Technology in bioanalysis, -omics, and computation have evolved over the past half century to allow for comprehensive assessments of the molecular to whole body pharmacology of diverse corticosteroids. Such studies have advanced pharmacokinetic and pharmacodynamic (PK/PD) concepts and models that often generalize across various classes of drugs. These models encompass the "pillars" of pharmacology, namely PK and target drug exposure, the mass-law interactions of drugs with receptors/targets, and the consequent turnover and homeostatic control of genes, biomarkers, physiologic responses, and disease symptoms. Pharmacokinetic methodology utilizes noncompartmental, compartmental, reversible, physiologic [full physiologically based pharmacokinetic (PBPK) and minimal PBPK], and target-mediated drug disposition models using a growing array of pharmacometric considerations and software. Basic PK/PD models have emerged (simple direct, biophase, slow receptor binding, indirect response, irreversible, turnover with inactivation, and transduction models) that place emphasis on parsimony, are mechanistic in nature, and serve as highly useful "top-down" methods of quantitating the actions of diverse drugs. These are often components of more complex quantitative systems pharmacology (QSP) models that explain the array of responses to various drugs, including corticosteroids. Progressively deeper mechanistic appreciation of PBPK, drug-target interactions, and systems physiology from the molecular (genomic, proteomic, metabolomic) to cellular to whole body levels provides the foundation for enhanced PK/PD to comprehensive QSP models. Our research based on cell, animal, clinical, and theoretical studies with corticosteroids have provided ideas and quantitative methods that have broadly advanced the fields of PK/PD and QSP modeling and illustrates the transition toward a global, systems understanding of actions of diverse drugs.

The aim of this study was to evaluate the efficacy of ¹⁷⁷Lu-prostate-specific membrane antigen (PSMA)-617 (¹⁷⁷Lu-PSMA) and selective internal radiation therapy (SIRT) for the treatment of liver metastases of castration-resistant prostate cancer. Methods: Safety and survival of patients with metastatic castration-resistant prostate cancer and liver metastases assigned to ¹⁷⁷Lu-PSMA alone (n = 31) or in combination with SIRT (n = 5) were retrospectively analyzed. Additionally, a subgroup (n = 10) was analyzed using morphologic and molecular response criteria. Results: Median estimated survival was 5.7 mo for ¹⁷⁷Lu-PSMA alone and 8.4 mo for combined sequential ¹⁷⁷Lu-PSMA and SIRT. ¹⁷⁷Lu-PSMA achieved discordant therapy responses with both regressive and progressive liver metastases in the same patient (best vs. worst responding metastases per patient: –35% vs. +63% diameter change; P < 0.05). SIRT was superior to ¹⁷⁷Lu-PSMA for the treatment of liver metastases (0% vs. 56% progression). Conclusion: The combination of ¹⁷⁷Lu-PSMA and SIRT is efficient and feasible for the treatment of advanced prostate cancer. ¹⁷⁷Lu-PSMA alone seems to have limited response rates in the treatment of liver metastases.

⁶⁸Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as ⁶⁸Ga-PSMA-11, are promising small molecules for targeting prostate cancer (PCa). Although this radiopharmaceutical was produced mostly by means of manual synthesis and automated synthesis modules, a sterile cold kit was recently introduced. The aim of our study was to evaluate the image quality of ⁶⁸Ga-PSMA-11 PET/CT (PSMA-PET) in a population of PCa patients after the injection of comparable activities of ⁶⁸Ga-PSMA-11 obtained with the 2 different synthetic procedures. A secondary aim was to identify secondary factors that may have an impact on image quality and, thus, final interpretation. Methods: Two different groups of 100 consecutive PCa patients who underwent PSMA-PET were included in the study. The first group of patients was imaged with ⁶⁸Ga-PSMA-11 obtained using synthesis modules, whereas the second group’s tracer activity was synthesized using a sterile cold kit. All PET images were independently reviewed by 2 nuclear medicine diagnosticians with at least 2 y of experience in PSMA-based imaging and unaware of the patients’ clinical history. The 2 reviewers independently rated the quality of each PSMA-PET scan using a 3-point Likert-type scale. In cases of discordance, the operators together reviewed the images and reached a consensus. Performance was evaluated on the basis of the expected biodistribution, lesion detection rate, and physiologic background uptake. Results: Overall, 104 of 200 (52%) PSMA-PET scans were positive for PCa-related findings. No significant differences in image quality between cold kits and synthesis modules were found (P = 0.13), although a higher proportion of images was rated as excellent by the observers for kits than for modules (45% vs. 34%). Furthermore, after image quality had been dichotomized as excellent or not excellent, multivariate regression analysis found several factors to be significantly associated with a not-excellent quality: an increase in patient age (+5 y: odds ratio [OR], 1.40; 95% confidence interval [CI], 1.12–1.75), an increase in patient weight (+5 kg: OR, 1.89; 95% CI, 1.53–2.32), an increase in ⁶⁸Ga-PSMA-11 uptake time (+10 min: OR, 1.45; 95% CI, 1.08–1.96), and a decrease in injected activity (–10 MBq: OR, 1.28; 95% CI, 1.07–1.52). Conclusion: No significant differences were identified between the 2 groups of patients undergoing PSMA-PET; therefore, we were not able to ascertain any significant influences of tracer production methodology on final scan quality. However, increased patient age, increased patient weight, decreased injected activity, and increased ⁶⁸Ga-PSMA-11 uptake time were significantly associated with an overall poorer image quality.

Neuroendocrinelike transdifferentiation of prostate cancer adenocarcinomas correlates with serum levels of chromogranin A (CgA) and drives treatment resistance. The aim of this work was to evaluate whether CgA can serve as a response predictor for ¹⁷⁷Lu-prostate-specific membrane antigen 617 (PSMA) radioligand therapy (RLT) in comparison with the established tumor markers. Methods: One hundred consecutive patients with metastasized castration-resistant prostate cancer scheduled for PSMA RLT were evaluated for prostate-specific antigen (PSA), lactate dehydrogenase (LDH), and CgA at baseline and in follow-up of PSMA RLT. Tumor uptake of PSMA ligand, a known predictive marker for response, was assessed as a control variable. Results: From the 100 evaluated patients, 35 had partial remission, 16 stable disease, 15 mixed response, and 36 progression of disease. Tumor uptake above salivary gland uptake translated into partial remission, with an odds ratio (OR) of 60.265 (95% confidence interval [CI], 5.038–720.922). Elevated LDH implied a reduced chance for partial remission, with an OR of 0.094 (95% CI, 0.017–0.518), but increased the frequency of progressive disease (OR, 2.717; 95% CI, 1.391–5.304). All patients who achieved partial remission had a normal baseline LDH. Factor-2 elevation of CgA increased the risk for progression, with an OR of 3.089 (95% CI, 1.302–7.332). Baseline PSA had no prognostic value for response prediction. Conclusion: In our cohort, baseline PSA had no prognostic value for response prediction. LDH was the marker with the strongest prognostic value, and elevated LDH increased the risk for progression of disease under PSMA RLT. Elevated CgA demonstrated a moderate impact as a negative prognostic marker in general but was explicitly related to the presence of liver metastases. Well in line with the literature, sufficient tumor uptake is a prerequisite to achieve tumor response.

Prostate-specific membrane antigen (PSMA)–targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT–nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with ²²⁵Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair–associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0–6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair–targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

In around 20% of men with prostate cancer, metastasis develops during the course of their disease. Accordingly, discovering and developing new potent treatment strategies for patients with metastatic prostate cancer has been a major research focus during the last few decades. Identifying disease progression, especially within clinical trials, is essential in determining drug effectiveness. One major remaining question is how best to define disease progression. The criteria of the Prostate Cancer Clinical Trials Working Group (PCWG2) include clinical and laboratory parameters, as well as conventional imaging modalities such as MRI, CT, and bone scan findings, but advanced molecular imaging techniques, especially prostate-specific membrane antigen (PSMA) PET findings, are not considered. This is a problem because PSMA PET is used not only for detecting biochemical recurrence but also for restaging and as an intermediate-endpoint biomarker in ongoing clinical trials. Therefore, response criteria and PSMA PET progression (PPP) criteria need to be established with some urgency. The intent of this article is therefore to define prostate cancer progression by PSMA PET criteria. Our PPP proposal is based on the same principles as were applied for the PCGW2 criteria but adds value by including PSMA PET criteria. PPP defines PSMA treatment response using 3 different criteria. The first is the appearance of 2 or more new PSMA-positive distant lesions. The second is the appearance of 1 new PSMA-positive lesion plus consistent clinical or laboratory data and recommended confirmation by biopsy or correlative imaging within 3 mo of PSMA PET. The third is an increase in size or PSMA uptake of 1 or more existing lesions by at least 30%, plus consistent clinical or laboratory data or confirmation by biopsy or correlative imaging within 3 mo of PSMA PET.

For effective clinical management of patients being treated with ²²³Ra, there is a need for radiographic response biomarkers to minimize disease progression and to stratify patients for subsequent treatment options. The objective of this study was to evaluate an automated bone scan index (aBSI) as a quantitative assessment of bone scans for radiographic response in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods: In a multicenter retrospective study, bone scans from patients with mCRPC treated with monthly injections of ²²³Ra were collected from 7 hospitals in Sweden. Patients with available bone scans before treatment with ²²³Ra and at treatment discontinuation were eligible for the study. The aBSI was generated at baseline and at treatment discontinuation. The Spearman rank correlation was used to correlate aBSI with the baseline covariates: alkaline phosphatase (ALP) and prostate-specific antigen (PSA). The Cox proportional-hazards model and Kaplan–Meier curve were used to evaluate the association of covariates at baseline and their change at treatment discontinuation with overall survival (OS). The concordance index (C-index) was used to evaluate the discriminating strength of covariates in predicting OS. Results: Bone scan images at baseline were available from 156 patients, and 67 patients had both a baseline and a treatment discontinuation bone scan (median, 5 doses; interquartile range, 3–6 doses). Baseline aBSI (median, 4.5; interquartile range, 2.4–6.5) was moderately correlated with ALP (r = 0.60, P < 0.0001) and with PSA (r = 0.38, P = 0.003). Among baseline covariates, aBSI (P = 0.01) and ALP (P = 0.001) were significantly associated with OS, whereas PSA values were not (P = 0.059). After treatment discontinuation, 36% (24/67), 80% (54/67), and 13% (9/67) of patients demonstrated a decline in aBSI, ALP, and PSA, respectively. As a continuous variable, the relative change in aBSI after treatment, compared with baseline, was significantly associated with OS (P < 0.0001), with a C-index of 0.67. Median OS in patients with both aBSI and ALP decline (median, 134 wk) was significantly longer than in patients with ALP decline only (median, 77 wk; P = 0.029). Conclusion: Both aBSI at baseline and its change at treatment discontinuation were significant parameters associated with OS. The study warrants prospective validation of aBSI as a quantitative imaging response biomarker to predict OS in patients with mCRPC treated with ²²³Ra.

The ₂ receptor is a potential in vivo target for measuring proliferative status in cancer. The feasibility of using N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-¹⁸F-fluoroethoxy)-5-methylbenzamide (¹⁸F-ISO-1) to image solid tumors in lymphoma, breast cancer, and head and neck cancer has been previously established. Here, we report the results of the first dedicated clinical trial of ¹⁸F-ISO-1 in women with primary breast cancer. Our study objective was to determine whether ¹⁸F-ISO-1 PET could provide an in vivo measure of tumor proliferative status, and we hypothesized that uptake would correlate with a tissue-based assay of proliferation, namely Ki-67 expression. Methods: Twenty-eight women with 29 primary invasive breast cancers were prospectively enrolled in a clinical trial (NCT 02284919) between March 2015 and January 2017. Each received an injection of 278–527 MBq of ¹⁸F-ISO-1 and then underwent PET/CT imaging of the breasts 50–55 min later. In vivo uptake of ¹⁸F-ISO-1 was quantitated by SUV_max and distribution volume ratios and was compared with ex vivo immunohistochemistry for Ki-67. Wilcoxon rank-sum tests assessed uptake differences across Ki-67 thresholds, and Spearman correlation tested associations between uptake and Ki-67. Results: Tumor SUV_max (median, 2.0 g/mL; range, 1.3–3.3 g/mL), partial-volume–corrected SUV_max, and SUV ratios were tested against Ki-67. Tumors stratified into the high–Ki-67 (≥20%) group had SUV_max greater than the low–Ki-67 (<20%) group (P = 0.02). SUV_max exhibited a positive correlation with Ki-67 across all breast cancer subtypes ( = 0.46, P = 0.01, n = 29). Partial-volume–corrected SUV_max was positively correlated with Ki-67 for invasive ductal carcinoma ( = 0.51, P = 0.02, n = 21). Tumor–to–normal-tissue ratios and tumor distribution volume ratio did not correlate with Ki-67 (P > 0.05). Conclusion: ¹⁸F-ISO-1 uptake in breast cancer modestly correlates with an in vitro assay of proliferation.

The incidence of abnormal cerebrospinal fluid (CSF) flow dynamics in children with central nervous system (CNS) tumors before intraventricular therapy has not been described. Methods: We performed a single-institution, retrospective review of patients with primary or metastatic CNS tumors treated between 2003 and 2018 (15 y). Patients underwent ¹¹¹In-diethylenetriaminepentaacetic acid injection into the CSF intraventricular space followed by nuclear medicine imaging at 90 min, 4 h, 24 h, and 48 h (if required). CSF flow was classified as normal, delayed, asymmetric, or obstructed. Results: In total, 278 CSF flow studies were performed on 224 patients, 202 of whom (90%) were less than 18 y old. Of these, 116 patients (52%) had metastatic CNS neuroblastoma, 57 (25%) had medulloblastoma, and 51 (23%) had other histologic types of CNS tumors. Of the 278 studies, 237 (85%) were normal, 9 (3%) required neurosurgical intervention, 25 (9%) were delayed, and 7 (3%) were asymmetric. Conclusion: Abnormal CSF flow and the necessity for neurosurgical intervention must be considered when attempting to ensure appropriate intraventricular therapy in the pediatric population.

Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may remain asymptomatic, leading to under-recognition of the related disease, coronavirus disease, 2019 (COVID-19), and to incidental findings in nuclear imaging procedures performed for standard clinical indications. Here, we report about our local experience in a region with high COVID-19 prevalence and dynamically increasing infection rates. Methods: Within the 8-d period of March 16–24, 2020, hybrid imaging studies of asymptomatic patients who underwent ¹⁸F-FDG PET/CT or ¹³¹I SPECT/CT for standard oncologic indications at our institution in Brescia, Italy, were analyzed for findings suggestive of COVID-19. The presence, radiologic features, and metabolic activity of interstitial pneumonia were identified, correlated with the subsequent short-term clinical course, and described in a case series. Results: Six of 65 patients (9%) who underwent PET/CT for various malignancies showed unexpected signs of interstitial pneumonia on CT and elevated regional ¹⁸F-FDG avidity. Additionally, 1 of 12 patients who received radioiodine for differentiated thyroid carcinoma also showed interstitial pneumonia on SPECT/CT. Five of 7 patients had subsequent proof of COVID-19 by reverse-transcriptase polymerase chain reaction. The remaining 2 patients were not tested immediately but underwent quarantine and careful monitoring. Conclusion: Incidental findings suggestive of COVID-19 may not be infrequent in hybrid imaging of asymptomatic patients in regions with an expansive spread of SARS-CoV-2. Nuclear medicine services should prepare accordingly.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N⁶-methyladenosine (m⁶A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m⁶A in 18S rRNA at position A₁₈₃₂. We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m⁶A in rRNA in stemness, differentiation, development, and diseases.

The proteostasis network is regulated by transcellular communication to promote health and fitness in metazoans. In Caenorhabditis elegans, signals from the germline initiate the decline of proteostasis and repression of cell stress responses at reproductive maturity, indicating that commitment to reproduction is detrimental to somatic health. Here we show that proteostasis and stress resilience are also regulated by embryo-to-mother communication in reproductive adults. To identify genes that act directly in the reproductive system to regulate somatic proteostasis, we performed a tissue targeted genetic screen for germline modifiers of polyglutamine aggregation in muscle cells. We found that inhibiting the formation of the extracellular vitelline layer of the fertilized embryo inside the uterus suppresses aggregation, improves stress resilience in an HSF-1-dependent manner, and restores the heat-shock response in the somatic tissues of the parent. This pathway relies on DAF-16/FOXO activation in vulval tissues to maintain stress resilience in the mother, suggesting that the integrity of the embryo is monitored by the vulva to detect damage and initiate an organismal protective response. Our findings reveal a previously undescribed transcellular pathway that links the integrity of the developing progeny to proteostasis regulation in the parent.

In this issue of Genes & Development, Lu and colleagues (pp. 663–677) have discovered a key new mechanism of alternative promoter choice that is involved in differentiation of spermatocytes. Promoter choice has strong potential as mechanism for differentiation of many different cell types.

Background and objective

Exercise is a cornerstone of management for type 2 diabetes; however, little is known about the cardiovascular (CV) response to submaximal functional exercise in people with type 2 diabetes. The aim of this study was to compare performance and CV response during a 6-minute walk test (6MWT) between people with type 2 diabetes and matched control subjects.

Management of cystic fibrosis has been revolutionised by the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. These compounds treat the underlying molecular basis of the disease by increasing activity of defective CFTR channels, which improves many clinical parameters and enhances patient quality of life [1]. Next-generation modulators, also known as triple combination therapy, promise to be highly efficacious in up to 90% of patients [2] and will likely dramatically change the landscape of cystic fibrosis disease. Studies examining individuals before and after initiation of CFTR modulators have revealed novel functions of CFTR and shown that CFTR modulators do not reverse all disease manifestations [3–5]. Thus, knowledge of the post-modulator cystic fibrosis disease state is crucial for understanding what continued therapies will be needed for people with cystic fibrosis and what new challenges may arise.

Background

The increasing incidence of life-threatening Pneumocystis pneumonia (PCP) in non-HIV immunocompromised patients is a global concern. Yet, no reports have examined the prognostic significance of pre-existing interstitial lung disease (ILD) in non-HIV PCP.

Introduction

Immunotherapy has become the standard of care in advanced non-small cell lung cancer (NSCLC). We aimed to quantify the economic impact, in France, of anti-PD-1 therapy for NSCLC.

Eligibility criteria for a biologic treatment for severe asthma include poor disease control despite a full medication plan according to Global Initiative for Asthma steps 4–5 [1]. Adherence to inhaled therapy should be verified as part of that prescription requirement [2]. In fact, it has been demonstrated that poor adherence is a major cause of uncontrolled asthma, regardless of its severity [3]. Furthermore, biologics do not exert a disease-modifying effect [4]; in contrast to allergen immunotherapy, which is able to permanently modulate the way the immune system reacts to allergens beyond the immunotherapy treatment course [5], biologic therapy withdrawal usually leads to asthma relapse [4]. Thus, a low adherence rate to inhaled treatment in patients undergoing biologic therapy raises some issues related to sustainability.

ABSTRACT

Bezlotoxumab is a human monoclonal antibody against Clostridium difficile toxin B, indicated to prevent recurrence of C. difficile infection (rCDI) in high-risk adults receiving antibacterial treatment for CDI. An exploratory genome-wide association study investigated whether human genetic variation influences bezlotoxumab response. DNA from 704 participants who achieved initial clinical cure in the phase 3 MODIFY I/II trials was genotyped. Single nucleotide polymorphisms (SNPs) and human leukocyte antigen (HLA) imputation were performed using IMPUTE2 and HIBAG, respectively. A joint test of genotype and genotype-by-treatment interaction in a logistic regression model was used to screen genetic variants associated with response to bezlotoxumab. The SNP rs2516513 and the HLA alleles HLA-DRB1*07:01 and HLA-DQA1*02:01, located in the extended major histocompatibility complex on chromosome 6, were associated with the reduction of rCDI in bezlotoxumab-treated participants. Carriage of a minor allele (homozygous or heterozygous) at any of the identified loci was related to a larger difference in the proportion of participants experiencing rCDI versus placebo; the effect was most prominent in the subgroup at high baseline risk for rCDI. Genotypes associated with an improved bezlotoxumab response showed no association with rCDI in the placebo cohort. These data suggest that a host-driven, immunological mechanism may impact bezlotoxumab response. Trial registration numbers are as follows: NCT01241552 (MODIFY I) and NCT01513239 (MODIFY II).

IMPORTANCE Clostridium difficile infection is associated with significant clinical morbidity and mortality; antibacterial treatments are effective, but recurrence of C. difficile infection is common. In this genome-wide association study, we explored whether host genetic variability affected treatment responses to bezlotoxumab, a human monoclonal antibody that binds C. difficile toxin B and is indicated for the prevention of recurrent C. difficile infection. Using data from the MODIFY I/II phase 3 clinical trials, we identified three genetic variants associated with reduced rates of C. difficile infection recurrence in bezlotoxumab-treated participants. The effects were most pronounced in participants at high risk of C. difficile infection recurrence. All three variants are located in the extended major histocompatibility complex on chromosome 6, suggesting the involvement of a host-driven immunological mechanism in the prevention of C. difficile infection recurrence.

ABSTRACT

Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as "virulence factor delivery bags" in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro. In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis.

IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

Extractables and leachables (E&L) are identified and quantified so that their impact on patient safety can be established and assessed. The uncertainty in the impact assessment is affected by the uncertainty in the substance's experimentally determined identity and concentration. Thus, these experimentally determined quantities must be reported not only in terms of their absolute result but also in terms of the uncertainty in the result, which is based on the amount and rigor of the information on which the result is based. In this way, the impact assessment can be tempered to account for the uncertainty in its input data. To facilitate the assignment and reporting of uncertainty, classification hierarchies are proposed and discussed for both identification and quantitation. Both hierarchies establish levels or degrees of identification and quantitation based on the uncertainty of the result and contain descriptions of the quality and quantity of information required to achieve a certain level within the hierarchy. The minimal levels that must be achieved to support impact assessment are also established.

This technology review, written by a small group of pharmaceutical microbiologists experienced in cell therapies, discussed a risk-based approach to microbiological contamination detection and control during gene and cell therapy production. Topics discussed include a brief overview of cell therapies, a risk analysis related to donor selection, cell collection and infectious agent testing, cell transformation and expansion, packaging, storage, and administration, and cell therapy microbial contamination testing and release.

Capture bioprocessing unit operations were previously shown to clear or kill several log₁₀ of a model mycoplasma Acholeplasma laidlawii in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: Mycoplasma orale and Mycoplasma arginini. Clearance of M. orale and M. arginini from protein A column purification was similar to that seen with A. laidlawii, though some between cycle carryover was evident, especially for M. orale. However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated M. arginini though detectable levels of M. orale remained. A small-scale model of a commercial low-pH hold step did inactivate live M. orale, but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with A. laidlawii. Additionally, ultraviolet-C irradiation was shown to be effective for A. laidlawii and M. orale inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared A. laidlawii. These data argue that M. orale and M. arginini overall would be largely cleared by early bioprocessing steps as shown previously for A. laidlawii, and that barrier technologies can effectively reduce the risk from media components. For some unit operations, M. orale and M. arginini may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients.

Glass is the favorite material for parenteral packaging because of its physico-chemical properties. Type I borosilicate glass is worldwide use at this scope, but it may have some issues related to breakage, corrosion and delamination that might compromise the drug quality, safety and efficacy. These issues can be mitigated and avoided starting from the appropriate selection of the most suitable raw material at the early stage of the glass container design. In this study, Type I borosilicate glass vials manufactured using two glass tubes having different chemical compositions, were studied and compared in terms of their resistance to corrosion. Testing design was applied with the aim to select the best practice approach comparing different storage simulation conditions: ageing treatment through autoclaving and stability testing (real-time and accelerated). Clear differences were found between the different glass types in terms of hydrolytic and corrosion resistance that highlighted the relation between chemical composition and glass chemical durability. Non-negligible differences were also observed using different storage conditions.

Technologies allowing genetic sequencing of the human microbiome are opening new realms to discovery. The host microbiota substantially impacts immune responses both in immune-mediated inflammatory diseases (IMIDs) and in tumors affecting tissues beyond skin and mucosae. However, a mechanistic link between host microbiota and cancer or IMIDs has not been well established. Here, we propose T helper 17 (T_H17) lymphocytes as the connecting factor between host microbiota and rheumatoid or psoriatic arthritides, multiple sclerosis, breast or ovarian cancer, and multiple myeloma. We theorize that similar mechanisms favor the expansion of gut-borne T_H17 cells and their deployment at the site of inflammation in extraborder IMIDs and tumors, where T_H17 cells are driving forces. Thus, from a pathogenic standpoint, tumors may share mechanistic routes with IMIDs. A review of similarities and divergences in microbiota-T_H17 cell interactions in IMIDs and cancer sheds light on previously ignored pathways in either one of the two groups of pathologies and identifies novel therapeutic avenues.

Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5' cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.

While flagella have been studied extensively as motility organelles, with a focus on internal structures such as the axoneme, more recent research has illuminated the roles of the flagellar surface in a variety of biological processes. Parasitic protists of the order Kinetoplastida, which include trypanosomes and Leishmania species, provide a paradigm for probing the role of flagella in host-microbe interactions and illustrate that this interface between the flagellar surface and the host is of paramount importance. An increasing body of knowledge indicates that the flagellar membrane serves a multitude of functions at this interface: attachment of parasites to tissues within insect vectors, close interactions with intracellular organelles of vertebrate cells, transactions between flagella from different parasites, junctions between the flagella and the parasite cell body, emergence of nanotubes and exosomes from the parasite directed to either host or microbial targets, immune evasion, and sensing of the extracellular milieu. Recent whole-organelle or genome-wide studies have begun to identify protein components of the flagellar surface that must mediate these diverse host-parasite interactions. The increasing corpus of knowledge on kinetoplastid flagella will likely prove illuminating for other flagellated or ciliated pathogens as well.

Acetylation is a conserved modification used to regulate a variety of cellular pathways, such as gene expression, protein synthesis, detoxification, and virulence. Acetyltransferase enzymes transfer an acetyl moiety, usually from acetyl coenzyme A (AcCoA), onto a target substrate, thereby modulating activity or stability. Members of the GCN5-N-acetyltransferase (GNAT) protein superfamily are found in all domains of life and are characterized by a core structural domain architecture. These enzymes can modify primary amines of small molecules or of lysyl residues of proteins. From the initial discovery of antibiotic acetylation, GNATs have been shown to modify a myriad of small-molecule substrates, including tRNAs, polyamines, cell wall components, and other toxins. This review focuses on the literature on small-molecule substrates of GNATs in bacteria, including structural examples, to understand ligand binding and catalysis. Understanding the plethora and versatility of substrates helps frame the role of acetylation within the larger context of bacterial cellular physiology.

The substantial discrepancy between the strong effects of functional foods and various drugs, especially traditional Chinese medicines (TCMs), and the poor bioavailability of these substances remains a perplexing problem. Understanding the gut microbiota, which acts as an effective bioreactor in the human intestinal tract, provides an opportunity for the redefinition of bioavailability. Here, we discuss four different pathways associated with the role of the gut microbiota in the transformation of parent compounds to beneficial or detrimental small molecules, which can enter the body’s circulatory system and be available to target cells, tissues, and organs. We further describe and propose effective strategies for improving bioavailability and alleviating side effects with the help of the gut microbiota. This review also broadens our perspectives for the discovery of new medicinal components.

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 srtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.

IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Diadenosine tetraphosphate (Ap₄A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap₄A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap₄A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap₄A in B. subtilis. YqeK shows a catalytic efficiency toward Ap₄A similar to that of the symmetrically cleaving Ap₄A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.

IMPORTANCE Elevation of Ap₄A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap₄A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap₄A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae. By identifying the player contributing to Ap₄A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

While airway clearance techniques (ACTs) are recommended for individuals with bronchiectasis, many trials have demonstrated inconsistent benefits or failed to reach their primary outcome. This review determined the most common clinical and patient-reported outcome measures used to evaluate the efficacy of ACTs in bronchiectasis. A literature search of five databases using relevant keywords and filtering for studies published in English, up until the end of August 2019, was completed. Studies included randomised controlled trials, using crossover or any other trial design, and abstracts. Studies were included where the control was placebo, no intervention, standard care, usual care or an active comparator. Adults with bronchiectasis not related to cystic fibrosis were included. Extracted data comprised study authors, design, duration, intervention, outcome measures and results. The search identified 27 published studies and one abstract. The most common clinical outcome measures were sputum volume (n=23), lung function (n=17) and pulse oximetry (n=9). The most common patient-reported outcomes were health-related quality of life (measured with St George's Respiratory Questionnaire, n=4), cough-related quality of life (measured with Leicester Cough Questionnaire, n=4) and dyspnoea (measured with Borg/modified Borg scale, n=8). Sputum volume, lung function, dyspnoea and health- and cough-related quality of life appear to be the most common clinical and patient-reported measures of airway clearance treatment efficacy.

Chitotriosidase (CHIT1) is a highly conserved and regulated chitinase secreted by activated macrophages; it is a member of the 18-glycosylase family (GH18). CHIT1 is the most prominent chitinase in humans, can cleave chitin and participates in the body's immune response and is associated with inflammation, infection, tissue damage and remodelling processes. Recently, CHIT1 has been reported to be involved in the molecular pathogenesis of pulmonary fibrosis, bronchial asthma, COPD and pulmonary infections, shedding new light on the role of these proteins in lung pathophysiology. The potential roles of CHIT1 in lung diseases are reviewed in this article.

Physician-led thoracic ultrasound (TUS) has substantially changed how respiratory disorders, and in particular pleural diseases, are managed. The use of TUS as a point-of-care test enables the respiratory physician to quickly and accurately diagnose pleural pathology and ensure safe access to the pleural space during thoracentesis or chest drain insertion. Competence in performing TUS is now an obligatory part of respiratory speciality training programmes in different parts of the world. Pleural physicians with higher levels of competence routinely use TUS during the planning and execution of more sophisticated diagnostic and therapeutic interventions, such as core needle pleural biopsies, image-guided drain insertion and medical thoracoscopy. Current research is gauging the potential of TUS in predicting the outcome of different pleural interventions and how it can aid in tailoring the optimum treatment according to different TUS-based parameters.

A new larger benthic porcelaneous foraminifer of soritid affinity is described as Tarburina zagrosiana n. gen., n. sp. from the late Maastrichtian of the Tarbur Formation, Zagros Zone, SW Iran. It occurs in foraminiferal–dasycladalean wackestones and packstones, in association with Loftusia ssp., dicyclinids/cuneolinids, Neobalkhania bignoti Cherchi & Schroeder, Gyroconulina columellifera Schroeder & Darmoian, Spirolina? farsiana Schlagintweit & Rashidi, Broeckina cf. dufrenoyi (d'Archiac), other benthic foraminifers, and dasycladalean algae. Due to its elongate test and marginal chamber subdivision by aligned vertical partitions, Tarburina n. gen. can be compared with representatives of the Praerhapydionininae. The interio-marginal slit-like foramina/aperture of Tarburina represents an outstanding feature in complex porcelaneous taxa. The monospecific genus Tarburina is considered a Maastrichtian newcomer within the Late Cretaceous Global Community Maturation cycle of larger benthic foraminifera. A biostratigraphic and palaeobiogeographical restriction seems possible, as reported for many other Late Cretaceous larger benthic foraminifera.

Assemblages of upper lower through upper Miocene Discoaster spp. have been quantified from Integrated Ocean Drilling Program (IODP) Site U1338 in the eastern equatorial Pacific Ocean. These assemblages can be grouped into five broad morphological categories: six-rayed with bifurcated ray tips, six-rayed with large central areas, six-rayed with pointed ray tips, five-rayed with bifurcated ray tips and five-rayed with pointed ray tips. Discoaster deflandrei dominates the assemblages prior to 15.8 Ma. The decline in abundance of D. deflandrei close to the early–middle Miocene boundary occurs together with the evolution of the D. variabilis group, including D. signus and D. exilis. Six-rayed discoasters having large central areas become a prominent member of the assemblages for a 400 ka interval in the late middle Miocene. Five- and six-rayed forms having pointed tips become prominent in the early late Miocene and show a strong antiphasing relationship with the D. variabilis group. Discoaster bellus completely dominates the Discoaster assemblages for a 400 ka interval in the middle late Miocene. Abundances of all discoasters, or discoasters at the species level, show only (surprisingly) weak correlations to carbonate contents or oxygen and carbon isotopes of bulk sediment when calculated over the entire sample interval.

ABSTRACTBackground:A home-based record (HBR) is a health document kept by the patient or their caregivers, rather than by the health care facility. HBRs are used in 163 countries, but they have not been implemented universally or consistently. Effective implementation maximizes both health impacts and cost-effectiveness. We sought to examine this research-to-practice gap and delineate the facilitators and barriers to the effective implementation and use of maternal and child health HBRs especially in low- and middle-income countries (LMICs).Methods:Using a framework analysis approach, we created a framework of implementation categories in advance using subject expert inputs. We collected information through 2 streams. First, we screened 69 gray literature documents, of which 18 were included for analysis. Second, we conducted semi-structured interviews with 12 key informants, each of whom had extensive experience with HBR implementation. We abstracted the relevant data from the documents and interviews into an analytic matrix. The matrix was based on the initial framework and adjusted according to emergent categories from the data.Results:We identified 8 contributors to successful HBR implementation. These include establishing high-level support from the government and ensuring clear communication between all ministries and nongovernmental organizations involved. Choice of appropriate contents within the record was noted as important for alignment with the health system and for end user acceptance, as were the design, its physical durability, and timely redesigns. Logistical considerations, such as covering costs sustainably and arranging printing and distribution, could be potential bottlenecks. Finally, end users' engagement with HBRs depended on how the record was initially introduced to them and how its importance was reinforced over time by those in leadership positions.Conclusions:This framework analysis is the first study to take a more comprehensive and broad approach to the HBR implementation process in LMICs. The findings provide guidance for policy makers, donors, and health care practitioners regarding best implementation practice and effective HBR use, as well as where further research is required.

ABSTRACTBackground:A significant number of girls are married as children, which negatively impacts their health, education, and development. Given the sheer numbers of girls at risk of child marriage globally, the challenge to eliminate the practice is daunting. Programs to prevent child marriage are typically small-scale and overlook the costs and scalability of the intervention.Implementation:This study tested and costed different approaches to preventing child marriage in rural Burkina Faso and Tanzania. The approaches tested were community dialogue, provision of school supplies, provision of a livestock asset, a model including all components, and a control arm. A quasi-experimental design was employed with surveys undertaken at baseline and after 2 years of intervention. We examined the prevalence of child marriage and school attendance controlling for background characteristics and stratified by age group. Programmatic costs were collected prospectively.Results:Among those in the community dialogue arm in Burkina Faso, girls aged 15 to 17 years had two-thirds less risk (risk ratio [RR]=0.33; 95% confidence interval [CI]=0.19, 0.60) of being married and girls aged 12 to 14 years had a greater chance of being in school (RR=1.18; 95% CI=1.07,1.29) compared to the control site. In Tanzania, girls aged 12 to 14 years residing in the multicomponent arm had two-thirds less risk of being married (RR=0.33; 95% CI=0.11, 0.99), and girls 15 to 17 in the conditional asset location had half the risk (RR=0.52; 95% CI=0.30, 0.91). All the interventions tested in Tanzania were associated with increased risk of girls 12 to 14 years old being in school, and the educational promotion arm was also associated with a 30% increased risk of girls aged 15 to 17 years attending school (RR=1.3; 95% CI=1.01, 1.67). Costs per beneficiary ranged from US$9 to US$117.Conclusion:The study demonstrates that minimal, low-cost approaches can be effective in delaying child marriage and increasing school attendance. However, community dialogues need to be designed to ensure sufficient quality and intensity of messaging. Program managers should pay attention to the cost, quality, and coverage of interventions, especially considering that child marriage persists in the most hard-to-reach rural areas of many countries.

ABSTRACTIntroduction:We evaluated a 2-way short message service (SMS) communication platform to improve continuation of pre-exposure prophylaxis (PrEP) for HIV prevention among Kenyan women who initiated PrEP within routine maternal child health (MCH) and family planning clinics.Methods:We adapted an existing SMS platform (Mobile WACh [mWACh]) to send PrEP-tailored, theory-based SMS and allow clients to communicate with a remote nurse. Women who did not have HIV and who were initiating PrEP at 2 MCH/family planning clinics in Kisumu County, Kenya, from February to October 2018, were offered enrollment into the mWACh-PrEP program; SMS communication was free. We evaluated acceptability, satisfaction, and implementation metrics. In a pre/postevaluation, we compared PrEP continuation at 1-month postinitiation among women who initiated PrEP in the period before (n=166) versus after mWACh-PrEP implementation, adjusting for baseline differences.Results:Of the 334 women who were screened for enrollment into the mWACh-PrEP program; 193 (58%) were eligible and of those, 190 (98%) accepted enrollment. Reasons for ineligibility (n=141) included no phone access (29%) and shared SIM cards (25%). Median age was 25 years (interquartile range=22–30), and 91% were MCH clients. Compared to women who initiated PrEP in the month before mWACh-PrEP implementation, women who enrolled in mWACh-PrEP were more likely to return for their first PrEP follow-up visit (40% vs. 53%; adjusted risk ratio [aRR]=1.26; 95% confidence interval [CI]= 1.06, 1.50; P=.008) and more likely to continue PrEP (22% vs. 43%; aRR=1.75; 95% CI=1.21, 2.55; P=.003). Among those who returned, 99% reported successful receipt of SMS through the mWACh-PrEP system and 94% reported that mWACh-PrEP helped them understand PrEP better. Concerns about PrEP use, how it works, and side effects accounted for the majority (80%) of issues raised by participants using SMS.Conclusions:Two-way SMS expanded support for PrEP and opportunities for dialogue beyond the clinic and enabled women to ask and receive answers in real time regarding PrEP, which facilitated its continued use.

ABSTRACTBackground:The focused assessment with sonography for HIV-associated tuberculosis (TB) (FASH) ultrasound protocol has been increasingly used to help clinicians diagnose TB. We sought to quantify the diagnostic utility of FASH for TB among individuals with HIV in Malawi.Methods:Between March 2016 and August 2017, 210 adults with HIV who had 2 or more signs and symptoms that were concerning for TB (fever, cough, night sweats, weight loss) were enrolled from a public HIV clinic in Lilongwe, Malawi. The treating clinicians conducted a history, physical exam, FASH protocol, and additional TB evaluation (laboratory diagnostics and chest radiography) on all participants. The clinician made a final treatment decision based on all available information. At the 6-month follow-up visit, we categorized participants based on clinical outcomes and diagnostic tests as having probable/confirmed TB or unlikely TB; association of FASH with probable/confirmed TB was calculated using Fisher's exact tests. The impact of FASH on empiric TB treatment was determined by asking the clinicians prospectively about whether they would start treatment at 2 time points in the baseline visit: (1) after the initial history and physical exam; and (2) after history, physical exam, and FASH protocol.Results:A total of 181 participants underwent final analysis, of whom 56 were categorized as probable/confirmed TB and 125 were categorized as unlikely TB. The FASH protocol was positive in 71% (40/56) of participants with probable/confirmed TB compared to 24% (30/125) of participants with unlikely TB (odds ratio=7.9, 95% confidence interval=3.9,16.1; P<.001). Among those classified as confirmed/probable TB, FASH increased the likelihood of empiric TB treatment before obtaining any other diagnostic studies from 9% (5/56) to 46% (26/56) at the point-of-care. For those classified as unlikely TB, FASH increased the likelihood of empiric treatment from 2% to 4%.Conclusion:In the setting of HIV coinfection in Malawi, FASH can be a helpful tool that augments the clinician's ability to make a timely diagnosis of TB.