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Introduction Type 1 diabetes (T1D) begins years before clinical presentation with hyperglycemia (1, 2). Following the initial breech of immune tolerance, the autoimmune response against β cells is thought to [...], Role of the funding source: Funding from the NIH was used for support of the participating clinical centers and the coordinating center. The funding source did not participate in the collection or the analysis of the data. BACKGROUND. The β cell killing that characterizes type 1 diabetes (T1D) is thought to begin years before patients present clinically with metabolic decompensation; however, this primary pathologic process of the disease has not been measured. METHODS. Here, we measured β cell death with an assay that detects p cell-derived unmethylated insulin (INS) DNA. Using this assay, we performed an observational study of 50 participants from 2 cohorts at risk for developing T1D from the TrialNet Pathway to Prevention study and of 4 subjects who received islet autotransplants. RESULTS. In at-risk subjects, those who progressed to T1D had average levels of unmethylated INS DNA that were elevated modestly compared with those of healthy control subjects. In at-risk individuals that progressed to T1D, the observed increases in unmethylated INS DNA were associated with decreases in insulin secretion, indicating that the changes in unmethylated INS DNA are indicative of β cell killing. Subjects at high risk for T1D had levels of unmethylated INS DNA that were higher than those of healthy controls and higher than the levels of unmethylated INS DNA in the at- risk progressor and at-risk nonprogressor groups followed for 4 years. Evaluation of insulin secretory kinetics also distinguished high-risk subjects who progressed to overt disease from those who did not. CONCLUSION. We conclude that a blood test that measures unmethylated INS DNA serves as a marker of active p cell killing as the result of T1D-associated autoimmunity. Together, the data support the concept that p cell killing occurs sporadically during the years prior to diagnosis of T1D and is more intense in the peridiagnosis period. TRIAL REGISTRATION. Clinicaltrials.gov NCT00097292. FUNDING. Funding was from the NIH, the Juvenile Diabetes Research Foundation, and the American Diabetes Association.

Relapsing fever, an arthropod-borne infection caused by several Borrelia spp. spirochetes, is transmitted by ticks and lice (1,2). In 1995, Fukunaga et al. (3) discovered a novel relapsing fever spirochete [...]

Human babesiosis is an emerging tick-borne disease in the United States, caused primarily by the intraerythrocytic protozoan Babesia microti and by other Babesia species (1,2). B. microti also may be [...]

Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted. [ABSTRACT FROM AUTHOR]

Babesia microti, the primary cause of human babesiosis in the United States, is transmitted by Ixodes scapularis ticks; transmission may also occur through blood transfusion and transplacentally. Most infected people experience a viral-like illness that resolves without complication, but those who are immunocompromised may develop a serious and prolonged illness that is sometimes fatal. The geographic expansion and increasing incidence of human babesiosis in the northeastern and midwestern United States highlight the need for high-throughput sensitive and specific assays to detect parasites in both ticks and humans with the goals of improving epidemiological surveillance, diagnosis of acute infections, and screening of the blood supply. Accordingly, we developed a B. microti-specific quantitative PCR (qPCR) assay (named BabMq18) designed to detect B. microti DNA in tick and human blood samples using a primer and probe combination that targets the 18S rRNA gene of B. microti. This qPCR assay was compared with two nonquantitative B. microti PCR assays by testing tick samples and was found to exhibit higher sensitivity for detection of B. microti DNA. The BabMq18 assay has a detection threshold of 10 copies per reaction and does not amplify DNA in I. scapularis ticks infected with Babesia odocoilei, Borrelia burgdorferi, Borrelia miyamotoi, or Anaplasma phagocytophilum. This highly sensitive and specific qPCR assay can be used for detection of B. microti DNA in both tick and human samples. Finally, we report the prevalence of B. microti infection in field-collected I. scapularis nymphs from three locations in southern New England that present disparate incidences of human babesiosis. [ABSTRACT FROM AUTHOR]

Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito indigenous to Asia, now an invasive species worldwide, is an important vector for several arboviruses. Genetic analysis using the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5) gene was carried out in populations from Cameroon (n = 50), Hawaii (n = 38), Italy (n = 20), the continental United States, Brazil, and its native range. Data for Brazil, the continental United States, and the native range was obtained from Birungi and Munstermann (2002). Direct sequencing was used to identity unique haplotypes. The limited phylogeographic partitioning of haplotypes with low levels of sequence divergence in both Cameroon and Hawaii was consistent with the population structure of Ae. albopictus in the United States and Brazil. Four new haplotypes were identified from the samples from Cameroon and Hawaii, adding to previously described haplotypes. Hawaii shared a haplotype with Cameroon that was unique to these two regions. Hawaii also had higher overall haplotype diversity than seen in previous continental United States, Brazil, or native range populations. Hawaiian, Cameroon, and Italian populations did not share haplotypes with Brazil, which validates the earlier mitochondrial DNA studies indicating a separate introduction of this species into Brazil. [ABSTRACT FROM AUTHOR]

Islet autoantibody (iAb)-positive individuals have a high risk of progression to type 1 diabetes (T1D), although the rate of progression is highly variable and factors involved in the rate of progression are largely unknown. The ratio of unmethylated/methylated insulin DNA levels (unmethylated INS ratio) has been shown to be higher in participants at high risk of T1D compared to healthy controls. We aimed to evaluate whether an unmethylated INS ratio may be a useful biomarker of beta cell death and rate of progression to T1D. In TrialNet participants who were followed in the Pathway to Prevention Study and progressed to diabetes (n = 57, median age of onset 15.3 years), we measured unmethylated INS ratio and autoantibodies by electrochemiluminescence (ECL) assays (ECL-IAA, ECL-GADA, and ECL-IA2) and radioimmunoassays (RIA) (mIAA, GADA, IA2A, and ZnT8A) longitudinally for 24 months prior to diagnosis. Linear models were used to test the association between unmethylated INS ratio and the age at T1D diagnosis and unmethylated INS ratio and iAb over time. Close to diabetes onset, the unmethylated INS ratio was associated with mIAA (p = 0.003), ECL-IAA (p = 0.002), and IA2A (p = 0.01) levels, but not with GADA, ECL-GADA, ECL-IA2, or ZnT8A levels. No significant associations were found at baseline (24 months prior to T1D diagnosis). Only mIAA levels were significantly associated with an unmethylated INS ratio over time, with a 0.24 change in the ratio for each 0.1 change in mIAA z-score (p = 0.02). Adjusting for a baseline unmethylated INS ratio, an increased rate of change in unmethylated INS ratio from baseline to diabetes onset was associated with a five-year decrease in age at T1D diagnosis (p = 0.04). [ABSTRACT FROM AUTHOR]

Context: There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death., Objective: To assess the performance of three unmethylated insulin DNA assays., Design and Participants: Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay., Results: All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays., Conclusions: The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stresses encountered during human islet isolation lead to unavoidable β-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, β-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less β-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.