Genomic characteristics of prospectively sequenced Mycobacterium tuberculosis from respiratory and non-respiratory sources.

Understanding the differences between Mycobacterium tuberculosis strains isolated from respiratory and non-respiratory sources may inform clinical care and control strategies. We examined demographic and genomic characteristics of all culture-confirmed M. tuberculosis cultures isolated from respiratory and non-respiratory sources in New South Wales, Australia, from January 2017 to December 2021, using logistic regression models. M. tuberculosis strains from 1,831 patients were sequenced; 64.7% were from respiratory, 32.1% from non-respiratory, and 2.2% from both sources. Female patients had more frequent isolation from a non-respiratory source (p = 0.03), and older adults (≧65 years) from a respiratory source (p < 0.0001). Lineage 2 strains were relatively over-represented among respiratory isolates (p = 0.01). Among 39 cases with sequenced isolates from both sources, 43.6% had 1-10 single nucleotide polymorphism differences. The finding that older adults were more likely to have M. tuberculosis isolated from respiratory sources has relevance for TB control given the expected rise of TB among older adults.

Advancing pathogen genomics in resource-limited settings.

Genomic sequencing has emerged as a powerful tool to enhance early pathogen detection and characterization with implications for public health and clinical decision making. Although widely available in developed countries, the application of pathogen genomics among low-resource, high-disease burden settings remains at an early stage. In these contexts, tailored approaches for integrating pathogen genomics within infectious disease control programs will be essential to optimize cost efficiency and public health impact. We propose a framework for embedding pathogen genomics within national surveillance plans across a spectrum of surveillance and laboratory capacities. We adopt a public health approach to genomics and examine its application to high-priority diseases relevant in resource-limited settings. For each grouping, we assess the value proposition for genomics to inform public health and clinical decision-making, alongside its contribution toward research and development of novel diagnostics, therapeutics, and vaccines.

Impact of Whole-Genome Sequencing of Mycobacterium tuberculosis on Treatment Outcomes for MDR-TB/XDR-TB: A Systematic Review.

The emergence and persistence of drug-resistant tuberculosis is a major threat to global public health. Our objective was to assess the applicability of whole-genome sequencing (WGS) to detect genomic markers of drug resistance and explore their association with treatment outcomes for multidrug-resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB). METHODS: Five electronic databases were searched for studies published in English from the year 2000 onward. Two reviewers independently conducted the article screening, relevant data extraction, and quality assessment. The data of the included studies were synthesized with a narrative method and are presented in a tabular format. RESULTS: The database search identified 949 published articles and 8 studies were included. An unfavorable treatment outcome was reported for 26.6% (488/1834) of TB cases, which ranged from 9.7 to 51.3%. Death was reported in 10.5% (194/1834) of total cases. High-level fluoroquinolone resistance (due to gyrA 94AAC and 94GGC mutations) was correlated as the cause of unfavorable treatment outcomes and reported in three studies. Other drug resistance mutations, like kanamycin high-level resistance mutations (rrs 1401G), rpoB Ile491Phe, and ethA mutations, conferring prothionamide resistance were also reported. The secondary findings from this systematic review involved laboratory aspects of WGS, including correlations with phenotypic DST, cost, and turnaround time, or the impact of WGS results on public health actions, such as determining transmission events within outbreaks. CONCLUSIONS: WGS has a significant capacity to provide accurate and comprehensive drug resistance data for MDR/XDR-TB, which can inform personalized drug therapy to optimize treatment outcomes.

A laboratory framework for ongoing optimization of amplification-based genomic surveillance programs.

IMPORTANCE: This study provides a laboratory framework to ensure ongoing relevance and performance of amplification-based whole genome sequencing to strengthen public health surveillance during extended outbreaks or pandemics. The framework integrates regular reviews of the performance of a genomic surveillance system and highlights the importance of ongoing monitoring and the identification and implementation of improvements to whole genome sequencing methods to enhance public health responses to pathogen outbreaks.

Exploring programmatic indicators of tuberculosis control that incorporate routine Mycobacterium tuberculosis sequencing in low incidence settings: a comprehensive (2017-2021) patient cohort analysis.

Background: Routine whole genome sequencing of Mycobacterium tuberculosis has been implemented with increasing frequency. However, its value for tuberculosis (TB) control programs beyond individual case management and enhanced drug resistance detection has not yet been explored. Methods: We analysed routine sequencing data of culture-confirmed TB cases notified between 1st January 2017 and 31st December 2021 in New South Wales (NSW), Australia. Genomic surveillance included evidence of local TB transmission, defined by single nucleotide polymorphism (SNP) clustering over a variable (0-25) SNP threshold, and drug resistance conferring mutations. Findings: M. tuberculosis sequences from 1831 patients were examined, representing 64.8% of all notified TB cases and 96.2% of culture-confirmed cases. Applying a traditional 5-SNP cluster threshold identified 62 transmission clusters with 183 clustered cases; 101/183 (55.2%) had 0 SNP differences. Cluster assessment over a 5-year period, using a 5-SNP threshold, provided a comprehensive overview of likely recent transmission within NSW, Australia, as an indicator of local TB control. Genotypic drug susceptibility testing (DST) was highly concordant with phenotypic DST and provided a 6.8% increase in antimycobacterial resistance detection. Importantly, it detected mutations missed by routine molecular tests. Lineage 2 strains were more likely to be drug resistant (p < 0.0001) and locally transmitted if drug resistant (p < 0.0001). Interpretation: Performing routine prospective WGS in a low incidence country like Australia, provides genomically informed programmatic indicators of local TB control. A rolling 5-year cluster assessment reflects epidemic containment and progress towards 'zero TB transmission'. Genomic DST also provides valuable information for clinical care and drug resistance surveillance. Funding: NHMRC Centre for Research Excellence in Tuberculosis (www.tbcre.org.au) and NSW Health Prevention Research Support Program.

Genomic markers of drug resistance in Mycobacterium tuberculosis populations with minority variants.

Minority variants of Mycobacterium tuberculosis harboring mutations conferring resistance can become dominant populations during tuberculosis (TB) treatment, leading to treatment failure. Our understanding of drug-resistant within-host subpopulations and the frequency of resistance-conferring mutations in minority variants remains limited. M. tuberculosis sequences recovered from liquid cultures of culture-confirmed TB cases notified between January 2017 and December 2021 in New South Wales, Australia were examined. Potential drug resistance-conferring minority variants were identified using LoFreq, and mixed populations of different M. tuberculosis strains (≥100 SNPs apart) were examined using QuantTB. A total of 1831 routinely sequenced M. tuberculosis strains were included in the analysis. Drug resistance-conferring minority variants were detected in 3.5% (65/1831) of sequenced cultures; 84.6% (55/65) had majority strains that were drug susceptible and 15.4% (10/65) had majority strains that were drug resistant. Minority variants with high-confidence drug resistance-conferring mutations were 1.5 times more common when the majority strains were drug resistant. Mixed M. tuberculosis strain populations were documented in 10.0% (183/1831) of specimens. Minority variants with high-confidence drug resistance-conferring mutations were more frequently detected in mixed M. tuberculosis strain populations (2.7%, 5/183) than in single strain populations (0.6%, 10/1648; P = 0.01). Drug-resistant minority variants require monitoring in settings that implement routine M. tuberculosis sequencing. The frequency with which drug-resistant minority variants are detected is likely influenced by pre-culture requirement. Culture-independent sequencing methods should provide a more accurate reflection of drug-resistant subpopulations.

SABRes: in silico detection of drug resistance conferring mutations in subpopulations of SARS-CoV-2 genomes.

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in 25,197 genomes generated over the course of the SARS-CoV-2 pandemic in Australia and identified 299 genomes containing resistance conferring mutations to the five antiviral therapeutics that retain effectiveness against currently circulating strains of SARS-CoV-2 - Sotrovimab, Bebtelovimab, Remdesivir, Nirmatrelvir and Molnupiravir. These genomes accounted for a 1.18% prevalence of resistant isolates discovered by SABRes, including 80 genomes with resistance conferring mutations found in viral subpopulations. Timely recognition of these mutations within subpopulations is critical as these mutations can provide an advantage under selective pressure and presents an important step forward in our ability to monitor SARS-CoV-2 drug resistance.

Implementation of evidence on management of pleural diseases: insights from a territory-wide survey of clinicians in Hong Kong.

BACKGROUND: Major advances in management of common pleural diseases have taken place in the past decade. However, pleural diseases are often managed by physicians of diverse training background and research on implementation of new knowledge is scanty. We aim to evaluate the practice pattern in pleural medicine among physicians in Hong Kong, for identification of possible gaps for clinical service improvement. METHODS: The Hong Kong Thoracic Society undertook a cross-sectional questionnaire survey in 2019, targeting clinicians of various subspecialties in internal medicine and levels of experience (basic and higher trainees, specialists) from twelve regional hospitals of diverse service scopes throughout Hong Kong. Respondents were selected by non-probability quota sampling. The questionnaire tool consisted of 46 questions covering diagnostic and therapeutic aspects of common pleural diseases. The responses were anonymous, and analysed independently using SPSS statistics software. RESULTS: The survey collected 129 responses, 47(36%) were from clinicians specialized in respiratory medicine. Majority of the respondents (98%) managed pleural diseases, including performing pleural procedures in their practice. Fifty-five percent of all the respondents had not received any formal training in transthoracic ultrasonography. A significant proportion of clinicians were unaware of pleuroscopy for investigation of exudative pleural effusion, indwelling pleural catheter for recurrent malignant pleural effusion, and combined intra-pleural Alteplase plus DNase for treatment of pleural infection (30%, 15% and 70% of non-respiratory clinicians respectively). Significant heterogeneity was found in the management of pleural infection, malignant pleural effusion and pneumothorax among respiratory versus non-respiratory clinicians. Contributing factors to the observed heterogeneity included lack of awareness or training, limited accessibility of drugs, devices, or dedicated service support. CONCLUSION: Significant heterogeneity in management of pleural diseases was observed among medical clinicians in Hong Kong. Continuous medical education and training provision for both specialists and non-specialists has to be strengthened to enhance the implementation of advances, improve quality and equity of healthcare provision in pleural medicine.

Improved Neutralisation of the SARS-CoV-2 Omicron Variant following a Booster Dose of Pfizer-BioNTech (BNT162b2) COVID-19 Vaccine.

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired over 30 mutations in the spike protein (with 15 in the receptor-binding domain), raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three, and six months post-two doses of Pfizer-BioNTech BNT162b2 had a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres were boosted. Despite this increase, neutralising antibody titres were reduced fourfold for Omicron compared to lineage A.2.2 SARS-CoV-2.

Transition program: Initial implementation with adults with neuromuscular conditions.

PURPOSE: To identify current medical and psychosocial needs and to examine the effectiveness of healthcare transition program for adult-aged patients with neuromuscular conditions transitioning from pediatric to adult services. DESIGN AND METHODS: At Neuromuscular Transition Clinic visit, 46 patients were evaluated and referred to adult-based providers, if did not currently have one, from an acquired list of interested clinicians. At mean follow-up of 22 months, 42 were interviewed by phone regarding referrals for Core Services (primary care, physiatry, dental care and gynecology), Medical Specialties and Rehabilitation Services. Mean age was 30 years with 62% males. Majority (74%) had cerebral palsy. Sixty percent were non-ambulatory. RESULTS: As per protocol, all were indicated to need Core Services. Eighty-three percent already had adult primary care provider. Most referrals were given for physiatry (62%), vocational training (100%), and occupational therapy (88%). At follow-up, visits were completed most frequently with adult provider for primary care (100%), occupational therapy (78%), and neurology (75%). Referred provider was seen 100% for physiatry, neurology, physical therapy, occupational therapy and vocational training. Of the total 125 referrals given across all services, 73 (58%) participants had completed a visit with an adult provider. CONCLUSIONS: As only about 60% transitioned to adult-based services after referral, healthcare transition remains challenging and requires tailoring of services according to patients' needs, staff and willing-and-available adult-based providers. PRACTICE IMPLICATIONS: Transitioning healthcare of patients with neuromuscular conditions from pediatric- to adult-based providers remains challenging. This clinical specialty requires tailoring of services based on patient's needs, and availability of adult-based providers and resources.

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture.

Background: Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model. Methods: Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33). Results: Intrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts. Discussion and Conclusion: The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

Understanding moral empathy: A verbatim-theatre supported phenomenological exploration of the empathy imperative.

OBJECTIVES: Several studies have measured a decline in empathy during medical training, speculating that factors within the formal, informal and hidden curricula are responsible for this phenomenon. Although the medical education literature describes the moral domain of empathy as most fundamental to the empathic response, most research into the decline has examined the cognitive, affective and behavioural domains. This study distinguishes itself by focusing on how moral empathy is affected through training. METHODS: Ten medical residents from core education specialties at McMaster University participated in lightly structured interviews concerning their training experiences. Interview transcripts were analysed by way of a descriptive phenomenological approach. Analyses afforded descriptions of the way medical training influences moral empathy. These descriptions were then used to generate a verbatim theatre play that was performed for an audience of residents, educators, learners, researchers and scholars. Following the play, audience participants completed a survey to member-check the descriptions and to glean other reflective experiences in resident training that impact moral empathy. The survey results informed revisions to the codebook that was subsequently used to re-analyse the interview transcripts. This resulted in a final, refined version of the influence of training on learner moral empathy. RESULTS: The findings suggest that a resident's sense of moral empathy relies upon the notion of an innate capacity for empathy, and is influenced by their clinical and classroom education, and specific experiences with patients during training. Importantly, these factors are rarely experienced as having a direct deleterious impact on residents' moral empathy but rather are experienced as challenges to their ability to act on their moral empathy. CONCLUSIONS: The study promotes reflection of what it means to experience empathy in the moral domain. The description offers a new perspective from which to view empathic declines that have been previously reported, while also highlighting a moral-behavioural tension that has implications for competency-based assessment and the way empathy is conceptualised in medical education.

Documenting elimination of co-circulating COVID-19 clusters using genomics in New South Wales, Australia.

OBJECTIVE: To adapt 'fishplots' to describe real-time evolution of SARS-CoV-2 genomic clusters. RESULTS: This novel analysis adapted the fishplot to depict the size and duration of circulating genomic clusters over time in New South Wales, Australia. It illuminated the effectiveness of interventions on the emergence, spread and eventual elimination of clusters and distilled genomic data into clear information to inform public health action.

Value of routine whole genome sequencing for Mycobacterium tuberculosis drug resistance detection.

Routine whole genome sequencing (WGS) of pathogens is becoming more feasible as sequencing costs decrease and access to benchtop sequencing equipment and bioinformatics pipelines increases. This study examined the added value gained from implementing routine WGS of all Mycobacterium tuberculosis isolates in New South Wales, Australia. Drug resistance markers inferred from WGS data were compared to commercial genotypic drug susceptibility testing (DST) assays and conventional phenotypic DST in all isolates sequenced between 2016 and 2019. Of the 1107 clinical M. tuberculosis isolates sequenced, 29 (2.6%) were multi-drug resistant (MDR); most belonged to Beijing (336; 30.4%) or East-African Indian (332; 30%) lineages. Compared with conventional phenotypic DST, WGS identified an additional 1% of isolates which were likely drug resistant, explained by mutations previously associated with treatment failure and mixed bacterial populations. However, WGS provided a 20% increase in drug resistance detection in comparison with commercial genotypic assays by identifying mutations outside of the classic resistance determining regions in rpoB, inhA, katG, pncA and embB genes. Gains in drug resistance detection were significant (p = 0.0137, paired t-test), but varied substantially for different phylogenetic lineages. In low incidence settings, routine WGS of M. tuberculosis provides better guidance for person-centered management of drug resistant tuberculosis than commercial genotypic assays.

Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome sequencing and agent-based modeling.

In January 2020, a novel betacoronavirus (family Coronaviridae), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the etiological agent of a cluster of pneumonia cases occurring in Wuhan City, Hubei Province, China1,2. The disease arising from SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), subsequently spread rapidly causing a worldwide pandemic. Here we examine the added value of near real-time genome sequencing of SARS-CoV-2 in a subpopulation of infected patients during the first 10 weeks of COVID-19 containment in Australia and compare findings from genomic surveillance with predictions of a computational agent-based model (ABM). Using the Australian census data, the ABM generates over 24 million software agents representing the population of Australia, each with demographic attributes of an anonymous individual. It then simulates transmission of the disease over time, spreading from specific infection sources, using contact rates of individuals within different social contexts. We report that the prospective sequencing of SARS-CoV-2 clarified the probable source of infection in cases where epidemiological links could not be determined, significantly decreased the proportion of COVID-19 cases with contentious links, documented genomically similar cases associated with concurrent transmission in several institutions and identified previously unsuspected links. Only a quarter of sequenced cases appeared to be locally acquired and were concordant with predictions from the ABM. These high-resolution genomic data are crucial to track cases with locally acquired COVID-19 and for timely recognition of independent importations once border restrictions are lifted and trade and travel resume.

Virtual Reality for Pediatric Needle Procedural Pain: Two Randomized Clinical Trials.

OBJECTIVE: To assess the efficacy and safety of a virtual reality distraction for needle pain in 2 common hospital settings: the emergency department (ED) and outpatient pathology (ie, outpatient laboratory). The control was standard of care (SOC) practice. STUDY DESIGN: In 2 clinical trials, we randomized children aged 4-11 years undergoing venous needle procedures to virtual reality or SOC at 2 tertiary Australian hospitals. In the first study, we enrolled children in the ED requiring intravenous cannulation or venipuncture. In the second, we enrolled children in outpatient pathology requiring venipuncture. In the ED, 64 children were assigned to virtual reality and 59 to SOC. In pathology, 63 children were assigned to virtual reality and 68 to SOC; 2 children withdrew assent in the SOC arm, leaving 66. The primary endpoint was change from baseline pain between virtual reality and SOC on child-rated Faces Pain Scale-Revised. RESULTS: In the ED, there was no change in pain from baseline with SOC, whereas virtual reality produced a significant reduction in pain (between-group difference, -1.78; 95% CI, -3.24 to -0.317; P = .018). In pathology, both groups experienced an increase in pain from baseline, but this was significantly less in the virtual reality group (between-group difference, -1.39; 95% CI, -2.68 to -0.11; P = .034). Across both studies, 10 participants experienced minor adverse events, equally distributed between virtual reality/SOC; none required pharmacotherapy. CONCLUSIONS: In children aged 4-11 years of age undergoing intravenous cannulation or venipuncture, virtual reality was efficacious in decreasing pain and was safe. TRIAL REGISTRATION: Australia and New Zealand Clinical Trial Registry: ACTRN12617000285358p.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1ß instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription.

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple 'one modification, one effect' system.

Immuno-targeting the multifunctional CD38 using nanobody.

CD38, as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein, we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved, identifying three separate epitopes on the carboxyl domain. Chromobodies, engineered by tagging the nanobody with fluorescence proteins, provide fast, simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin, PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines, with half maximal effective concentration reaching as low as 10(-11) molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma.