Establishing Healthy Breath Baselines With Tin Oxide Sensors: Fundamental Building Blocks for Noninvasive Health Monitoring.

INTRODUCTION: Volatile organic compounds (VOCs) in breath serve as a source of biomarkers for medical conditions relevant to warfighter health including Corona Virus Disease and other potential biological threats. Electronic noses are integrated arrays of gas sensors that are cost-effective and miniaturized devices that rapidly respond to VOCs in exhaled breath. The current study seeks to qualify healthy breath baselines of exhaled VOC profiles through analysis using a commercialized array of metal oxide (MOX) sensors. MATERIALS AND METHODS: Subjects were recruited/consented through word of mouth and using posters. For each sample, breath was analyzed using an array of MOX sensors with parameters that were previously established. Data were also collected using a lifestyle questionnaire and from a blood test to assess markers of general health. Sensor data were processed using a feature extraction algorithm, which were analyzed through statistical approaches to identify correlations with confounding factors. Reproducibility was also assessed through relative standard deviation values of sensor features within a single subject and between different volunteers. RESULTS: A total of 164 breath samples were collected from different individuals, and 10 of these volunteers provided an additional 9 samples over 6 months for the longitudinal study. First, data from different subjects were analyzed, and the trends of the 17 extracted features were elucidated. This revealed not only a high degree of correlation between sensors within the array but also between some of the features extracted within a single sensor. This helped guide the removal of multicollinear features for multivariate statistical analyses. No correlations were identified between sensor features and confounding factors of interest (age, body mass index, smoking, and sex) after P-value adjustment, indicating that these variables have an insignificant impact on the observed sensor signal. Finally, the longitudinal replicates were analyzed, and reproducibility assessment showed that the variability between subjects was significantly higher than within replicates of a single volunteer (P-value = .002). Multivariate analyses within the longitudinal data displayed that subjects could not be distinguished from one another, indicating that there may be a universal healthy breath baseline that is not specific to particular individuals. CONCLUSIONS: The current study sought to qualify healthy baselines of VOCs in exhaled breath using a MOX sensor array that can be leveraged in the future to detect medical conditions relevant to warfighter health. For example, the results of the study will be useful, as the healthy breath VOC data from the sensor array can be cross-referenced in future studies aiming to use the device to distinguish disease states. Ultimately, the sensors may be integrated into a portable breathalyzer or current military gear to increase warfighter readiness through rapid and noninvasive health monitoring.

Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

Granulomas in Pediatric Liver Biopsies: Single Center Experience.

BACKGROUND: Granulomas in pediatric liver biopsies (GPLB) are rare with the largest pediatric cohort reported over 25 years ago. METHODS: Single-center retrospective study of GPLB. RESULTS: Seventeen liver biopsies from 16 patients with granulomas were identified (9 boys, 56%) with a median age of 13 years (range: 1-18) for which the most common indication was the presence of a nodule/mass (47%). Significant comorbidities were seen in 13 patients (81%) and included: liver transplant (25%), history of a neoplasm (25%), autoimmune hepatitis (6%), Crohn disease (6%), bipolar disorder (6%), severe combined immunodeficiency (6%), and sickle cell disease (6%). Eleven patients were taking multiple medications at the time of biopsy. Granulomas were more commonly pan-acinar (11 cases) followed by subcapsular (4 cases), portal (1 case), and periportal (1 case). Necrosis was seen in 10 cases (59%). GMS stain was positive in 2 cases for Histoplasma-like yeast; microbiological cultures were negative in all cases (no: 4). A 18S and 16S rRNA gene sequencing performed in 15 cases revealed only 1 with a pathogenic microorganism, Mycobacterium angelicum. CONCLUSION: In our experience, GPLB are heterogenous with only 3 cases having an identifiable infectious etiology and many of the remaining cases being associated to multiple medications, suggesting drug-induced liver injury as possible etiology.

Soluble Immune Checkpoints Are Dysregulated in COVID-19 and Heavy Alcohol Users With HIV Infection.

Immune checkpoints (ICPs) consist of paired receptor-ligand molecules that exert inhibitory or stimulatory effects on immune defense, surveillance, regulation, and self-tolerance. ICPs exist in both membrane and soluble forms in vivo and in vitro. Imbalances between inhibitory and stimulatory membrane-bound ICPs (mICPs) in malignant cells and immune cells in the tumor immune microenvironment (TIME) have been well documented. Blockades of inhibitory mICPs have emerged as an immense breakthrough in cancer therapeutics. However, the origin, structure, production regulation, and biological significance of soluble ICPs (sICPs) in health and disease largely remains elusive. Soluble ICPs can be generated through either alternative mRNA splicing and secretion or protease-mediated shedding from mICPs. Since sICPs are found in the bloodstream, they likely form a circulating immune regulatory system. In fact, there is increasing evidence that sICPs exhibit biological functions including (1) regulation of antibacterial immunity, (2) interaction with their mICP compartments to positively or negatively regulate immune responses, and (3) competition with their mICP compartments for binding to the ICP blocking antibodies, thereby reducing the efficacy of ICP blockade therapies. Here, we summarize current data of sICPs in cancer and infectious diseases. We particularly focus on sICPs in COVID-19 and HIV infection as they are the two ongoing global pandemics and have created the world's most serious public health challenges. A "storm" of sICPs occurs in the peripheral circulation of COVID-19 patients and is associated with the severity of COVID-19. Similarly, sICPs are highly dysregulated in people living with HIV (PLHIV) and some sICPs remain dysregulated in PLHIV on antiretroviral therapy (ART), indicating these sICPs may serve as biomarkers of incomplete immune reconstitution in PLHIV on ART. We reveal that HIV infection in the setting of alcohol misuse exacerbates sICP dysregulation as PLHIV with heavy alcohol consumption have significantly elevated plasma levels of many sICPs. Thus, both stimulatory and inhibitory sICPs are present in the bloodstream of healthy people and their balance can be disrupted under pathophysiological conditions such as cancer, COVID-19, HIV infection, and alcohol misuse. There is an urgent need to study the role of sICPs in immune regulation in health and disease.

Persistent SARS-CoV-2 infectivity greater than 50 days in a case series of allogeneic peripheral blood stem cell transplant recipients.

The coronavirus disease 19 (COVID-19) pandemic has infected tens of millions across the world, but there is a significant gap in our understanding about COVID-19 in the hematopoietic stem transplant (HSCT) recipient population. Prolonged viral shedding is frequently observed with severe acute respiratory syndrome coronavirus-2 (SARSCoV-2), but studies suggest viral loads decline 10 days after symptom onset. Current CDC guidance suggests that severely ill and immunocompromised hosts are no longer infectious after 20 days from symptom onset. Cycle threshold (Ct) values are inversely proportional to the viral load and are used to detect SARS-CoV-2 RNA concentration. Specimens with reverse transcriptase PCR (RT-PCR) Ct values > 33-34 have been associated with inability to culture virus, and have been used as a surrogate for diminished infectivity. We report two cases of  allogeneic peripheral blood stem cell transplant (PBSCT) recipients who had prolonged durations of infectivity with SARSCov-2, based on culture positivity and persistently low Ct values for greater than 50 days.

An unusual presentation of cutaneous histoplasmosis as a recurrent solitary and spontaneously healing lesion in an immunocompetent patient.

Infection with Histoplasma capsulatum typically manifests as a self-limiting pulmonary disease in immunocompetent patients. Systemic symptoms such as cutaneous lesions are associated with immunodeficient states. Our patient was an immunocompetent 68-year-old male who presented with a plaque on his left infraorbital area that was concerning for malignancy. Histological examination of the lesion revealed granulomatous inflammation and small yeast forms suggestive of H. capsulatum. The lesion resolved spontaneously and recurred 1 year later. On recurrence, histological examination again revealed yeast forms consistent with H. capsulatum. Serum and urine testing for H. capsulatum antigen were negative. Next-generation sequencing detected H. capsulatum, which supported the diagnosis of a cutaneous infection. The patient was prescribed and started treatment with itraconazole for 1 year after recurrence of the lesion, and he has not reported further disease recurrence to date. This case is unique because of the presentation of a primary cutaneous recurrent H. capsulatum lesion, and it demonstrated the utility of laboratory testing in its diagnosis.

Multicenter Evaluation of the QIAstat-Dx Respiratory Panel for Detection of Viruses and Bacteria in Nasopharyngeal Swab Specimens.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

Current Concepts in the Epidemiology, Diagnosis, and Management of Histoplasmosis Syndromes.

Histoplasmosis is a global disease endemic to regions of all six inhabited continents. The areas of highest endemicity lie within the Mississippi and Ohio River Valleys of North America and parts of Central and South America. As a result of climate change and anthropogenic land utilization, the conditions suitable for Histoplasma capsulatum are changing, leading to a corresponding change in epidemiology. The clinical manifestations of histoplasmosis are protean, variably resembling other common conditions such as community-acquired pneumonia, tuberculosis, sarcoidosis, Crohn's disease, or malignancy. Making a successful diagnosis is contingent on a thorough understanding of epidemiology, common clinical presentations, and best testing practices for histoplasmosis. While most subclinical or self-limited diseases do not require treatment in immunocompetent patients, all immunocompromised patients and those with progressive disseminated disease or chronic pulmonary disease should be treated. Liposomal amphotericin B is the preferred agent for severe or disseminated disease, while itraconazole is adequate for milder cases and "step-down" therapy following response to amphotericin B. In this review, we discuss the current evidence-based approaches to the epidemiology, diagnosis, and management of histoplasmosis.

A Novel Mechanism for Zika Virus Host-Cell Binding.

Zika virus (ZIKV) recently emerged in the Western Hemisphere with previously unrecognized or unreported clinical presentations. Here, we identify two putative binding mechanisms of ancestral and emergent ZIKV strains featuring the envelope (E) protein residue asparagine 154 (ASN154) and viral phosphatidylserine (PS). Synthetic peptides representing the region containing ASN154 from strains PRVABC59 (Puerto Rico 2015) and MR_766 (Uganda 1947) were exposed to neuronal cells and fibroblasts to model ZIKV E protein/cell interactions and bound MDCK or Vero cells and primary neurons significantly. Peptides significantly inhibited Vero cell infectivity by ZIKV strains MR_766 and PRVABC59, indicating that this region represents a putative binding mechanism of ancestral African ZIKV strains and emergent Western Hemisphere strains. Pretreatment of ZIKV strains MR_766 and PRVABC59 with the PS-binding protein annexin V significantly inhibited replication of PRVABC59 but not MR_766, suggesting that Western hemisphere strains may additionally be capable of utilizing PS-mediated entry to infect host cells. These data indicate that the region surrounding E protein ASN154 is capable of binding fibroblasts and primary neuronal cells and that PS-mediated entry may be a secondary mechanism for infectivity utilized by Western Hemisphere strains.

A Comprehensive Systems Biology Approach to Studying Zika Virus.

Zika virus (ZIKV) is responsible for an ongoing and intensifying epidemic in the Western Hemisphere. We examined the complete predicted proteomes, glycomes, and selectomes of 33 ZIKV strains representing temporally diverse members of the African lineage, the Asian lineage, and the current outbreak in the Americas. Derivation of the complete selectome is an 'omics' approach to identify distinct evolutionary pressures acting on different features of an organism. Employment of the M8 model did not show evidence of global diversifying selection acting on the ZIKV polyprotein; however, a mixed effect model of evolution showed strong evidence (P<0.05) for episodic diversifying selection acting on specific sites. Single nucleotide polymorphisms (SNPs) were predictably frequent across strains relative to the derived consensus sequence. None of the 9 published detection procedures utilize targets that share 100% identity across the 33 strains examined, indicating that ZIKV escape from molecular detection is predictable. The predicted O-linked glycome showed marked diversity across strains; however, the N-linked glycome was highly stable. All Asian and American strains examined were predicted to include glycosylation of E protein ASN154, a modification proposed to mediate neurotropism, whereas the modification was not predicted for African strains. SNP diversity, episodic diversifying selection, and differential glycosylation, particularly of ASN154, may have major biological implications for ZIKV disease. Taken together, the systems biology perspective of ZIKV indicates: a.) The recently emergent Asian/American N-glycotype is mediating the new and emerging neuropathogenic potential of ZIKV; and b.) further divergence at specific sites is predictable as endemnicity is established in the Americas.

An unusual presentation of leishmaniasis in a human immunodeficiency virus-positive individual.

INTRODUCTION: Leishmaniasis is a neglected tropical disease caused by vector-borne protozoa of the genus Leishmania. Cutaneous and mucocutaneous forms result in disfiguration or mutilation, whilst visceral leishmaniasis (VL) affects multiple organs and is fatal if untreated. Notably, Leishmania are capable of establishing a chronic infection, which may reactivate years after initial infection when the host becomes immune-suppressed. CASE PRESENTATION: A 24-year-old human immunodeficiency virus (HIV)-positive male presented for excision of anal condylomas. At the time of his current condyloma excision, the patient had no additional symptoms or cutaneous findings, but was noted to have been only intermittently compliant with his antiretroviral therapy. Microscopic examination of the haematoxylin and eosin-stained anal condyloma tissue revealed koilocytic change, ulceration and brisk histiocytic inflammation containing numerous small intracellular bodies suggestive of Leishmania amastigotes. A bone marrow biopsy was performed and demonstrated similar intracellular forms. Anal condyloma tissue and bone marrow aspirate were sent to the Centers for Disease Control and Prevention's Parasitic Diseases Branch for confirmation of Leishmania and speciation. Specific immunohistochemical staining for Leishmania in the tissue section was positive and the species was confirmed as Leishmania donovani by PCR. Subsequently, the patient resumed highly active antiretroviral therapy and received anti-Leishmania therapy. CONCLUSION: Whilst the presentation of VL in HIV-positive patients is often similar to those without HIV, here we describe an unusual initial presentation of leishmaniasis in an HIV-positive patient where the parasite was found in an anal condyloma. VL is a critical diagnosis that should be considered and pursued when leishmaniasis is encountered in seemingly illogical clinical settings.

Francisella philomiragia Bacteremia in a Patient with Acute Respiratory Insufficiency and Acute-on-Chronic Kidney Disease.

Francisella philomiragia is a very uncommon pathogen of humans. Diseases caused by it are protean and have been reported largely in near-drowning victims and those with chronic granulomatous disease. We present a case of F. philomiragia pneumonia with peripheral edema and bacteremia in a renal transplant patient and review the diverse reports of F. philomiragia infections.

Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes.

A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 µg/ml. This concentration significantly reduced bacterial viability, while 40 µg/ml killed all cells of the A. baumannii ATCC 19606(T) and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606(T) and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-µg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606(T) or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media.

Large granules in the peripheral blood smear and bone marrow aspirate of a 3-year-old male with lymphadenopathy and fever.

A 3-year-old male with oculocutaneous albinism presented with lymphadenopathy and fever. Serological testing revealed Epstein-Barr virus (EBV)-specific immunoglobulin M (IgM) and a diagnosis of infectious mononucleosis was made. A complete blood count and peripheral blood smear demonstrated mild anemia, thrombocytopenia, and neutropenia with leukocytes that contained large azurophilic and eosinophilic granules. Bone marrow examination demonstrated increased hemophagocytic histiocytes along with granulocytes that contained large eosinophilic granules. In addition to hemophagocytic lymphohistiocytosis, presumably due to acute EBV infection, the patient was diagnosed with Chediak-Higashi syndrome based on the pathognomonic granules within peripheral leukocytes and precursors. The differential diagnosis of a young patient with oculocutaneous albinism presenting with an acute viral infection includes a relatively narrow range of genetic syndromes based solely on the history of albinism. This case demonstrates the application of clinical laboratory data to presumptively diagnose Chediak-Higashi syndrome in the midst of a presentation of hemophagocytic lymphohistiocytosis secondary to acute EBV infection.