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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIREvariants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIREvariant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendric cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109–base pair frame-shifting pseudoexon. We also found low-level AIREexpression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE–expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIREtranscript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIREvariant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

Standard 2-tier testing (STTT), incorporating a screening enzyme immunoassay (EIA) or an immunofluorescence assay (IFA) that reflexes to IgM and IgG immunoblots, has been the primary diagnostic test for Lyme disease since 1995. In 2019, the Food and Drug Administration approved a modified 2-tier test strategy using 2 EIAs: offering a faster, less expensive, and more sensitive assay compared with STTT. New technologies examine early immune responses to Borrelia burgdorferi have the potential to diagnose Lyme disease in the first weeks of infection when existing serologic testing is not recommended due to low sensitivity.

AbstractBackground:Monoclonal antibody (mAb) treatment for coronavirus disease 2019 (COVID-19) has been underutilized due to logistical challenges, lack of access, and variable treatment awareness among patients and health-care professionals. The use of telehealth during the pandemic provides an opportunity to increase access to COVID-19 care.Methods:This is a single-center descriptive study of telehealth-based patient self-referral for mAb therapy between March 1, 2021, and October 31, 2021, at Baltimore Convention Center Field Hospital (BCCFH).Results:Among the 1001 self-referral patients, the mean age was 47, and most were female (57%). White (66%), and had a primary care provider (PCP) (62%). During the study period, self-referrals increased from 14/mo in March to 427 in October resulting in a 30-fold increase. Approximately 57% of self-referred patients received a telehealth visit, and of those 82% of patients received mAb infusion therapy. The median time from self-referral to onsite infusion was 2 d (1-3 IQR).Discussion:Our study shows the integration of telehealth with a self-referral process improved access to mAb infusion. A high proportion of self-referrals were appropriate and led to timely treatment. This approach helped those without traditional avenues for care and avoided potential delay for patients seeking referral from their PCPs.

Lyme disease is the most common tick-borne disease in North America. Though human infection is mostly transmitted in a limited geography, the range has expanded in recent years. One notable area of recent expansion is in the mountainous region of southwestern Virginia. The ecological factors that facilitate or constrain the range of human Lyme disease in this region remain uncertain. To evaluate this further, we obtained ecological data, including remotely sensed data on forest structure and vegetation, weather data, and elevation. These data were aggregated within the census block groups of a 9,153 km2area around the cities of Blacksburg and Roanoke, VA, an area with heterogeneous Lyme disease transmission. In this geographic area, 755 individuals were reported to have Lyme disease in the 10 yr from 2006 to 2015, and these cases were aggregated by block group. A zero-inflated negative binomial model was used to evaluate which environmental variables influenced the abundance of Lyme disease cases. Higher elevation and higher vegetation density had the greatest effect size on the abundance of Lyme disease. Measures of forest edge, forest integrity, temperature, and humidity were not associated with Lyme disease cases. Future southward expansion of Lyme disease into the southeastern states may be most likely in ecologically similar mountainous areas.

Climate change is a worsening global crisis that will continue negatively impacting population health and well-being unless adaptation and mitigation interventions are rapidly implemented. Climate change-related cardiovascular disease is mediated by air pollution, increased ambient temperatures, vector-borne disease and mental health disorders. Climate change-related cardiovascular disease can be modulated by climate change adaptation; however, this process could result in significant health inequity because persons and populations of lower socioeconomic status have fewer adaptation options. Clear scientific evidence for climate change and its impact on human health have not yet resulted in the national and international impetus and policies necessary to slow climate change. As respected members of society who regularly communicate scientific evidence to patients, clinicians are well-positioned to advocate on the importance of addressing climate change. This narrative review summarizes the links between climate change and cardiovascular health, proposes actionable items clinicians and other healthcare providers can execute both in their personal life and as an advocate of climate policies, and encourages communication of the health impacts of climate change when counseling patients. Our aim is to inspire the reader to invest more time in communicating the most crucial public health issue of the 21st century to their patients.

ABSTRACTThe primary objective of this study was to determine whether patients with putative late-onset Lyme arthritis based upon synovial fluid Borrelia burgdorferiIgM and IgG immunoblot testing offered by commercial laboratories satisfied conventional criteria for the diagnosis of Lyme arthritis. Secondary objectives included assessing the prior duration and responsiveness of associated antibiotic therapy. We conducted a retrospective analysis of 11 patients referred to an academic medical center infectious disease clinic during the years 2007 to 2009 with a diagnosis of Lyme disease based upon previously obtained synovial fluid B. burgdorferiimmunoblot testing. Ten of the 11 (91%) patients with a diagnosis of late-onset Lyme arthritis based upon interpretation of synovial fluid B. burgdorferiimmunoblot testing were seronegative and did not satisfy published criteria for the diagnosis of late-onset Lyme arthritis. None of the 10 patients had a clinical response to previously received antibiotics despite an average course of 72 days. Diagnosis of Lyme arthritis should not be based on synovial fluid B. burgdorferiimmunoblot testing. This unvalidated test does not appear useful for the diagnosis of Lyme disease, and this study reinforces the longstanding recommendation to use B. burgdorferiimmunoblot testing only on serum samples and not other body fluids. Erroneous interpretations of “positive” synovial fluid immunoblots may lead to inappropriate antibiotic courses and delays in diagnosis of other joint diseases.

ABSTRACTLyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferiis definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.

ABSTRACTAccurate diagnosis of pneumococcal pneumonia in the acute-care setting remains a challenge due to the inadequate sensitivity of conventional diagnostic tests. Sputum cultures, which are likely to have the highest diagnostic yields of all specimen types, have been considered unreliable, due to their inability to differentiate colonization from infection. Our objective was to evaluate the potential clinical utility of a rapid quantitative real-time PCR assay using sputum samples for Streptococcus pneumoniaein adult patients with community-acquired pneumonia (CAP). A prospective clinical observational study of consecutively enrolled emergency department patients with CAP was performed; only those patients with excess good-quality sputum samples were included for evaluation. Sputum samples were tested for the presence of S. pneumoniaeby using a quantitative PCR that targets the pneumolysin gene. PCR findings were compared with those of a composite reference standard comprising Gram staining of sputum samples and sputum/blood cultures. The area under the curve (AUC) and a log-transformed threshold, which provides the maximal sensitivity and specificity, were calculated. Of 487 subjects enrolled, 129 were evaluable. Receiver operating characteristic curve analysis demonstrated an AUC of 0.87. Sensitivity and specificity were 90.0 percent and 80.0 percent, respectively; positive and negative predictive values were 58.7 percent and 96.2 percent, respectively. We have demonstrated that a quantitative rapid pneumolysin PCR assay has favorable accuracy for diagnosis of pneumococcal pneumonia in adult patients with CAP; this assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute-care setting, where rapid pathogen identification may assist in selection of the most appropriate antibiotics.

ABSTRACTB. burgdorferiinvasiveness correlates with ospCgenotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites, B. burgdorferi ospCheterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patient's home were isolated. 3' ospCsequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same ospCwere seen in only one locality, and five of six sites contained two or more B. burgdorferi ospCclones. Four invasive patient isolates and six small mammal isolates clustered with “noninvasive” reference ospCgenotypes. A high degree of ospCdiversity exists among B. burgdorferiisolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive ospCtypes contradicts the ospCinvasiveness hypothesis. Alternative genetic markers for B. burgdorferidisseminated disease should be investigated.

B. burgdorferi invasiveness correlates with ospC genotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites, B. burgdorferi ospC heterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patient's home were isolated. 3' ospC sequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same ospC were seen in only one locality, and five of six sites contained two or more B. burgdorferi ospC clones. Four invasive patient isolates and six small mammal isolates clustered with "noninvasive" reference ospC genotypes. A high degree of ospC diversity exists among B. burgdorferi isolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive ospC types contradicts the ospC invasiveness hypothesis. Alternative genetic markers for B. burgdorferi disseminated disease should be investigated.

ABSTRACTTo understand the molecular determinants of measles virus (MV) virulence, we have used the SCID-hu thymus/liver xenograft model (SCID-hu thy/liv) in which in vivo MV virulence phenotypes are faithfully duplicated. Stromal epithelial and monocytic cells are infected by MV in thymus implants, and virulent strains induce massive thymocyte apoptosis, although thymocytes are not infected. To determine whether passage of an avirulent vaccine strain in human tissue increases virulence, we studied a virus isolated from thymic tissue 90 days after infection with the vaccine strain Moraten (pMor-1) and a virus isolated from an immunodeficient child with progressive vaccine-induced disease (Hu2). These viruses were compared to a minimally passaged wild-type Edmonston strain (Ed-wt) and the vaccine strain Moraten. pMor-1, Hu2, and Ed-wt displayed virulent phenotypes in thymic implants, with high levels of virus being detected by 3 days after infection (105.2, 102.8, and 103.4, respectively) and maximal levels being detected between 7 and 14 days after infection. In contrast, Moraten required over 14 days to grow to detectable levels. pMor-1 produced the highest levels of virus throughout infection, suggesting thymic adaptation of this strain. Similar to other virulent strains, Ed-wt, Hu2, and pMor-1 caused a decrease in the number of viable thymocytes as assessed by trypan blue exclusion and fluorescence-activated cell sorter analysis. Thymic architecture was also disrupted by these strains. Sequence analysis of the hemagglutinin (H) and matrix (M) genes showed no common changes in Hu2 and pMor-1. M sequences were identical in pMor-1 and Mor and varied in H at amino acid 469 (threonine to alanine), a position near the base of propeller 4 in the propeller blade/stem model of H structure. Further study will provide insights into the determinants of virulence.

To identify associated clinical parameters, concurrent respiratory tract infections, and the association between macrolide-based therapy and mortality in patients with community-acquired pneumonia ascribed to atypical.

ABSTRACTAn understanding of the determinants of measles virus (MV) virulence has been hampered by the lack of an experimental model of infection. We have previously demonstrated that virulence phenotypes in human infections are faithfully reproduced by infection of human thymus/liver (thy/liv) implants engrafted into SCID mice, where the virus grows primarily in stromal cells but induces thymocyte apoptosis (P. G. Auwaerter et al., J. Virol. 70:3734–3740, 1996). To begin to elucidate the roles of the C protein, V protein, and the 5' untranslated region of the F gene (F 5'UTR) in MV infection in vivo, the replication of strains bearing mutations of these genes was compared to that of the parent sequence-tagged Edmonston strain (EdTag). Growth curves show that mutants fall into two phenotypic classes. One class of mutants demonstrated kinetics of growth similar to that of EdTag, with decreased peak titers. The second class of mutants manifested peak titers similar to that of EdTag but had different replication kinetics. Abrogation of V expression led to delayed and markedly prolonged replication. Additionally, thymocyte survival was prolonged and implant architecture was preserved throughout the course of infection. In contrast, massive bystander thymocyte death occurred after infection with EdTag and all other mutants. A mutant which overexpressed V in Vero cells (V+) had the opposite phenotype of the A mutant not expressing V (V-). V+ grew more rapidly than EdTag with 100-fold-greater levels of virus production 3 days after infection. These results suggest that C, V, and the F 5'UTR are accessory factors required for efficient virus replication in vivo. In addition, thymocyte survival after V- infection suggests this protein may play multiple roles in pathogenesis of MV infection of thymus. Since these recombinant mutant viruses grew identically to the parent virus in Vero cells, the data show that thy/liv implants are an excellent model for investigating the determinants of MV virulence.

Measles produces immune suppression which contributes to an increased susceptibility to other infections. Recently, high titered measles vaccines have been linked to increased long-term mortality among some female recipients. Because the mechanisms by which wild-type or attenuated live-vaccine strains of measles virus alter subsequent immune responses are not fully understood, this prompted an examination of the changes within the peripheral blood T cell receptor Vβrepertoire following measles immunization. Twenty-four 6- and 9-month-old infants were studied at 2 weeks and 3 months following immunization by semiquantitative reverse transcription-polymerase chain reaction. There was a significant increase in Vβ2 expression (P< 0.05), and a decrease in the Vβ4 subset (P< 0.03) 2 weeks following vaccination with subsequent return to baselines at 3 months in vaccine recipients who seroconverted. These data suggest that measles virus may affect immune responses in part by altering the T cell receptor repertoire.