 78 HLA antigen specificities. In the two patient groups, HLA class I and class II specificities were equally distributed, and a similar pattern was observed when the analysis was carried out for each HLA antigen locus  in the early-onset group, compared to 1.1 years  in the late-onset group. AMR-free survival did not differ between early- and late-onset groups  and in functional clusters . No significant differences were observed between the two groups  in the early-onset group, compared to 5.5 years (range 3.6–6.5) in the late-onset group (p = ns)  . As the t groups .The problem of clarifying whether HLA antibodies developing at different posttransplant intervals could have different cytotoxic capabilities and graft tissue damage potential has relevance in view of the need to establish the optimal terms of posttransplant DSA surveillance strategy, particularly concerning monitoring length. dnDSA appearance, does not differ in the early- and late-onset HLA-antibody groups. In previous studies, it had been shown that DSAs developing within the first year after transplantation resulted in early graft failure, whereas late-onset DSAs, although also detrimental, seemed to require a longer time to finally cause graft damage and loss [ dnDSAs emerging in the late posttransplant period. Through this longitudinal detection approach, we demonstrated that DSAs in the two patient groups displayed equivalent damaging capabilities. Indeed, early- and late-onset dnDSAs did not differ in the biological properties, such as high MFI values and complement-binding ability, recently demonstrated to be the main determinants of antibody-mediated graft damage and loss [ dnDSAs were comparably represented in both early- and late-onset groups. This apparent discrepancy could be in part explained by the fact that our study exclusively analyzed nonsensitized recipients. Indeed, in a first set alloresponse condition, the ubiquitous cellular expression of class I HLA antigens within the kidney graft tissue may be balanced by the greater stimulating capability of the highly polymorphic class II molecules, in particular HLA DQ antigens [ dnDSAs, we demonstrated in both patient groups that C3d binding was almost exclusively a property of class II, whereas C1q binding was expressed in a similar percentage by both classes. This finding gives additional strength to previous data demonstrating that, in nonsensitized low-risk kidney recipients, class II specific and, in particular, anti-DQ de novo antibodies are the principal effectors of graft loss in all posttransplant phases [ early and late dnDSA damaging capacity was further supported by the observation that the two study groups displayed a similar histological pattern of tissue graft damage. In this regard, it is worth underlining that, in our cohort represented by recipients of grafts from young donors, the susceptibility to HLA-antibody mediated insult is only marginally influenced by organ ageing [Our study, carried out in a homogeneous patient population not including sensitized recipients, demonstrates that the time interval to AMR development and graft loss, evaluated from the firstand loss , 9. Thesand loss , 29 and and loss . In our and loss , 30–32. and loss . The sizand loss , we founantigens –15, 22. t phases , 30. Then ageing . dnDSA production in some patients and not in others, as well as the biological factors influencing their development at different times after transplant, in the presence of the same degree of HLA mismatching and the same immunosuppressive regimen, are not completely understood. In this study, younger recipient age appeared to favor an earlier dnDSA production, likely reflecting a propensity to display a stronger alloreactivity, that suggests a note of caution in immunosuppressive therapy minimization in the pediatric setting.At present, the reasons for dnDSAs at late phases of posttransplant follow-up should not differ from that applied in the early-onset dnDSA patient group.Based on our findings, management of patients found positive forThus, monitoring of HLA antibodies throughout the entire posttransplant course is recommended, despite high costs and organization difficulties, in order to identify patients at risk for AMR and graft loss.


Online recruitment is feasible, low-cost, and can provide high-quality epidemiological data. However, little is known about the feasibility of recruiting postpartum women online, or sample representativeness.The current study investigates the feasibility of recruiting a population of postpartum women online for health research and examines sample representativeness.Two samples of postpartum women were compared: those recruited online as participants in a brief survey of new mothers (n=1083) and those recruited face-to-face as part of a nationally representative study (n=579). Sociodemographic, general health, and mental health characteristics were compared between the two samples.Obtaining a sample of postpartum women online for health research was highly efficient and low-cost. The online sample over-represented those who were younger (aged 25-29 years), were in a de facto relationship, had higher levels of education, spoke only English at home, and were first-time mothers. Members of the online sample were significantly more likely to have poor self-rated health and poor mental health than the nationally representative sample. Health differences remained after adjusting for sociodemographic differences.Potential exists for feasible and low-cost e-epidemiological research with postpartum populations; however, researchers should consider the potential influence of sample nonrepresentativeness. Internet-based recruitment and data collection for epidemiological research has been shown to offer benefits in terms of feasibility and accessibility  ,2. ImporOne specific population-group in which Internet-based research and data collection may be highly enabling, but where little research is available, is postpartum women (first 12 months after birth). Previous research investigating postpartum parents has relied largely on recruitment via hospitals and child/maternal health clinics, requiring considerable time and financial investment. However, there may be significant emerging opportunities to recruit this population more efficiently online. Internet usage in this group is likely to be high, given postpartum women are often socially isolated at home, restricted in their mobility, and time-poor -10. StudThe current study investigates the feasibility of recruiting a large sample of postpartum women online for health research, and examines sample representativeness. Sociodemographic and health characteristics were compared between an online sample and another postpartum sample that was recruited face-to-face in the context of a nationally representative household survey.babycentre [Research: Mums Wanted! Researchers are looking for Australian women with a young baby to do a brief survey ”. No incentive was provided for participation. Mental health was not mentioned, in an effort to minimize health selection. Individuals who clicked on the advertisement were redirected to the online survey, which took approximately 15 minutes to complete. The Living with a Young Baby Survey (LYBS) was completed by 1083 respondents. The LYBS was approved by The Australian National University Human Research Ethics Committee.Participants were recruited during January and February 2015 for an anonymous online survey investigating women’s general postpartum health and psychological wellbeing. Online recruitment involved two processes: (1) advertisement on a popular Australian pregnancy/infant-focused website bycentre , and (2)Comparative data were drawn from waves (time-points) 11 and 13 of the Household Income Labour Dynamics in Australia Study (HILDA), as at these time-points the K-10 Psychological Distress Scale was included in the survey. HILDA is a longitudinal nationally representative household panel survey that has been conducted annually since 2001 . At baseSociodemographic measures included location (state and remoteness), language spoken at home, age, relationship status, number of children, and level of education completed. General self-rated health was measured by asking, “In general, how would you say your own health is?” Possible responses were, “excellent”, “very good”, “good”, “fair”, or “poor” [Psychological distress was measured using the K-10 Kessler Psychological Distress Scale [Low), 16-21 (Moderate), 22-29 (High), and 30-50 (Very High), consistent with previous research [r “poor” . Psycholss Scale . The K-1research ,20.Descriptive statistics for sociodemographic and health variables from both samples are presented. The data reported for the HILDA sample was weighted by the cross-sectional population weights included in waves 11 and 13. Chi-square tests of association identified characteristics that differed between the LYBS and HILDA samples. Univariate logistic regression provided estimates of the magnitude of differences in characteristics between the LYBS and HILDA samples (odds ratios). Multivariate logistic regression  tested whether differences in sociodemographic characteristics accounted for differences in general health and mental health between the samples. The weighted HILDA data provides the best possible comparison group available: postpartum women can be identified, self-rated health and the K-10 are reported (comparable measures to the LYBS), and the sample is representative of the Australian population.babycentre website took place over 9 days, and 264 surveys were completed. There was no cost associated with advertising on the babycentre website. Online recruitment from Facebook took place over 4 days, and 819 surveys were completed. Information provided by the Facebook Ads Manager shows that across the 4 days the advertisement was clicked on 2647 times, and the total audience reach was 38,765. Given that 819 surveys were completed, we estimate a conversion rate of click-to-completed-survey of 30.94% (819/2647). The total cost for advertising on Facebook was Aus $448.68 (the cost per click on the advertisement was Aus $0.17), and cost per survey completion was Aus $0.55.Online recruitment from the not completed high school (-5.5%). Mothers in the online LYBS had poorer general health and higher psychological distress than those in the HILDA study. Multivariate logistic regression , and the findings may be unique to the Australian context. While the monetary figures clearly demonstrate that online recruitment is low-cost, we did not specifically conduct cost-effective analyses. Researchers adopting Internet methods to investigate postpartum health should consider the implications of sample nonrepresentation in relation to their specific research aims .


A 37-year-old Caucasian woman presented with acute decrease in central vision in her right eye and was found to have subfoveal choroidal neovascularization (CNV) due to presumed ocular histoplasmosis syndrome (POHS). Her visual acuity improved from 20/70 to 20/20 at her 6-month follow-up, after 3 consecutive monthly intravitreal bevacizumab injections were initiated at her first visit. Although no CNV activity was seen on fluorescein angiography (FA) or spectral-domain optical coherence tomography (SD-OCT) at her 2-month, 4-month, and 6-month follow-up visits, persistent flow in the CNV lesion was detected on optical coherence tomography angiography (OCTA). OCTA shows persistent vascular flow as well as changes in vascular flow in CNV lesions associated with POHS, indicating the continued presence of patent vessels and changes in these CNV lesions, even when traditional imaging of the lesion with OCT and FA indicates stability of the lesion with no disease activity. Additional cases with longitudinal follow-up are needed to assess how OCTA should be incorporated into clinical practice. Presumed ocular histoplasmosis syndrome (POHS) can lead to central vision loss with development of choroidal neovascularization (CNV). Herein, we report a case of POHS related CNV that was successfully treated with intravitreal bevacizumab. The evolution of the CNV lesion on multimodal imaging, including optical coherence tomography angiography (OCTA), will be presented.A 37-year-old Caucasian woman, with no past medical or ocular history, presented with acute-onset and decreased central vision in the right eye. Her left eye was asymptomatic. Visual acuities of the right eye and left eye measured 20/70 and 20/20, respectively, with no improvement with pinhole. The anterior segment was normal with no intraocular inflammation in both eyes. Dilated fundus examination of both eyes showed peripapillary hyper- and hypopigmentation and punched-out chorioretinal lesions in the midperipheral retina consistent with a diagnosis of POHS Figures . AdditioAt her 1-month follow-up, the visual acuity of the right eye improved from 20/70 to 20/40, with resolution of subretinal hemorrhage and near resolution of CME. Intravitreal bevacizumab #2 was given.At her 2-month follow-up, the visual acuity of the right eye improved to 20/32, with no subretinal fluid (SRF) or CME on SD-OCT and resolution of leakage on FA . HoweverAt her 4-month follow-up, the visual acuity of the right eye improved to 20/25, with stable findings on SD-OCT and FA. Although there was persistent flow within the CNV lesion on OCTA, observation was opted given her excellent visual acuity.At her 6-month follow-up, her right eye visual acuity further improved to 20/20, with no CNV activity seen on SD-OCT or FA. The area of vascular flow on OCTA appeared to have enlarged slightly, but cautious observation was opted for again given her excellent visual acuity.Our patient's fundus examination was consistent with classic POHS. Although she did not have a history of spelunking or living in the Mississippi/Ohio river valleys, she endorsed exposure to farm animals. The CNV in her right eye responded well to intravitreal bevacizumab treatment. Her visual acuity improving from Snellen 20/70 to 20/20 over a span of 6 months, with only 3 monthly injections initiated at her presentation. Our experience was consistent with that of published case series, which showed intravitreal antivascular endothelial growth factors (VEGF) injections to be effective for POHS related CNV; on average, patients' visual acuity improved requiring relatively few treatments (range 2.6 to 7 injections per patient per year) –4 as comPreviously, Wang et al.  reportedThe serial multimodal images obtained for our patient also provided several interesting observations. First, although no CNV activity was seen on FA or SD-OCT at her follow-up visits, persistent flow of the CNV lesion was detected on OCTA. This suggests that OCTA is more sensitive than FA or SD-OCT in detecting the presence of persistent CNV related to POHS. This observation parallels the work published by de Oliveira Dias et al. , in whicTo the authors' knowledge, this is the first reported case of OCTA findings in CNV related to POHS. Our case suggests that OCTA could be useful in the management of POHS related CNV, similar to neovascular AMD, in that OCTA seems to be more sensitive than FA and SD-OCT in detecting subtle changes in CNV lesions. What remains unknown is whether the treatment based on subtle OCTA findings, in the absence of fluid on SD-OCT, ultimately translates to better outcomes. Additional cases of CNV related to POHS with longitudinal follow-up imaged with OCTA are needed to assess how OCTA should be incorporated into clinical management decisions.


Exploring clinically effective methods to reduce ischemia-reperfusion (IR) injury in humans is critical. Several drugs have shown protective effects, but studies using other interventions have been rare. Electroacupuncture (EA) has induced similar protection in several animal studies but no study has investigated how the effects could be translated and reproduced in humans. This study aimed to explore the potential effect and mechanisms of EA in IR-induced endothelial dysfunction in humans.This is a prospective, randomized, crossover, sham-controlled trial consisting of two protocols. Protocol 1 was a crossover study to investigate the effect of EA on IR-induced endothelial dysfunction. Twenty healthy volunteers were randomly assigned to EA or sham EA (sham). Flow mediated dilation (FMD) of the brachial artery (BA), nitroglycerin-mediated endothelial independent dilation, blood pressure before and after IR were measured. In protocol 2, seven volunteers were administered COX-2 inhibitor celecoxib  for five days. After consumption, volunteers underwent FMD before and after IR identical to protocol 1.In protocol 1, baseline BA diameter, Pre-IR BA diameter and FMD were similar between the two groups (p = NS). After IR, sham group showed significantly blunted FMD . However, EA protected this blunted FMD . In protocol 2, this protective effect was completely abolished by pre-treatment with celecoxib .EA may prevent IR-induced endothelial dysfunction via a COX-2 dependent mechanism. Previous studies have emphasized the importance of vascular endothelial cells, which are particularly susceptible to and actively participate in the pathophysiology of tissue injury induced by ischemia-reperfusion (IR). In the clinic, the chronic dysfunction of these endothelial cells leads to atherosclerosis and also acts as an important independent predictor of negative prognosis for coronary artery diseases . As it cIn pharmaceuticals, several molecules have been identified as mediators in maintaining endothelial function. Nitric oxide (NO) is considered one of the most significant as it is known to be involved in several of the protective mechanisms , includiIn complementary and alternative medicine, acupuncture is commonly used to alleviate pain but it has also been widely explored in the treatment of hypertension  and manyA preliminary study in patients with hypertension suggested that acupuncture improved FMD , but it st to August 23rd 2014, healthy nonsmoking volunteers (25 to 40 years of age) were recruited and those currently taking medication for hypertension, diabetes or other chronic diseases, suffering from hepatic, renal, thyroid, or cerebrovascular diseases, currently pregnant, or has body mass index greater than 25 kg/m2 were excluded. After trial commencement, we further excluded those taking dietary supplements or enrolled in another clinical trial. Twenty one people were assessed for eligibility and twenty people fulfilled the inclusion criteria. All of them completed two sessions of treatment for protocol 1. Among the participants, seven volunteered to further take part in the celebrex experimental group (protocol 2) (From May 1tocol 2) .th, 2014, No. 1403–01) and was developed in accordance with the Declaration of Helsinki. The study protocol was explained in detail to eligible participants, and written informed consent was obtained from all subjects. ₩ 100,000 (approximately $ 90) was given to each participant per session as compensation for their time. The study was registered at Clinical Trials.gov: NCT02255006 . There was a delay in registering this study due to language and administrative issues. The authors confirm that all ongoing and related trials for this intervention are registered.This study was a prospective, randomized, subject and assessor blind, crossover and placebo-controlled clinical trial conducted in the Division of Cardiology, Department of Internal Medicine, and the Department of Acupuncture and Moxibustion, Kyung Hee University Medical Centre, Seoul, Republic of Korea. Data were collected in the Division of Cardiology, Kyung Hee University Medical Centre. This study protocol was approved by the institutional review board (IRB) of Kyung Hee University Hospital  were enrolled in the trial. All subjects were asked to abstain from caffeine and drugs, including supplemental vitamins, for the duration of the study. Initially, endothelium-dependent FMD of the brachial artery (BA) and nitroglycerin-mediated endothelial independent dilation (NMD) were measured before and after IR  . EA was Participants were randomly assigned to either EA or sham EA using a computerized random allocation sequence with a permutable block design. A member who had no contact with any of the participants notified only the acupuncturist of the participant allocation right before treatment by phone. Assessors were concealed to all of the allocations, and the participants were naturally blinded because the point of real EA and sham EA were very close, the study was conducted in a semi-lit evaluation room limiting view of the treatment area, and most were acupuncture naïve. The acupuncturists only came into the study room to insert the needles and to apply EA while refraining from any conversation.Protocol 2 was performed in a non-randomized, non-blinded manner. After completing protocol 1, seven volunteers were further recruited to participate in protocol 2. They underwent a fifteen-day wash-out period. Volunteers were administered the selective COX-2 inhibitor, celecoxib  200 mg twice a day for five days. The last dose of celecoxib was administered on the morning of FMD measurement. Subjects underwent FMD measurement before and after IR, as described above. EA was performed for 20 minutes as in the previously described method.For active EA treatment, unilateral PC5, PC6, ST36, and ST37 were used . PC5 andDisposable, sterile needles  and a low-frequency electrical stimulator  were used. Each needle was inserted at a 90 degree angle to a depth of 1cm at PC5 and PC6 and to a depth of 2 cm at ST36 and ST37. Then, the practitioner rotated the needle several times and induced de-qi . Thereafter, needles were connected to the pole and electrical stimulation was applied with a 2 Hz continuous wave current. Intensity was increased until participants felt stimulation but not discomfort, and their muscles twitched slightly. EA was performed after ten minutes of inducing ischemia to the end of reperfusion, 20 minutes in total. Acupuncture treatment was conducted by Doctors of Korean Medicine educated by the College of Korean Medicine for six years and licensed by the Korean Ministry of Health and Welfare; with more than four years of clinical experience. All practitioners were trained to follow the study protocol.For sham EA treatment, four non-acupuncture points  were inserted to a depth of 0.5 cm at a 90 degree angle. Manual manipulation was not conducted, and de-qi sensations were not induced. Needles were connected to the pole and identical sounds were generated as in the active EA group, but electrical stimulation was not delivered to the sham EA group. After each session, any bleeding, bruises, or unintended effects due to EA were identified and documented. These EA and sham EA methods have been used successfully in previous clinical trials , 18.Subjects were asked to rest for ten minutes in the supine position prior to each study protocol. The FMD and NMD measurements were performed by two independent operators. Brachial artery images were obtained using a commercially available system  equipped with a 14-MHz linear array transducer. The ECG-gated, end-diastolic, longitudinal, B-mode images were digitally stored on the hard disk of the instrument for on-line and off-line analysis. For FMD measurement, the baseline BA diameter was averaged from six separate images taken at five-second intervals. Subsequently, a pneumatic cuff placed at the level of the lower arm was inflated to 200 mmHg for five minutes. After wrist-cuff deflation, the BA diameter was re-examined and averaged from six separate images taken at five-second intervals. FMD was calculated as the percent maximum increase in arterial diameter. After a ten-minute rest period to allow restoration of baseline conditions, NMD was again assessed by obtaining two-dimensional images before and three minutes after administration of 50 μg of GTN in order to characterize endothelial-independent vasodilation. The artery diameter was calculated from the trailing edge of the intima-blood interface to the leading edge semi-automatically using a modified version of Image J software  as well as custom-designed software. Baseline and post-IR (reactive hyperemia) blood flows were measured using pulsed-wave Doppler as an average velocity-time integral for the first five cardiac cycles after cuff deflation and were multiplied by heart rate and vessel cross-sectional area. The reproducibility of this method in our laboratory was previously reported several times ,20. A dit-test or Wilcoxon signed rank test. Between-group differences and the interaction of IR and group randomization were studied with a two-way analysis of variance. Post hoc comparisons were performed using the Bonferroni correction. A p-value less than 0.05 was considered statistically significant. Multivariate analysis (adjusted age and gender) was performed to determine whether acupuncture could protect endothelial dysfunction defined as FMD < 6%. All analyses were performed with SPSS software  by a statistician blinded to participant allocation. The CONSORT checklist, original, and translated version of the trial protocol are available . EA and sham EA had no effect on baseline BA diameter before and after IR , p = NS.In the sham EA group, BA diameter and blood flow values were not different from baseline values before and after IR (p = NS). However, post-IR FMD was significantly decreased compared with the pre-IR level  in the EA group. In contrast, EA restored the FMD reduction that was observed in the sham EA group after IR injury  . IR did th and 75th percentile levels, respectively; and the horizontal lines within the boxes indicate the median levels. FMD: flow-mediated dilatation, IR: ischemia-reperfusion.FMD was significantly blunted after IR, demonstrating that celecoxib prevented electroacupuncture-induced endothelial protection against IR. Boxes show interquartile ranges; the lower and upper boundaries of the boxes indicate the 25To the best of our knowledge, this study is the first to demonstrate the protective effects of EA against endothelial dysfunction and to verify the mechanistic significance of COX-2 in a human forearm IR model. We observed a significant blunting of FMD after IR in the sham control group, whereas EA prevented it. We observed a significant blunting of FMD after IR in the sham control group, whereas EA prevented it. Since EA treatment was performed on the contralateral side of IR injury, it also revealed that a big part of its mechanism relied on central pathways.Previous animal studies have suggested that the mechanisms leading to such preconditioning—mimetic properties of EA is likely multifactorial, with modulation of the autonomic nervous system (ANS), endothelial nitric oxide synthase (eNOS), and inducible NOS all coming to play ,21,22. EIn non-acupuncture related clinical studies, the proposed endothelial protection mechanism has also emphasized the role of NO in the physiology of both ischemic and pharmacological preconditioning via a direct and cGMP-mediated opening of KATP channels ,27. In eTo further clarify the proposed mechanisms, specific inhibitors are utilized before inducing ischemia. Past studies have revealed that intravenous PG I2 given before ischemia reduced infarct size and administration of COX-2 inhibitors before infarction hindered the infarct size-limiting effects of late ischemic preconditioning . AdminisAdding to the current field of knowledge, our study newly demonstrated that the endothelial protection afforded by EA disappears after administration of celecoxib, a COX-2 inhibitor, and suggests that COX-2 may be involved in the signaling pathway leading to EA-mediated protection in a human forearm IR model. In the area of pain research, where EA has been most actively explored, EA also induced its anti-nociceptive effects by modulation of COX-2 expression in the nervous system related to pain control. In a recent study, EA alleviated mechanical and thermal hypersensitivity after lumbar L5 spinal nerve ligation by inhibiting COX-2 expression in the spinal dorsal horn and this inhibition was as effective as the COX-2 inhibitor celecoxib . Along tOur study had a small sample size and was conducted at a single center. Endothelial dysfunction induced by IR injury was limited to the conduit circulation and our assessment did not include the coronary circulation or distal microcirculation, which are also of importance in clinical IR injury. The data were obtained from a sample of healthy volunteers who likely did not have impaired endothelium-dependent responses. Also, among the volunteers in protocol 2, EA non-responders may have been included. Future studies are necessary to investigate the potential clinical implications in greater detail.This pilot trial is the first human evidence verifying the effects of EA on endothelial pharmacological preconditioning and suggests that it may be induced via a COX-2-dependent mechanism.Kyung Hee University Medical Centre Ethics Board.S1 FigPC5 & PC6, ST36 & ST37, and sham electroacupuncture points. PC: pericardium, ST: stomach.(TIF)Click here for additional data file.S2 FigDiagram showing how electroacupuncure was conducted and how flow-mediated dilatation was measured on subjects. FMD: flow-mediated dilatation, IR: ischemia-reperfusion.(TIF)Click here for additional data file.S1 FileConsolidated Standards of Reporting Trials checklist.(DOC)Click here for additional data file.S2 FileOriginal Korean version of trial protocol.(DOC)Click here for additional data file.S3 FileTranslated version of original trial protocol.(DOCX)Click here for additional data file.S4 FileIndividual data points of the study.(XLSX)Click here for additional data file.


We screened serum samples referred to the national reference laboratory in Guatemala that were positive for chikungunya or dengue viruses in June 2015. Co-infection with both viruses was detected by reverse transcription PCR in 46 (32%) of 144 samples. Specimens should be tested for both arboviruses to detect co-infections. Chikungunya virus (CHIKV) and dengue virus (DENV) are arboviruses currently circulating in Southeast Asia, Central and West Africa, the Pacific islands, and the Americas, and their transmission can occur simultaneously  diagnostic algorithms indicate that DENV-positive samples do not need to be tested for CHIKV or other viruses and vice versa  for either virus. Clinical data for patients were obtained from surveillance databases to compare patients with monoinfections or coinfections with DENV or CHIKV. All samples were obtained from patients During June 2015, a total of 523 samples were tested for CHIKV at the NHL; 328 (63%) were positive for CHIKV RNA by RT-PCR. A total of 514 samples were also tested by RT-PCR for DENV; 75 (9%) were positive . SeventySamples selected were consecutive positive samples from the beginning of the month; there were no additional exclusion or inclusion criteria. We included as many samples as possible within the time available. RNA was extracted by using a Viral RNA Mini Kit  and tested by using the US Centers for Disease Control  multiplex DENV RT-PCR and the CDC CHIKV 6856F/6981c/6919-FAM RT-PCR (Twenty-five (33.8%) of 74 DENV-positive samples were positive for CHIKV, and 21 (30%) of 70 CHIKV-positive samples were positive for DENV RNA. Co-infection with CHIKV was detected in 4/9 samples containing DENV-1, 40/76 samples containing DENV-2, 0/3 samples containing DENV-3 and 2/7 samples containing DENV-4.Clinical characteristics of patients with CHIKV and DENV monoinfections and co-infections were similar . PatientOur findings must be viewed with caution because of the limitations of this study. The study was not powered to analyze clinical associations with a high robustness because of the small number (n = 46) of co-infected samples tested. Also, because samples were selected at a reference laboratory and only symptomatic cases were tested, the prevalence of co-infection might be different in a less selected population.The PAHO algorithm recommends screening clinical specimens by using consecutive assays and ending screening once a pathogen is identified. CDC in turn recommends simultaneously conducting RT-PCRs for DENV, CHIKV, and Zika virus. Emergence of Zika virus in Latin America has further complicated diagnosis of arboviral infections, and simultaneous co-infections with all 3 arboviruses have been reported  of 74 DENV-positive samples were positive for CHIKV, and 21 (30%) of 70 CHIKV-positive samples were positive for DENV. Because Guatemala reported 49,043 cases of DENV and CHIKV infections in 2015, it is likely that the algorithm missed a large number of co-infections. Patients with single and dual infections had similar clinical manifestations in this limited study.The frequency of arbovirus coinfections in Guatemala is high. Simultaneous screening for DENV, CHIKV, and Zika virus in disease-endemic areas would improve the quality of arboviral surveillance and potentially aid in clinical management of the disease.


Nivolumab, an anti-programmed cell death-1 monoclonal antibody, has improved the survival of patients with malignant melanoma. Despite its efficacy, nivolumab inconsistently induces thyroid dysfunction as an immune-related adverse event (irAE). This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEs.After excluding 10 patients, data of 24 patients with malignant melanoma  were retrospectively analyzed.P < 0.01). There were no significant differences between patients with and without thyroid irAEs regarding age, sex, tumor stage, response to nivolumab therapy, baseline thyroid function, antithyroid peroxidase antibody (anti-TPO Ab) and antithyroglobulin antibody (anti-Tg Ab).Thyroid irAEs were observed in seven patients (29%). Three patients had hypothyroidism after preceding transient thyrotoxicosis, and the other four patients had hypothyroidism without thyrotoxicosis. Levothyroxine-Na replacement was required in three patients. Antithyroid antibody (ATA) titer was elevated in one of four assessable patients. The average (±SD) time to onset of thyroid irAE was 33.6 (±21.9) weeks. The administration period of nivolumab was longer in patients with thyroid irAEs than in those without thyroid irAEs (Thyroid dysfunction was a common irAE of nivolumab in malignant melanoma. Neither anti-TPO Ab nor anti-Tg Ab was associated with the risk for nivolumab-induced thyroid dysfunction. A conventional ATA-independent mechanism might be involved in thyroid irAEs. Further studies are required to clarify the mechanism and identify the predictive factors of thyroid irAEs. Nivolumab, an anti-programmed cell death-1 (PD-1) antibody, has emerged as a breakthrough medication for several advanced malignancies ; howeverAlthough thyroid dysfunction with thyroiditis is the most frequently reported nivolumab-induced endocrinopathy , 2, the .Here, we report the clinical characteristics and parameters of nivolumab-induced thyroid dysfunction in patients with melanoma. Thirty-four patients who were treated with nivolumab at our hospital were examined. This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEsWe should take into consideration thyroid irAEs for successful treatment with this beneficial medication because we observed the occurrence of nivolumab-induced thyroid dysfunction more frequently in our study than in previous reports , 11. FurThe study retrospectively recruited all of 34 patients with advanced malignant melanoma who were treated with nivolumab  at Kyusyu University Hospital from September 1, 2014 to September 30, 2016 . None of the patients had a history of any disease that required immunomodulating agents or pretreatment with other checkpoint inhibitors such as ipilimumab, which is an anti-cytotoxic T-lymphocyte associated protein-4 antibody.The baseline clinical features of the patients are summarized in Table t test . Significance was defined as a P-value < 0.05.All statistical analyses were performed using JMP® 13 . Patient, tumor, and treatment variables were compared using the Fisher’s exact , Mann–Whitney–Wilcoxon , and the unpaired two-sample P < 0.01). The administration durations in patients without thyroid irAEs were all shorter than 33.6 weeks, which was the average administration period in patients with thyroid irAEs.There were no significant differences between patients with and without thyroid irAEs regarding sex, age, tumor stage, response to nivolumab therapy, and baseline thyroid function Table . The aveThyroid irAEs were observed in seven of 24 patients (29%), with four having grade 1 irAEs and three having grade 2 irAEs, when we evaluated them every 3 weeks  titers were assessed in 17 patients  of patients developed nivolumab-induced thyroid irAEs. Second, ATA titers would possibly not be useful for predicting the risk for nivolumab-induced thyroid dysfunction. Third, thyroid-specific autoimmunity induced by nivolumab might be independent of its antitumor efficacy.A higher prevalence of thyroid dysfunction in patients with advanced melanoma treated with nivolumab was observed in this study than in previous reports. Recent pooled analysis including two phase III trials of nivolumab  reportedAnti-TPO Ab and anti-Tg Ab titers would possibly not be useful for predicting the risk for nivolumab-induced thyroid dysfunction. In our study, only 25% 1/4) of patients with thyroid irAEs had elevated anti-Tg Ab titers, with none having elevated anti-TPO Ab titers (Table /4 of patThyroid-specific autoimmunity induced by nivolumab might be independent of the antitumor efficacy of nivolumab. Some studies suggest that the occurrence of irAEs in patients treated with immune checkpoint inhibitors may be correlated with an amelioration of malignancies , 21, 22.FOXP3 play a role in genetic susceptibility to autoimmune thyroid diseases [FOXP3 was reported to have an association with the severity of Hashimoto’s disease [We proposed that autoimmune thyroid dysfunction without both anti-TPO and anti-Tg Ab was induced by nivolumab. Several environmental and genetic factors have been reported as triggers of autoimmune thyroid diseases . The gendiseases , and the disease . PD-1 is disease . NivolumPD-1 may be a key regulator for preventing a subtype of autoimmune thyroiditis, which was found in the Japanese population. The prevalence of Hashimoto’s thyroiditis correlated with the Th1 response and cellular immunity was reported to be higher in Caucasian than in Japanese, while the prevalence of Graves’ disease correlated with the Th2 response was reported higher in Japanese than in Caucasians –31. As tThis study has some limitations. First, the number of patients treated with nivolumab was small; thus, studies with larger number of patients will be necessary to clarify the features of nivolumab-induced thyroid irAEs. Second, we did not evaluate ATA in all patients before the onset of irAE, and the TRAb titer was not examined in patients with thyroid irAEs. Third, treatment duration, especially that in non-thyroid irAE group, was short because the occurrence of irAEs can be delayed . FinallyWhen advanced melanoma patients were treated with nivolumab, 29% of them developed thyroid dysfunction. Anti-TPO and anti-Tg Ab titers were not useful for predicting the risk for nivolumab-induced thyroid dysfunction. Therefore, constant monitoring and awareness of thyroid irAEs are recommended, particularly in patients undergoing long-term nivolumab therapy. Nivolumab may induce thyroid irAEs by evoking non-conventional thyroid-specific autoimmunity. Further studies are required to clarify the characteristics and predictive factors of thyroid irAEs.