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Purpose The clinical utility of echocardiography in the setting of a positive blood culture in paediatric patients presenting with osteomyelitis (OM) and/or septic arthritis (SA). Methods Retrospective review between 2013 and 2019: Patients < 18 years with OM, SA or combined infection (OM+SA) were included. Patients were excluded for immunodeficiency, loss of follow-up or penetrating infection. Charts with positive blood cultures were reviewed for echocardiography on that admission. Demographic variables were compared utilizing the Student's t-test and Fisher's exact test. A multivariable linear regression model was constructed to examine the association between echocardiography and length of stay, controlling for age, sex, fever, white blood cell (WBC) on admission, antibiotic administration and surgery performed. Results Of 157 patients with OM, SA or combined infection, 44 had a positive blood culture. In all, 26 had an echocardiogram, and none showed endocarditis. Echocardiography was independently associated with a 6.2-day length of stay increase. WBC count and surgical intervention demonstrated a trend toward significance in length of stay, with each WBC unit increase associated with a 0.53-day increase. Surgical intervention was associated with an average 6.3-day length of stay decrease. Conclusion No patient had a positive echocardiogram, and no changes in management were initiated. However, an echocardiogram increased stay by 6.2 days. In addition to costs associated with increased stay, patients were billed between $1460 and $1700 per echocardiogram. The utility of echocardiograms in the setting of bacteremia associated with musculoskeletal infections in the paediatric population should be re-examined, and guidelines should be updated to reflect the cost-benefit analysis. Level of Evidence III

Background Genomic RNA of severe acute respiratory syndrome-associated coronavirus type 2 (SARS-CoV-2) has been detected in the breast milk of lactating women, but its pathological significance has remained uncertain due to the small size of prior studies. Methods Breast milk from 110 lactating women was analyzed by reverse transcription-polymerase chain reaction (285 samples) and viral culture (160 samples). Those containing SARS-CoV-2 viral RNA (vRNA) were examined for the presence of subgenomic RNA (sgRNA), a putative marker of infectivity. Results Sixty-five women had a positive SARS-CoV-2 diagnostic test, 9 had symptoms but negative diagnostic tests, and 36 symptomatic women were not tested. SARS-CoV-2 vRNA was detected in the milk of 7 (6%) women with either a confirmed infection or symptomatic illness, including 6 of 65 (9%) women with a positive SARS-CoV-2 diagnostic test. Infectious virus was not detected in any culture and none had detectable sgRNA. In control experiments, infectious SARS-CoV-2 could be cultured after addition to breastmilk despite several freeze–thaw cycles, as it occurs in the storage and usage of human milk. Conclusions SARS-CoV-2 RNA can be found infrequently in the breastmilk after recent infection, but we found no evidence that breastmilk contains an infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants. Impact This article goes beyond prior small studies to provide evidence that infectious SARS-CoV-2 is not present in the milk of lactating women with recent infection, even when SARS-CoV-2 RNA is detected. Recent SARS-CoV-2 infection or detection of its RNA in human milk is not a contraindication to breastfeeding.

Pharmacological interactions limit treatment options for children living with human immunodeficiency virus (HIV) and tuberculosis (TB). We found that 12 mg/kg twice daily raltegravir chewable tablets (administered after crushing) safely achieved pharmacokinetic targets in children living with HIV aged 4 weeks to Clinical Trials Registration NCT01751568.

CD8

Objectives:Drug–drug interactions limit current antiretroviral treatment options for HIV-infected children with tuberculosis (TB). Rifampicin (RIF) induces UDP-glucuronosyltransferase activity, accelerating the clearance of raltegravir (RAL). We sought to establish an optimal and well tolerated dose

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naı̈ve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naı̈ve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naı̈ve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naı̈ve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

BackgroundSARS-CoV-2 infections of infants and toddlers are usually mild but can result in life-threatening disease. SARS-CoV-2 RNA been detected in the breast milk of lactating women, but the potential role of breastfeeding in transmission to infants has remained uncertain.MethodsBreast milk specimens were examined for the presence of the virus by RT-PCR and/or culture. Specimens that contained viral RNA (vRNA) were examined for the presence of subgenomic coronavirus RNA (sgRNA), a putative marker of infectivity. Culture methods were used to determine the thermal stability of SARS-CoV-2 in human milk.ResultsBreast milk samples from 110 women (65 confirmed with a SARS-CoV-2 diagnostic test, 36 with symptoms but without tests, and 9 with symptoms but a negative SARS-CoV-2 diagnostic test) were tested by RT-PCR (285 samples) and/or viral culture (160 samples). Although vRNA of SARS-CoV-2 was detected in the milk of 7 of 110 (6%) women with either a confirmed infection or symptomatic illness, and in 6 of 65 (9%) of women with a positive SARS-CoV-2 diagnostic test, virus was not detected in any culture. None of the 7 milk specimens with detectable vRNA contained sgRNA. Notably, when artificially added to human milk in control experiments, infectious SARS-CoV-2 could be cultured despite several freeze-thaw cycles, as occurs in the storage and usage of human milk.ConclusionsSARS-CoV-2 RNA can be found infrequently in the breastmilk of women with recent infection, but we found no evidence that breastmilk contains infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants.Key PointsQuestionSARS-CoV-2 RNA has been detected in a small number of human milk samples collected from recently infected women. The role of breastfeeding in transmission of the virus to infants has remained uncertain due to the small number of specimens analyzed in any study published thus far.FindingsIn a total study group of 110 women, SARS-CoV-2 RNA was detected in milk from 6 of 65 women (9.2%) with recent confirmed infection. Neither infectious virus nor subgenomic RNA (a marker of virus infectivity) were detected in any of the samples.MeaningWe found no evidence that infectious SARS-CoV-2 is present milk from recently infected women, even if SARS-CoV-2 PCR tests are positive, providing reassurance of the safety of breastfeeding.

To The Editor, Currently, the U.S. Centers for Disease Control and Prevention, American Academy of Pediatrics and the World Health Organization advise that women who are infected with SARS-CoV-2 may choose to breastfeed with appropriate protections to prevent transmission of the virus through respiratory droplets. However, the potential for exposure to SARS-CoV-2 through breastfeeding is currently unknown. To date, case reports on breastmilk samples from a total of 24 SARS-CoV-2-infected women have been published. Of those, viral RNA was detected in ten breastmilk samples from four women. In some but not all cases, environmental contamination as the source of the virus or retrograde flow from an infected infant could not be ruled out. We established a quantitative RT-PCR assay for SARS-CoV-2 in breastmilk with a limit of detection of 250 copies per mL and validated it by spiking breastmilk from uninfected women with known amounts of viral RNA. In addition, we established tissue culture methods to detect replication-competent SARS-CoV-2 in breastmilk. No viral RNA nor culturable virus was detected after Holder pasteurization of breastmilk samples that had been spiked with replication-competent SARS-CoV-2 (see Supplement). Between March 27 and May 6, 2020, we collected and analyzed 64 serial breastmilk samples from 18 SARS-CoV-2-infected women residing in the U.S. (see Supplement for clinical characteristics). Breastmilk samples were collected before and after women had a positive SARS-CoV-2 RT-PCR test and all but one woman had symptomatic disease (see Figure). One of the 64 breastmilk samples had detectable SARS-CoV-2 RNA by RT-PCR. The positive sample was collected on the day of symptom onset but one sample 2 days prior to symptom onset and two subsequent samples, collected 12 and 41 days later, tested negative for viral RNA. In addition, a subset of 26 breastmilk samples from nine women were tested for the presence of replication-competent virus using our established culture methods, and all were negative including the one sample that tested positive for viral RNA by RT-PCR. Although SARS-CoV-2 RNA was detected in one milk sample from one of eighteen infected women, the viral culture for that sample was negative. This suggests that SARS-CoV-2 RNA does not represent replication-competent virus and that breastmilk itself is likely not a source of infection for the infant. Furthermore, when control breastmilk samples spiked with replication-competent SARS-CoV-2 virus were treated by Holder pasteurization, a process commonly performed by donor milk banks, no replication-competent virus nor viral RNA was detectable. Further research to confirm these findings is needed, as well as an examination of convalescent milk for the presence of antibodies against SARS-CoV-2.

To The Editor, Currently, the U.S. Centers for Disease Control and Prevention, American Academy of Pediatrics and the World Health Organization advise that women who are infected with SARS-CoV-2 may choose to breastfeed with appropriate protections to prevent transmission of the virus through respiratory droplets. However, the potential for exposure to SARS-CoV-2 through breastfeeding is currently unknown. To date, case reports on breastmilk samples from a total of 24 SARS-CoV-2-infected women have been published. Of those, viral RNA was detected in ten breastmilk samples from four women. In some but not all cases, environmental contamination as the source of the virus or retrograde flow from an infected infant could not be ruled out. We established a quantitative RT-PCR assay for SARS-CoV-2 in breastmilk with a limit of detection of 250 copies per mL and validated it by spiking breastmilk from uninfected women with known amounts of viral RNA. In addition, we established tissue culture methods to detect replication-competent SARS-CoV-2 in breastmilk. No viral RNA nor culturable virus was detected after Holder pasteurization of breastmilk samples that had been spiked with replication-competent SARS-CoV-2 (see Supplement). Between March 27 and May 6, 2020, we collected and analyzed 64 serial breastmilk samples from 18 SARS-CoV-2-infected women residing in the U.S. (see Supplement for clinical characteristics). Breastmilk samples were collected before and after women had a positive SARS-CoV-2 RT-PCR test and all but one woman had symptomatic disease (see Figure). One of the 64 breastmilk samples had detectable SARS-CoV-2 RNA by RT-PCR. The positive sample was collected on the day of symptom onset but one sample 2 days prior to symptom onset and two subsequent samples, collected 12 and 41 days later, tested negative for viral RNA. In addition, a subset of 26 breastmilk samples from nine women were tested for the presence of replication-competent virus using our established culture methods, and all were negative including the one sample that tested positive for viral RNA by RT-PCR. Although SARS-CoV-2 RNA was detected in one milk sample from one of eighteen infected women, the viral culture for that sample was negative. This suggests that SARS-CoV-2 RNA does not represent replication-competent virus and that breastmilk itself is likely not a source of infection for the infant. Furthermore, when control breastmilk samples spiked with replication-competent SARS-CoV-2 virus were treated by Holder pasteurization, a process commonly performed by donor milk banks, no replication-competent virus nor viral RNA was detectable. Further research to confirm these findings is needed, as well as an examination of convalescent milk for the presence of antibodies against SARS-CoV-2.

We describe a case of life-threatening disseminated coccidioidomycosis in a previously healthy child. Like most patients with disseminated coccidioidomycosis, this child had no genomic evidence of any known, rare immune disease. However, comprehensive immunologic testing showed exaggerated production of interleukin-4 and reduced production of interferon-γ. Supplementation of antifungal agents with interferon-γ treatment slowed disease progression, and the addition of interleukin-4 and interleukin-13 blockade with dupilumab resulted in rapid resolution of the patient's clinical symptoms. This report shows that blocking of type 2 immune responses can treat infection. This immunomodulatory approach could be used to enhance immune clearance of refractory fungal, mycobacterial, and viral infections. (Supported by the Jeffrey Modell Foundation and the National Institutes of Health.).

Enteroviruses cause a wide variety of diseases with neurologic, respiratory, skin, and gastrointestinal findings. The purpose of this review is to clarify changes in the classification of enteroviruses, provide information about recent disease outbreaks, and to summarize progress toward the treatment and prevention of these infections.Enteroviruses are now classified into four distinct species. New variants of coxsackievirus B1, enterovirus-A71, and enterovirus-D68 (EV-D68) have emerged as causes of recent outbreaks in the United States and other countries, including more severe disease manifestations than previously described. EV-D68 now commonly circulates in the United States, and has been linked to severe respiratory disease and associated with acute flaccid myelitis (AFM). Overcoming enormous political and logistical challenges, fewer than 100 cases of polio have been reported in 2015, and the initiation of 'endgame' strategies appears imminent. Unfortunately, treatment for enterovirus infections remains supportive, although the recently completed pleconaril trial in newborns suggests that antiviral therapy may reduce mortality in neonatal disease.Clinicians should be aware of the respiratory and neurological manifestations associated with EV-D68 and the potential for severe disease seen with other recently described enterovirus variants. Healthcare professionals should recognize the utility of rapid diagnostic methods and progress toward prevention and treatment of enterovirus infections.

A series of novel pyrazolopyridine compounds have been designed and prepared by a general synthetic route. Their activities against the replication of poliovirus-1, EV-A71, and CV-B3 enteroviruses were evaluated. The comprehensive understanding of the structure–activity relationship was obtained by utilizing the variation of four positions, namely, N1, C6, C4, and linker unit. From the screened analogues, the inhibitors with the highest selectivity indices at 50% inhibition of viral replication (SI50) were those with isopropyl at the N1 position and thiophenyl-2-yl unit at C6 position. Furthermore, the C4 position offered the greatest potential for improvement because many different N-aryl groups had better antiviral activities and compatibilities than the lead compound JX001. For example, JX040 with a 2-pyridyl group was the analogue with the most potent activity against non-polio enteroviruses, and JX025, possessing a 3-sulfamoylphenyl moiety, had the best activity against polioviruses. In addition, ana...

Infections with organisms producing extended-spectrum beta-lactamases are associated with well established risk factors and poor outcomes in adults, but these are less well defined in children. Our case-control analysis showed that infections with extended-spectrum beta-lactamases producing organisms are linked to prolonged antibiotic usage and are possibly associated with prolonged length of stay and worse overall outcomes in hospitalized children. Efforts to limit the duration and narrow the spectrum of antimicrobial therapy may assist in controlling infections due to these organisms.

Some perinatally infected children do not regain normal CD4(+) T-cell counts despite suppression of HIV-1 plasma viremia by antiretroviral therapy (ART). The frequency, severity and significance of these discordant treatment responses remain unclear.We examined the persistence of CD4(+) lymphocytopenia despite virologic suppression in 933 children (≥ 5 years of age) in the USA, Latin America and the Caribbean.CD4(+) T-cell trajectories were examined and Kaplan-Meier methods used to estimate median time to CD4(+) T-cell count at least 500 cells/μl.After 1 year of virologic suppression, most (99%) children achieved a CD4(+) T-cell count of at least 200 cells/μl, but CD4(+) T-cell counts remained below 500 cells/μl after 1 and 2 years of virologic suppression in 14 and 8% of children, respectively. Median times to first CD4(+) T-cell count at least 500 cells/μl were 1.29, 0.78 and 0.46 years for children with less than 200, 200-349 and 350-499 cells/μl at the start of virologic suppression. New AIDS-defining events occurred in nine children, including four in the first 6 months of virologic suppression. Other infectious and HIV-related diagnoses occurred more frequently and across a wide range of CD4(+) cell counts.ART improved CD4(+) cell counts in most children, but the time to CD4(+) cell count of at least 500 cells was highly dependent upon baseline immunological status. Some children did not reach a CD4(+) T-cell count of 500 cells/μl despite 2 years of virologic suppression. AIDS-defining events occurred in 1% of the population, including children in whom virologic suppression and improved CD4(+) T-cell counts were achieved.

Purpose Coccidioidomycosis (CM) is a systemic fungal disease caused by the dimorphic fungi Coccidioides immitis and Coccidioides posadasii. In its endemic areas of the United States, CM is growing as a public health challenge with a marked increase in incidence in the last 15 years. Although Coccidioides infection is asymptomatic in most cases, symptomatic pulmonary disease occurs in ~40% and disseminated coccidioidomycosis (DCM) occurs in ~1% of previously healthy children and adults. DCM is markedly more common in immunocompromised people, who often experience life-threatening disease despite use of antifungal medications. Although options for antifungal therapy have improved, lifelong therapy is needed for those who develop coccidioidal meningitis. The purpose of this article was to review the state of antifungal therapy and recent studies of host–pathogen interactions in CM in light of advances in immunomodulatory therapy. Methods The study included a review of PubMed and abstracts of the Coccidioidomycosis Study Group (years 2000–2019). Findings Current therapy for CM relies upon azole and polyene antifungal agents. Murine models and studies of DCM in patients with monogenic primary immunodeficiency states and acquired immunodeficiency have revealed the importance of both innate and adaptive immune responses in the control of infections with Coccidioides species. In particular, defects in sensing of fungi and induction of cellular immune responses have been frequently reported. More recently, polymorphisms in key signaling pathways and in the generation of Th17 and Th1 immune responses have been linked with DCM. Implications Antifungal therapy is sufficient to control disease in most cases of CM, but treatment failure occurs in cases of severe pulmonary disease and nonmeningeal disseminated disease. Lifelong therapy is recommended for meningitis in view of the very high risk of recurrence. Corticosteroid therapy is advised by some experts for severe pulmonary disease and for some neurologic complications of DCM. DCM is only rarely the result of a severe monogenic immunodeficiency. Case studies suggest that reorienting cellular immune responses or augmenting effector immune responses may help resolve DCM. Systematic investigation of immunotherapy for coccidioidomycosis is advisable and may help to address the recent marked increase in reports of the disease in endemic areas.

Object The object of this study was to determine if etiology and age at surgery were linked with fevers and altered white blood cell and CSF laboratory values after cerebral hemispherectomy. Methods Seizure etiologies (n = 76) were classified into hemimegalencephaly (HME), cortical dysplasia (CD), infarcts (stroke), Rasmussen encephalitis (RE), history of infections, and Sturge-Weber syndrome (SWS) and were compared with clinical variables, maximum daily temperature (Tmax), and blood and CSF studies through Day 12 posthemispherectomy. Results The Tmax on Days 2–4 and 9–12 postsurgery were higher for HME and RE cases than for stroke cases. Patients with RE showed positive correlations, whereas those with SWS had negative correlations between Tmax and age at surgery. Blood WBC counts on postsurgery Days 3, 6, and 9–12 were higher in the HME and CD cases than in the stroke and RE cases. The percentage of blood polymorphonuclear cells (%bloodPMNs) was higher in the RE cases than in the HME, CD, and SWS cases. The RE, HME, and CD cases showed positive correlations between %bloodPMNs and age at surgery. The percentage of blood monocytes (%bloodMono) was higher in the patients with HME than in those with stroke or RE. The HME and CD cases showed negative correlations between %bloodMono and age at surgery. The CSF red blood cell counts were higher in the RE than in the CD and stroke cases. The percentage of CSF monocytes was higher in patients with CD than in those with stroke and RE. The percentage of CSF lymphocytes positively correlated with age at surgery. Conclusions Seizure etiology and age at surgery were associated with developing fevers and altered blood and CSF values after pediatric cerebral hemispherectomy. These findings indicate that besides infections, other clinical variables have an impact on developing fevers and abnormal laboratory values posthemispherectomy. Cultures appear to be the most reliable predictor of infections.

Object The authors sought to determine if clinical epilepsy variables, maximum daily temperature (Tmax), and blood and CSF findings were associated with the risk of developing hydrocephalus after first-time resection-disconnection hemispherectomy. Methods Patients who underwent cerebral hemispherectomy in whom a standardized perioperative protocol was used, including the use of ventriculostomies (n = 79), were classified into those who developed and those who did not develop hydrocephalus requiring CSF shunts. The authors compared these 2 groups for clinical variables, Tmax, and blood and CSF studies through postoperative Day 12. Results In this cohort, 30% of the patients required CSF shunts, of which 8% developed late hydrocephalus up to 3 years posthemispherectomy. Multivariate analysis found that etiology was associated with developing posthemispherectomy hydrocephalus. Higher shunt rates were observed for patients with hemimegalencephaly (40%; n = 15) and a history of CNS infection (100%; n = 4) compared with cortical dysplasia (17%; n = 23) and Rasmussen encephalitis (17%; n = 12). In univariate analysis, other factors associated with developing hydrocephalus were elevated maximum daily temperatures, elevated white blood cell counts, decreased CSF protein, and increased CSF red blood cell counts. Conclusions The findings of the study indicate that etiology was the factor most strongly associated with developing posthemispherectomy hydrocephalus. These findings suggest that there are variable mechanisms for developing hydrocephalus after cerebral hemispherectomy depending on the procedure, and in resection-disconnection operations the mechanism may involve changes in CSF bulk flow that varies by histopathology.

Fevers and leukocytosis after pediatric craniotomy trigger diagnostic evaluation and antimicrobial therapy for possible brain infection. This study determined the incidence and predictors of infection in infants and children undergoing epilepsy neurosurgery.We reviewed the postoperative course of 100 consecutive surgeries for pediatric epilepsy, comparing those with and without infections for clinical variables and daily maximum temperatures, blood white blood cell (WBC) and differential and cerebrospinal fluid (CSF) studies.Infections were the most common adverse events after these surgeries. Four patients (4%) had CSF infections and 12 had non-CSF infections (including 1 with distinct CSF and bloodstream infections). Most (88%) infections occurred before postoperative day 12 and were associated with larger resections involving ventriculostomies. Fevers (T ≥ 38.5°C) were observed in the first 12 days postsurgery in 43% of cases, and were associated with patients undergoing hemispherectomy and multilobar resections. Fevers in the first 3 days postsurgery identified infections with 73% sensitivity, 69% specificity and 70% accuracy; 2 (13%) patients with infections never developed fevers. Peripheral blood WBC15,000 was found in 49% of patients and 5 cases of infections never had elevated WBC counts. WBC differential, CSF protein, red blood cell, WBC and red blood cell/WBC ratios were poor predictors of infections. Longer hospital stays were associated with infections and hemispherectomy and multilobar resections. Patients with and without infections were equally likely to be seizure free after surgery.Fevers and elevated blood WBC counts were common after pediatric epilepsy surgery, but CSF infections were uncommon. Positive cultures and other confirmatory microbiologic tests should drive changes in antimicrobial therapy after surgery.

We aimed to investigate the influence of class I and class II HLA specificities and of the concordance between maternal and infant HLA on vertical HIV-1 transmission. HLA typing of samples from mothers and infants enrolled in the Ariel study, a perinatal HIV-1 transmission cohort including 203 mother-infant pairs, was performed by serological and molecular methods. HLA effects were evaluated alone and by multivariate modeling considering also other known predictors of perinatal HIV-1 transmission (maternal viral load, antiretroviral therapy, duration of rupture of membranes, and histological chorioamnionitis). Modest associations were seen with specific HLA markers (increased risk with infant B67 and B58 and maternal DR1; decreased risk with maternal B12), but these were not statistically significant after adjusting for multiple comparisons. Mother-infant concordance at any class I locus was a strong predictor of transmission (odds ratio [OR], 4.16; p = 0.028). Transmission was not associated with class II concordance. Class I HLA concordance retained its importance after adjusting for maternal viral load, antiretroviral therapy, duration of rupture of membranes or histological chorioamnionitis. In multivariate modeling, only class I concordance (OR, 3.59; p = 0.069) and chorioamnionitis (OR, 3.79; p = 0.030) were retained as independent predictors of transmission. HLA alleles, and in particular the class I concordance between maternal and neonatal HLA, may regulate the risk of perinatal HIV-1 transmission. AIDS Res Hum Retroviruses

Plague is a disease caused by Yersinia pestis. Septicemic and pneumonic plague have a high mortality rate if untreated. Here we describe the challenges of accurately diagnosing a nonfatal pediatric case of septicemic plague with involvement of multiple organs; to our knowledge, the first documented case of multifocal plague osteomyelitis.

Supranormal thymic output up to 2 decades after HIV-1 infection Christian R. Aguilera-Sandoval a , Otto O. Yang a,b , Nebojsa Jojic c , Pietro Lovato d , Diana Y. Chen a , Maria Ines Boechat a , Paige Cooper a , Jun Zuo a , Christina Ramirez e , Marvin Belzer f , Joseph A. Church f and Paul Krogstad a Objectives: AIDS is caused by CD4 þ T-cell depletion. Although combination anti- retroviral therapy can restore blood T-cell numbers, the clonal diversity of the recon- stituting cells, critical for immunocompetence, is not well defined. Methods: We performed an extensive analysis of parameters of thymic function in perinatally HIV-1-infected (n ¼ 39) and control (n ¼ 28) participants ranging from 13 to 23 years of age. CD4 þ T cells including naive (CD27 þ CD45RA þ ) and recent thymic emigrant (RTE) (CD31 þ /CD45RA þ ) cells, were quantified by flow cytometry. Deep sequencing was used to examine T-cell receptor (TCR) sequence diversity in sorted RTE CD4 þ T cells. Results: Infected participants had reduced CD4 þ T-cell levels with predominant depletion of the memory subset and preservation of naive cells. RTE CD4 þ T-cell levels were normal in most infected individuals, and enhanced thymopoiesis was indicated by higher proportions of CD4 þ T cells containing TCR recombination excision circles. Memory CD4 þ T-cell depletion was highly associated with CD8 þ T-cell activation in HIV-1-infected persons and plasma interlekin-7 levels were corre- lated with naive CD4 þ T cells, suggesting activation-driven loss and compensatory enhancement of thymopoiesis. Deep sequencing of CD4 þ T-cell receptor sequences in well compensated infected persons demonstrated supranormal diversity, providing additional evidence of enhanced thymic output. Conclusion: Despite up to two decades of infection, many individuals have remarkable thymic reserve to compensate for ongoing CD4 þ T-cell loss, although there is ongoing viral replication and immune activation despite combination antiretroviral therapy. The longer term sustainability of this physiology remains to be determined. Copyright s 2016 Wolters Kluwer Health, Inc. All rights reserved. AIDS 2016, 30:701–711 Keywords: adolescents, CD4 þ T cells, immune reconstitution, perinatal HIV infection, recent thymic emigrant, T-cell receptor, thymopoiesis, T-cell receptor recombination excision circles a David Geffen School of Medicine, University of California, b AIDS Healthcare Foundation, Los Angeles, California, USA, Microsoft Corporation, d Department of Computer Science, University of Verona, Verona, Italy, e Fielding School of Public Health, University of California, and f Children’s Hospital Los Angeles and the Keck School of Medicine at University of Southern California, Los Angeles, California, USA. Correspondence to Paul Krogstad, MD, UCLA AIDS Institute, David Geffen School of Medicine at UCLA, 615 Charles E. Young Drive South, BSRB 173, Los Angeles, CA, USA. Tel: +1 310 825 5235; fax: +1 310 206 4764; e-mail: pkrogstad@mednet.ucla.edu Received: 2 September 2015; revised: 24 November 2015; accepted: 16 December 2015. c DOI:10.1097/QAD.0000000000001010 ISSN 0269-9370 Copyright Q 2016 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

Antiviral drugs do not currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. We conducted high-throughput molecular screening and identified a structurally diverse set of compounds that inhibit the replication of coxsackievirus B3, a commonly encountered enterovirus. These compounds did not interfere with the function of the viral internal ribosome entry site or with the activity of the viral proteases, but they did drastically reduce the synthesis of viral RNA and viral proteins in infected cells. Sequence analysis of compound-resistant mutants suggests that the viral 2C protein is targeted by most of these compounds. These compounds demonstrated antiviral activity against a panel of the most commonly encountered enteroviruses and thus represent potential leads for the development of broad-spectrum anti-enteroviral drugs.

Our objective was to determine whether monitoring HIV-1 DNA concentration or new resistance mutations in peripheral blood mononuclear cells (PBMCs) during effective antiretroviral therapy (ART) predicts virologic failure. A retrospective analysis used blood specimens and clinical data from three nevirapine containing arms of a four-arm, open-label, randomized trial comparing ART regimens in HIV-1-infected children who had failed mono- or dual-nucleoside therapy. Sensitive assays compared cell-associated HIV-1 DNA concentrations and nevirapine (NVP) and lamivudine (3TC) resistance mutations in children with plasma HIV-1 RNA

In this study, we investigated the possibility of differential effects of protease inhibitor (PI)-containing (PI(+)) and PI-sparing (PI(-)) antiretroviral therapies (ART) on CD8(+) T cell apoptosis. We retrospectively analyzed both PD-1 expression and CD8(+) T cell apoptosis in a cross-sectional study of HIV-positive adolescents and young adults (mean age = 17.4 years), with perinatally or behaviorally acquired HIV infection. Fifty-one specimens of cryopreserved peripheral blood mononuclear cells (PBMCs) were analyzed using 7-color flow cytometry: 20 from patients receiving PI(+) ART, 14 from PI(-) ART, and 17 from the untreated. The results showed that percentages of PD-1(+) CD8(+) T cells were strongly correlated with plasma viral loads regardless of treatment (p = 0.0001). The percentage of PD-1(+) CD8(+) T cells was also positively associated with percentages of Annexin V(+) CD8(+) T cells (p = 0.04) in the PI(+)-treated group. The fraction of apoptotic (Annexin V(+)) CD8(+) T cells was associated with viral load in the patients receiving ART that contained one or more protease inhibitors (p = 0.029), but not in the PI(-) or untreated groups. In summary, we found a direct correlation between PD-1 expression on CD8(+) T cells and HIV levels that was not affected by types of medications used in the ART of those adolescents, suggesting that virological success is necessary for PD-1 downregulation. CD8(+) T cell apoptosis was linked to high levels of PD-1 expression and HIV viremia. However, there was a higher degree of apoptosis among viremic patients receiving PI therapy, suggesting an immunologically adverse effect of continuing PI(+) therapy after virological failure.

We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus–cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure–activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus–cell but not cell–cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus–cell fusion.

Following hematopoietic stem cell transplantation (HSCT), thymic reconstitution of peripheral T lymphocytes is essential to avoid a chronically immunodeficient state and disease recurrence. The purpose of this study was to determine if children and adolescents with treatment refractory SSc, awaiting HSCT, have sufficient thymic function to reconstitute T lymphocyte function after transplantation. Thirteen children with systemic scleroderma were enrolled and assessed by physical exam, chest MRI, measurement of autoantibodies, B and T cell immuno-phenotyping, and quantization of T cell receptor rearrangement excision circles (TREC) as a marker of thymopoiesis. MRI detected thymic tissue in 9/13 children. TREC levels were detectable in all but one child but were significantly reduced (p

Enteroviruses elicit protective mucosal immune responses that could be harnessed as part of a strategy to prevent sexual transmission of the human immunodeficiency virus-1 (HIV-1). We report the construction of replication competent recombinant vectors of coxsackievirus B3 (CVB3) that express one or more portions of the HIV-1 Gag protein. Vectors containing the capsid domain of Gag were initially genetically unstable with protein expression lost after brief passage in tissue culture. Codon modification to increase the G/C content of the HIV-1 capsid sequence resulted in enhanced genetic stability of CVB3 vectors during in vitro passage. Cells infected with a vector expressing the matrix (MA) subunit of the HIV-1 Gag protein were susceptible to lysis by CD8 T cell clones specific for the SL9 epitope found within MA. These studies suggest that CVB3 vectors may be useful as vaccine vector candidates, if hurdles in class I antigen presentation and stability can be overcome.

It has been over 25 years since the fi rst diagnosis of what would be known as AIDS. Although great strides in anti-HIV therapeutics have been made, there is still a great need for antiretrovirals that are effective against drug-resistant HIV. Enfuvirtide (ENF) is the fi rst of a new class of fusion inhibitors to be approved by the US Food and Drug Administration for use in combination with other antiretroviral agents among HIV-1 infected patients with previous treatment experience. The inclusion of enfuvirtide in an optimized antiretroviral background regimen for the treatment of HIV-1 infected (treatment-experienced) patients followed the success of two critical clinical trials (TORO: T20 vs Optimized Regimen Only I and II). Even though injection-site reactions persisted in these trials, improved virological and immunological responses were observed among patients. Challenges associated with ENF treatment include the high cost of the drug, injection-site reactions, determining the optimal time to initiate treatment, and the potential for the selection of drug resistant mutants and viral evolution. ENF is a promising novel treatment for HIV infected individu- als whose choices for effective treatment are limited by previous treatment and resistance. Understanding the implications of viral fi tness and evolution in the presence of ENF treat- ment is crucial in determining effective and safe treatment regimens, particularly among treatment-experienced patients.

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8 + T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.

Outcomes for infants with severe combined immunodeficiency (SCID) would be improved by universal newborn screening, but there are not yet screening tests of sufficient accuracy for the disorder. In a pilot study, we assessed the ability of a two-tiered strategy to improve accuracy. Dried blood samples from patients were assessed with two tests for lymphopenia: interleukin-7, a T-cell growth cytokine, and TRECs, a byproduct of T-cell receptor recombination. IL-7 screening has a specificity of 96.1% and TRECs have a specificity of 92.3%. Combining these tests in a two-tiered strategy increases specificity to 100% (97-100% CI). Sensitivity was 85% for IL-7 screening and 100% for TREC screening. A two-tiered strategy may be of sufficient accuracy to enable universal SCID screening, and should be assessed in a prospective trial.

We studied changes in 60 immunological parameters after the administration of highly active antiretroviral therapy (HAART) in 192 clinically stable antiretroviral drug-experienced HIV-1-infected children 4 months-17 years old. The studied immunological parameters included standard lymphocyte subsets and lymphocyte surface markers of maturation and activation. The most significant changes during the 48-week study period were seen for CD8(+), CD8(+)CD62L(+)CD45RA(+), CD8(+)CD38(+)HLA-DR(+), and CD4(+) T cell percentages (P < .0001 for all parameters). These changes suggest that significant decreases in the expression of activation markers and increases in the expression of naive markers in the CD8(+) T cell population may be related to better virologic control in these HIV-1-infected children, who had relatively stable immune function at the initiation of HAART. At week 44 of HAART, the major immunological parameters in these HIV-1-infected children moved from baseline values to about halfway to two-thirds of the way toward the values in healthy, uninfected children.

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of ≥98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.

Background. The study reviews the incidence, timing, and outcome of infectious enteritis (IE) after intestinal transplantation (ITx). Methods. A retrospective review of all patients who underwent ITx at a single institution between 1991 and 2003 was undertaken using database and medical records. Standard statistical analyses were performed. Results. Of 33 ITxrecipients, 13 (39%) developed 20 culture- or biopsy-proven episodes of IE. Recipient demographics included the following: 10 males, median age 34 (10-585) months, 11 liver + intestine grafts, and two isolated intestine grafts. Infections were diagnosed a median of 76 days (32-1,800 days) after ITx. There were 14 viral (one cytomegalovirus, eight rotavirus, four adenovirus, one Epstein-Barr virus), three bacterial (Clostridium difficile), and three protozoal (one Giardia lamblia, two Cryptosporidium) infections. The bacterial infections tended to present earlier than the viral infections, and the most frequent presenting symptom was diarrhea. Complete resolution was achieved in 17 (94%) incidences with the appropriate antimicrobial or conservative therapy. It was interesting that there were seven rejection episodes documented by biopsy at the approximate time of diagnosis of IE. There were two graft losses: one because of adenoviral enteritis and one because of rejection after rotavirus enteritis. Three-year patient survival is 74% with no deaths directly attributable to IE. Conclusions. IE can occur in 39% of recipients after ITx. Viral agents are the cause in two thirds of the cases. With supportive care and appropriate treatment, resolution is possible in the majority of cases. Differentiating rejection and infection on histopathology can be difficult and relies on cultures and immunostaining.

In children, aplastic anemia (AA) is a common complication associated with fulminant hepatic failure (FHF). The objective of this study was to determine whether specific pretransplantation clinical and laboratory characteristics can be used to distinguish between patients with FHF who are at higher risk of developing AA. We performed a retrospective case-control study to evaluate the clinical and laboratory characteristics of those patients who presented with evidence of FHF and eventually developed aplastic anemia. We identified nine patients with AA, and all had the indeterminate form of FHF and underwent liver transplantation (LTx). The AA patients were compared with a control group of 47 patients with indeterminate FHF that underwent transplantation and did not develop AA. We found that males were over-represented in the group of patients that developed AA (p = 0.01). Furthermore, during the pretransplant period, the AA group had a significantly lower white count (p = 0.005), absolute lymphocyte count (p = 0.004), and platelet count (p = 0.019) when compared with controls. We conclude that evidence of early bone marrow dysfunction is apparent before liver transplantation and the development of AA in a subset of patients with the indeterminate form of FHF.

This report details how social media communication was used in a group of teens to diagnose cutaneous leishmaniasis that they acquired during a trip to Israel. Their posts quickly brought the cluster to the attention of the teens and their parents, leading to prompt recognition of the true etiology of their lesions and appropriate treatment.

Circular DNA molecules known as T-cell receptor rearrangement excision circles (TREC) arise during T-cell development and are present in cells that have recently emigrated from the thymus. In cross-sectional studies, the number of peripheral blood lymphocytes bearing TREC decreases with age, consistent with an anatomically demonstrated loss of thymic epithelial tissue. TREC numbers increase following hematopoietic stem cell transplantation and during therapy for human immunodeficiency virus (HIV) infection. Quantitation of TREC has therefore been proposed as a parameter of thymic activity. In this study, we used real-time PCR to quantify TREC in peripheral blood samples obtained longitudinally from HIV-seronegative adolescents. TREC values in peripheral blood T cells were very stable throughout adolescence, once thought to be a time of rapid involution of the thymus. In addition, in a cross-sectional analysis, we examined TREC values in a cohort of HIV-positive adolescents and found evidence of ongoing thymopoiesis in perinatally infected individuals, despite lifelong infection. These data demonstrate the utility of TREC assessment in adolescents and that HIV infection does not uniformly result in accelerated thymic involution in childhood.

The relative potency and tolerability of multidrug regimens used to treat infants and children infected with human immunodeficiency virus type 1 (HIV-1) are largely unknown. In Pediatric AIDS Clinical Trials Group (PACTG) Protocol 377, 181 infants and children were assigned to receive stavudine (d4T) plus nevirapine (NVP) and ritonavir (RTV); d4T plus lamivudine (3TC) and nelfinavir (NFV); d4T plus NVP and NFV; or d4T plus 3TC, NVP, and NFV. Eleven additional children received d4T and NVP plus NFV given twice daily. All subjects had not previously received protease inhibitors or nonnucleoside reverse-transcriptase inhibitors and all had been immunologically stable while receiving reverse-transcriptase inhibitor therapy. After 48 weeks of therapy, 17 (41%) of 41 subjects receiving d4T-NVP-RTV, 13 (30%) of 44 receiving d4T-NVP-NFV, 21 (42%) of 50 receiving d4T-3TC and NFV (3 times daily), and 22 (52%) of 42 receiving d4T-3TC-NVP-NFV were still receiving their assigned therapy and had HIV-1 RNA suppression to

We have identified 14 patients with diverse primary immunodeficiencies who have developed progressive neurodegeneration of unknown etiology. All patients had received immunoglobulin replacement therapy for a mean duration of 6.5 years (range of 0.5-13.5 years) at the time of first neurological symptoms. Diagnostic tests of blood and cerebrospinal fluid analyses included chemistry, cultures, PCR for viral genomes, and cytology. In addition, neuroimaging and electrophysiologic studies were performed. Brain tissue histology (n = 5) revealed nonspecific encephalitis with microglial infiltration and neuronal loss. Twelve patients died 6 months to 15 years (median 4.3 years) after onset of neurologic findings. No evidence of any infectious disease that could have explained our patients' progressive encephalopathy was found either during their lifetimes or postmortem. These patients may have had an unusual manifestation of primary immunodeficiency diseases, an autoimmune reaction against neuronal tissue, a yet undefined infectious agent, or a complication of IVIG therapy. To help determine the etiology of this rare complication, an international surveillance system for primary immunodeficiency patients who develop progressive neurodegeneration of unknown cause is recommended.

Background Neonatal enterovirus sepsis has high mortality. Antiviral therapy is not available. Methods Neonates with suspected enterovirus sepsis (hepatitis, coagulopathy, and/or myocarditis) with onset at ≤15 days of life were randomized 2:1 to receive oral pleconaril or placebo for 7 days. Serial virologic (oropharynx, rectum, urine, serum), clinical, pharmacokinetic, and safety evaluations were performed. Results Sixty-one subjects were enrolled (43 treatment, 18 placebo), of whom 43 were confirmed enterovirus infected (31 treatment, 12 placebo). There was no difference in day 5 oropharyngeal culture positivity (primary endpoint; 0% in both groups). However, enterovirus-infected subjects in the treatment group became culture negative from all anatomic sites combined faster than placebo group subjects (median 4.0 versus 7.0 days, P = .08), and fewer subjects in the treatment group remained polymerase chain reaction (PCR)-positive from the oropharynx when last sampled (23% versus 58%, P = .02; median, 14.0 days). By intent to treat, 10/43 (23%) subjects in the treatment group and 8/18 (44%) in the placebo group died (P = .02 for 2-month survival difference); among enterovirus-confirmed subjects, 7/31 (23%) in the treatment group died versus 5/12 (42%) in the placebo group (P = .26). All pleconaril recipients attained concentrations greater than the IC90 after the first study day, but 38% were less than the IC90 during the first day of treatment. One subject in the treatment group and three in the placebo group had treatment-related adverse events. Conclusions Shorter times to culture and PCR negativity and greater survival among pleconaril recipients support potential efficacy and warrant further evaluation.