Clinical exome sequencing of 1000 families with complex immune phenotypes: Toward comprehensive genomic evaluations.

BACKGROUND: Prospective genetic evaluation of patients at this referral research hospital presents clinical research challenges. OBJECTIVES: This study sought not only a single-gene explanation for participants' immune-related presentations, but viewed each participant holistically, with the potential to have multiple genetic contributions to their immune phenotype and other heritable comorbidities relevant to their presentation and health. METHODS: This study developed a program integrating exome sequencing, chromosomal microarray, phenotyping, results return with genetic counseling, and reanalysis in 1505 individuals from 1000 families with suspected or known inborn errors of immunity. RESULTS: Probands were 50.8% female, 71.5% were ≥18 years, and had diverse immune presentations. Overall, 327 of 1000 probands (32.7%) received 361 molecular diagnoses. These included 17 probands with diagnostic copy number variants, 32 probands with secondary findings, and 31 probands with multiple molecular diagnoses. Reanalysis added 22 molecular diagnoses, predominantly due to new disease-gene associations (9 of 22, 40.9%). One-quarter of the molecular diagnoses (92 of 361) did not involve immune-associated genes. Molecular diagnosis was correlated with younger age, male sex, and a higher number of organ systems involved. This program also facilitated the discovery of new gene-disease associations such as SASH3-related immunodeficiency. A review of treatment options and ClinGen actionability curations suggest that at least 251 of 361 of these molecular diagnoses (69.5%) could translate into ≥1 management option. CONCLUSIONS: This program contributes to our understanding of the diagnostic and clinical utility whole exome analysis on a large scale.

Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy.

Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.

Cardiovascular Biomarker Profile on Antiretroviral Therapy Is Not Influenced by History of an IRIS Event in People With HIV and Suppressed Viremia.

Immune reconstitution inflammatory syndrome (IRIS) is characterized by release of proinflammatory cytokines and tissue inflammation occurring early after antiretroviral therapy (ART) initiation. The role of previous IRIS events in persistent chronic inflammation in people with HIV is currently unclear. In this retrospective analysis of 143 participants who maintained suppression of HIV viremia, we compared biomarkers related to inflammation, coagulation, and cardiovascular risk after 3 years on ART in participants with and without a history of IRIS. There was no evidence of higher levels of persistent chronic inflammation in people with HIV who had a history of an IRIS event. ClinicalTrials.gov Identifier . NCT00286767.

Targeted therapy guided by single-cell transcriptomic analysis in drug-induced hypersensitivity syndrome: a case report.

Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a potentially fatal multiorgan inflammatory disease associated with herpesvirus reactivation and subsequent onset of autoimmune diseases1-4. Pathophysiology remains elusive and therapeutic options are limited. Cases refractory to corticosteroid therapy pose a clinical challenge1,5 and approximately 30% of patients with DiHS/DRESS develop complications, including infections and inflammatory and autoimmune diseases1,2,5. Progress in single-cell RNA sequencing (scRNA-seq) provides an opportunity to dissect human disease pathophysiology at unprecedented resolutions6, particularly in diseases lacking animal models, such as DiHS/DRESS. We performed scRNA-seq on skin and blood from a patient with refractory DiHS/DRESS, identifying the JAK-STAT signaling pathway as a potential target. We further showed that central memory CD4+ T cells were enriched with DNA from human herpesvirus 6b. Intervention via tofacitinib enabled disease control and tapering of other immunosuppressive agents. Tofacitinib, as well as antiviral agents, suppressed culprit-induced T cell proliferation in vitro, further supporting the roles of the JAK-STAT pathway and herpesviruses in mediating the adverse drug reaction. Thus, scRNA-seq analyses guided successful therapeutic intervention in the patient with refractory DiHS/DRESS. scRNA-seq may improve our understanding of complicated human disease pathophysiology and provide an alternative approach in personalized medicine.

Case Report: Ocular toxoplasmosis in a WHIM syndrome immunodeficiency patient.

A patient with WHIM syndrome immunodeficiency presented with sudden painless right eye blindness associated with advanced retinal and optic nerve damage. Toxoplasma gondii was detected by PCR in vitreous fluid but not serum.  The patient was treated with pyrimethamine/sulfadiazine for 6 weeks due to evidence of active ocular inflammation and then received prophylaxis with trimethoprim-sulfamethoxazole due to his immunosuppression.  Vision did not return; however, the infection did not spread to involve other sites.  Toxoplasmosis is rare in primary immunodeficiency disorders and is the first protozoan infection reported in WHIM syndrome.

Beyond Blood Smears: Qualification of Plasmodium 18S rRNA as a Biomarker for Controlled Human Malaria Infections.

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.

Protracted course of disseminated adenovirus disease with necrotizing granulomas in the liver.

A 52- year-old male with chronic lymphocytic leukemia was hospitalized with disseminated adenovirus disease. More than a month following recovery, hepatic necrotizing granulomas secondary to adenovirus were found. This case illustrates the protracted course that adenovirus disease may take and emphasizes an unusual presentation with hepatic necrotizing granulomas.

Norovirus, astrovirus, and sapovirus among immunocompromised patients at a tertiary care research hospital.

We estimated the prevalence of astrovirus, sapovirus, and norovirus among patients enrolled in research protocols and receiving medical care at the Clinical Center of the National Institutes of Health, Bethesda, MD, a clinical research hospital with a large immunocompromised patient population. We identified patients whose fecal specimens were submitted to the Clinical Center for testing on the Biofire FilmArray Gastrointestinal Panel from September 15, 2015 through November 30, 2016. Among 442 patients with fecal specimens submitted for multiplex testing, 11% had norovirus identified, 2% had astrovirus, and 2% had sapovirus. Like norovirus, astrovirus was detected in multiple sequential samples from a single patient, consistent with chronic infection or the occurrence of multiple reinfections. Coinfection with non-viral gastrointestinal pathogens was detected in 31% of patients with positive results for norovirus, astrovirus, or sapovirus. Norovirus remains common in this immunocompromised patient population, and both sapovirus and astrovirus are present.

Diagnostic challenges of prolonged post-treatment clearance of Plasmodium nucleic acids in a pre-transplant autosplenectomized patient with sickle cell disease.

BACKGROUND: Autosplenectomy, as a result of sickle cell disease, is an important risk factor for severe malaria. While molecular methods are helpful in providing rapid and accurate infection detection and species identification, the effect of hyposplenism on result interpretation during the course of infection should be carefully considered. CASE PRESENTATION: A 32-year old autosplenectomized Nigerian male with severe sickle cell disease was referred to the National Institutes of Health for allogenic hematopoietic stem cell transplant. Despite testing negative for malaria by both smear and PCR 2 weeks after arrival in the USA, the patient developed fever and diffuse bilateral lower rib cage and upper abdominal pain 2 weeks later and subsequently tested positive for Plasmodium falciparum. Parasitaemia was tracked over time by microscopy and nucleic acid tests to evaluate the therapeutic response in the setting of hyposplenism. The patient showed prompt resolution of patent infection by microscopy but remained positive by molecular methods for > 30 days after treatment initiation. CONCLUSION: While molecular testing can provide sensitive Plasmodium nucleic acid detection, the persistence of Plasmodium nucleic acids following adequate treatment in functionally asplenic patients can lead to a diagnostic dilemma. In such patients, clinical response and peripheral blood smears should guide patient management following treatment. Nonetheless, in pre-transplant patients at high-risk for pre-existing Plasmodium infections, highly sensitive molecular assays can be useful to rule out infection prior to transplantation.

Pulmonary Histoplasma Infection After Allogeneic Hematopoietic Stem Cell Transplantation: Case Report and Review of the Literature.

Histoplasmosis causes a wide spectrum of clinical illness, including disseminated infection in the immunocompromised. We report a case of pulmonary histoplasmosis in an allogeneic stem cell transplant recipient and review the literature on this topic. Histoplasmosis in this patient population is uncommon, but it is associated with poor outcome.

Pneumocystis colonization in asthmatic patients not receiving oral corticosteroid therapy.

Pneumocystis jirovecii can colonize patients with chronic obstructive pulmonary disease. To determine if colonization occurs in asthma patients, sputum samples from 10 patients with mild asthma, who were not receiving oral corticosteroids, were evaluated by a sensitive real-time PCR assay that targets a multicopy gene of P. jirovecii. 2 patients (20%) had Pneumocystis DNA detected; 1 patient had 3 positive samples over an 11-day period. Thus, Pneumocystis colonization occurs in asthma patients, and further studies are warranted to evaluate its role in airways disease. TRIAL REGISTRATION NUMBER: NCT01113034.

High rates of cerebral toxoplasmosis in HIV patients presenting with meningitis in Accra, Ghana.

Background: Data on adult meningitis among patients infected with the human immunodeficiency virus (HIV) is scarce in western sub-Saharan Africa, including Ghana. Methods: HIV-infected adults with a provisional diagnosis of meningitis were consecutively enrolled, between August 2014 and January 2016. After patient data collection, cerebrospinal fluid (CSF) was obtained and evaluated for microbiological aetiologies, cell counts and biochemistry. Caregiver clinicians provided limited data for inpatients at the end-point of discharge or death. Results: Complete data sets from 84 patients were analysed (inpatients=63, outpatients=21). Median age was 40 years with 56% (47/84) being females. Only 30% (25/84) of the patients were on antiretroviral therapy (ART). CD4+ T-cell count was available for 81% (68/84) of patients and 61.9% (52/84) had counts below 150 cells/µL [median and interquartile range=56 (13.8-136)]. Microbiological aetiologies were detected in 60.7% (51/84) patients with the following distribution-Toxoplasmosis (25%), Epstein-Barr virus (28.6%), Cytomegalovirus and Cryptococcus (2.4%) each. Co-infection was identified in 20.7% (17/84) of the patients. Conclusion: Patients presenting with symptoms of meningitis had advanced HIV/AIDS, a quarter of whom had cerebral toxoplasmosis or infection with EBV. A high index of suspicion, laboratory exclusion of cryptococcal meningitis and prompt patient management with anti-toxoplasmosis empiric therapy may thus be required for optimal treatment.

Epidemiology of Norovirus Infection Among Immunocompromised Patients at a Tertiary Care Research Hospital, 2010-2013.

Background. Noroviruses are a major cause of infectious gastroenteritis worldwide, and viruses can establish persistent infection in immunocompromised individuals. Risk factors and transmission in this population are not fully understood. Methods. From 2010 through 2013, we conducted a retrospective review among immunocompromised patients (n = 268) enrolled in research studies at the National Institutes of Health Clinical Center and identified a subset of norovirus-positive patients (n = 18) who provided stool specimens for norovirus genotyping analysis. Results. Norovirus genome was identified by reverse-transcription quantitative polymerase chain reaction in stools of 35 (13%) of the 268 immunocompromised patients tested, and infection prevalence was 21% (11 of 53) in persons with primary immune deficiencies and 12% (20 of 166) among persons with solid tumors or hematologic malignancies. Among 18 patients with norovirus genotyping information, norovirus GII.4 was the most prevalent genotype (14 of 18, 78%). Persistent norovirus infection (≥6 months) was documented in 8 of 18 (44%) individuals. Phylogenetic analysis of the GII.4 capsid protein sequences identified at least 5 now-displaced GII.4 variant lineages, with no evidence of their nosocomial transmission in the Clinical Center. Conclusions. Norovirus was a leading enteric pathogen identified in this immunocompromised population. Both acute and chronic norovirus infections were observed, and these were likely community-acquired. Continued investigation will further define the role of noroviruses in these patients and inform efforts toward prevention and treatment.

Protection against malaria at 1 year and immune correlates following PfSPZ vaccination.

An attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine, PfSPZ Vaccine, is highly protective against controlled human malaria infection (CHMI) 3 weeks after immunization, but the durability of protection is unknown. We assessed how vaccine dosage, regimen, and route of administration affected durable protection in malaria-naive adults. After four intravenous immunizations with 2.7 × 10(5) PfSPZ, 6/11 (55%) vaccinated subjects remained without parasitemia following CHMI 21 weeks after immunization. Five non-parasitemic subjects from this dosage group underwent repeat CHMI at 59 weeks, and none developed parasitemia. Although Pf-specific serum antibody levels correlated with protection up to 21-25 weeks after immunization, antibody levels waned substantially by 59 weeks. Pf-specific T cell responses also declined in blood by 59 weeks. To determine whether T cell responses in blood reflected responses in liver, we vaccinated nonhuman primates with PfSPZ Vaccine. Pf-specific interferon-γ-producing CD8 T cells were present at ∼100-fold higher frequencies in liver than in blood. Our findings suggest that PfSPZ Vaccine conferred durable protection to malaria through long-lived tissue-resident T cells and that administration of higher doses may further enhance protection.

Clinical Performance of Check-Direct CPE, a Multiplex PCR for Direct Detection of bla(KPC), bla(NDM) and/or bla(VIM), and bla(OXA)-48 from Perirectal Swabs.

We evaluated the clinical performance of Check-Direct CPE for carbapenemase detection directly from 301 perirectal swabs (258 patients) in a nonoutbreak setting. Culture of a PCR-confirmed, carbapenemase-containing organism, or history of colonization with such organism within the previous 2 weeks, was used as the reference standard. Check-Direct CPE demonstrated a sensitivity value, specificity value, positive predictive value (PPV), and negative predictive value (NPV) of 100% (all bla(KPC)), 88%, 21%, and 100%, respectively. False positives accounted for 79% (n = 34) of samples for which a cycle threshold (C(T)) value was reached. Simulated studies to evaluate specimen pooling as an approach to minimize costs showed no difference in C(T) values for pooled groups of three or five that each contained a single specimen spiked with ∼1,500 CFU bla(KPC) Klebsiella pneumoniae; however, the detection rate dropped to 60% at a seeded concentration of ∼150 CFU. When data were pooled, C(T) values for bla(KPC) were higher for heavy-feces-containing than for light-feces-containing liquid-suspended specimens. Furthermore, C(T) values for liquid-suspended specimens were 4 to 5 C(T) values lower (i.e., represented greater sensitivity) than those seen in direct swab analysis. Culture was equivalent to or better than Check-Direct CPE for 13/15 (87%) isolates tested in a limit-of-detection analysis. Detection of a carbapenemase gene at a C(T) cutoff value of ≤35 was culture confirmed in 23/24 (96%) of cases; however, C(T) values of >35 overlapped broadly between culture-positive (n = 21) and culture-negative (n = 36) specimens. Check-Direct CPE will likely prove most useful in high-prevalence areas or in outbreak settings where rapid carbapenemase detection is critical for infection control management.

Combination systemic and intravitreal antiviral therapy in the management of acute retinal necrosis syndrome.

BACKGROUND AND OBJECTIVE: Acute retinal necrosis (ARN) may lead to severe visual loss because of its rapid progression and high likelihood of retinal detachment (RD). This study investigates whether combination systemic and intravitreal antiviral therapy is superior to systemic antiviral therapy alone. PATIENTS AND METHODS: Single-center, interventional, comparative case series of patients with ARN treated with combination systemic antiviral and intravitreal foscarnet injection therapy or systemic antiviral therapy alone. Survival analysis and incidence rates of visual acuity (VA) gain of two lines or greater, severe visual loss of 20/200 or worse, and RD were assessed. RESULTS: Twelve patients received combination therapy and 12 received systemic therapy alone. Patients receiving combination therapy were more likely to gain two or more lines of VA and showed decreased incidences of severe visual loss and RD. CONCLUSION: Combination oral and intravitreal antiviral therapy may improve the likelihood for VA gain and decrease the risk of RD in patients with ARN. Clinicians should consider administering combination systemic and intravitreal antiviral therapy for patients with the ARN syndrome.

First report of Westerdykella dispersa as a cause of an angioinvasive fungal infection in a neutropenic host.

Angioinvasive fungal infections (AFIs) are an important cause of morbidity and mortality among immunocompromised patients. However, clinicomicrobiological characteristics and treatment of many AFI agents remain poorly defined. We report the first human case of infection with Westerdykella dispersa, an emergent cause of AFI, which was successfully treated in a neutropenic pediatric patient.