High Clinical Impact of Broad-Range Fungal PCR in Suspected Fungal Sinusitis.

Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.

The Vaginal Microbiome of Transgender Men.

BACKGROUND: Hormonal changes influence the composition of vaginal flora, which is directly related to the health of an individual. Transgender men prescribed testosterone experience a vaginal hormone composition that differs from cisgender women. To the author's knowledge, there are no clinical studies evaluating the influence that testosterone administration has on the vaginal microbiome. METHODS: Vaginal swabs were self-collected by a cohort of self-identified healthy transgender men prescribed testosterone for at least 1 year (n = 28) and from cisgender women who were used as the comparator (n = 8). Participants completed a questionnaire to indicate the mode and dose of testosterone administration, sexual history, and vaginal health. Serum was collected for hormone analysis. Bacterial community profiles were assessed with broad-range PCR primers targeting the V3-V4 hypervariable region of the 16S bacterial rRNA, next-generation sequencing, and analysis by phylogenetic placement. RESULTS: Compared to cisgender women, the vaginal floras of transgender men were less likely to have Lactobacillus as their primary genus. Intravaginal estrogen administration was positively associated with the presence of Lactobacillus in transgender men (P = 0.045). Transgender men had a significantly increased relative abundance of >30 species and a significantly higher α diversity (P = 0.0003). The presence of Lactobacillus was significantly associated with a lower α diversity index (P = 0.017). CONCLUSIONS: The vaginal microbiome of transgender men who were assigned a female sex at birth and use testosterone may differ from that of cisgender women. Intravaginal estrogen administration may reduce these differences by promoting colonization with Lactobacillus species and decreasing α diversity.

Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion.

Several large-scale studies of human genetic variation have provided insights into processes such as recombination that have shaped human diversity. However, regions such as low-copy repeats (LCRs) have proven difficult to characterize, hindering efforts to understand the processes operating in these regions. We present a detailed study of genetic variation and underlying recombination processes in two copies of an LCR (NF1REPa and NF1REPc) on chromosome 17 involved in the generation of NF1 microdeletions and in a third copy (REP19) on chromosome 19 from which the others originated over 6.7 million years ago. We find evidence for shared hotspots of recombination among the LCRs. REP19 seems to contain hotspots in the same place as the nonallelic recombination hotspots in NF1REPa and NF1REPc. This apparent conservation of patterns of recombination hotspots in moderately diverged paralogous regions contrasts with recent evidence that these patterns are not conserved in less-diverged orthologous regions of chimpanzees.

Coinfection of Fusobacterium nucleatum and Actinomyces israelii in mastoiditis diagnosed by next-generation DNA sequencing.

Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii, resolving these diagnostic discrepancies.

Using body cameras to quantify the duration of a Code Stroke and identify workflow issues: a continuous observation workflow time study.

OBJECTIVE: 'Code Stroke' (Code) is used in health services to streamline hyperacute assessment and treatment delivery for patients with ischaemic stroke. However, there are few studies that detail the time spent on individual components performed during a Code. We sought to quantify the time taken for each process during a Code and investigate associations with modifiable and non-modifiable factors. DESIGN: Continuous observation workflow time study. SETTING AND PARTICIPANTS: Recordings of 100 Codes were performed at a high-volume primary stroke centre in Melbourne, Australia, between January and June 2020 using a body camera worn by a member of the stroke team. MAIN OUTCOME MEASURES: The main measures included the overall duration of Codes and the individual processes within the Code workflow. Associations between variables of interest and process times were explored using linear regression models. RESULTS: 100 Codes were captured, representing 19.2% of all Codes over the 6 months. The median duration of a complete Code was 54.2 min (IQR 39.1-74.7). Administrative work performed after treatment is completed (median 21.0 min (IQR 9.8-31.4)); multimodal CT imaging (median 13.0 min (IQR 11.5-15.7)), and time between decision and thrombolysis administration (median 8.1 min (IQR 6.1-10.8)) were the longest components of a Code. Tenecteplase was able to be prepared faster than alteplase (median 1.8 vs 4.9 min, p=0.02). The presence of a second junior doctor was associated with shorter administrative work time (median 10.3 vs 25.1 min, p<0.01). No specific modifiable factors were found to be associated with shorter overall Code duration. CONCLUSIONS: Codes are time intensive. Time spent on decision-making was a relatively small component of the overall Code duration. Data from body cameras can provide granular data on all aspects of Code workflow to inform potential areas for improvement at individual centres.

Molecular diagnosis of subcutaneous Pythium insidiosum infection by use of PCR screening and DNA sequencing.

Pythium insidiosum is an emerging human pathogen classified among brown algae and diatoms that can cause significant morbidity and mortality in otherwise healthy individuals. Here we describe a pediatric patient with pythiosis acquired in the southern United States, diagnosed by molecular screening and DNA sequencing of internal transcribed spacer region 1.

Practical utility of the ACT-FAST triage algorithm from a primary stroke centre perspective.

BACKGROUND: Rapid reperfusion in ischaemic stroke with emergent large vessel occlusion (ELVO) reduces morbidity and mortality. Limited distribution of endovascular clot retrieval (ECR) capable comprehensive stroke centres (CSCs) necessitates development of pre-hospital models of care to provide equitable and economical access to reperfusion therapy. We examine the time metrics of the traditional secondary transfer strategy in comparison to the direct bypass strategy and the potential utility of the ACT-FAST prehospital triage algorithm on a large volume Melbourne primary stroke centre (PSC). METHOD: Retrospective analysis of consecutive patients presenting to a PSC from 1 January 2020 to 31 December 2020. Clinical records were interrogated for ACT-FAST positive patients. Time metrics were established using Google Maps traffic modelling and local/published door-to-needle, door-in-door out and door-to-groin data. RESULTS: 88 patients during the study period were ACT-FAST positive. Of these, 49/88 (56%) cases had ELVO ischaemic strokes, 24/88 (27%) cases had intracranial haemorrhages and the remaining 15/88 (17%) had non ELVO ischaemic strokes or mimics (seizure, complex migraine, etc). 28/88 (32%) cases met indication for and were subsequently transferred to a CSC for consideration of ECR. The modelled median scene to groin time for the direct bypass strategy is 94 min whereas the median scene to groin time for the secondary transfer strategy is 109 min, giving a difference of 15 min. CONCLUSION: Time savings to groin puncture for the direct bypass strategy is substantially less than previous estimates and suggests that the secondary transfer strategy continues to be a viable pathway for a high efficiency PSC.

Examining Mid-Upper Arm Circumference Malnutrition z-Score Thresholds.

BACKGROUND: Anthropometric z-scores used commonly for diagnosis and determining degree of malnutrition, specifically body mass index (BMIz), weight-for-length (WLz), and mid-upper arm circumference (MUACz), are not wholly concordant, yet the proposed thresholds for classification are identical. This study was designed to critically examine MUACz thresholds and their ability to correctly classify nutrition status. METHODS: This was a 2-year, prospective single-center study of children ≤18 years seen by registered dietitians within a large pediatric institution. The sensitivity, specificity, and predictive performance of the malnutrition classification thresholds were estimated against clinician-based classification. RESULTS: Sixty-one dietitians enrolled 10,401 patients with distributions of z-scores for weight (-0.5 ± 1.9), length (-0.8 ± 1.6), BMI or WL (-0.1 ± 1.8), and MUAC (-0.4 ± 1.5), suggesting participants were smaller and shorter than the reference U.S. POPULATION: Distributions of MUACz were broad and overlapped between nutrition classification groups, an observation that extended to BMIz and WLz as well. Consequently, existing thresholds do not accurately classify 100% of children. Misclassification rates increase, with increasing severity ranging from 8% in children with no malnutrition to 71% in children with severe malnutrition. Algorithm- and manually-based refinement of thresholds result in mixed improvements and can be explored by the reader with the associated supplement. CONCLUSION: The sensitivity of proposed MUACz thresholds systematically decreases with increasing severity of malnutrition and will require optimization if we aim to limit the number of children at risk of misclassification. Indicators for overnutrition remain to be addressed but are explored herein.

User-Informed Medical Device Development: A Case Study for Pediatric Malnutrition Assessment.

Background. Mid-upper arm circumference (MUAC) has been extensively used to classify pediatric malnutrition. Recently, MUAC z score was recommended; however, the logistics of implementation were not addressed. This study examines the usability of a device that provides MUAC and corresponding z score range in a single device. Methods. This was a single-center study of nutrition services providers. The device was applied to children aged 2 months to 18 years admitted as inpatients or seen as outpatients. Surveys incorporated benchmarking questions, assessed the ease with which respondents could perform 6 critical tasks, and provided an open-ended question to elicit feedback. Users were surveyed monthly until saturation was reached. Survey data were analyzed in aggregate and cross-tabulated by the respondents' experience with the device. Thematic analysis of the open-ended responses followed a structured approach. Results. Sixty device users responded to the survey 280 times. Respondents were female (100%) with a mean age of 45.2 ± 13.2 years and 9.6 ± 8.0 years in practice. Increasing device use was accompanied by significantly shortened measuring times (P < .001) and shifts in ease of performance for 5 of 6 critical tasks (P < .05). Open-ended response themes related largely to design and materials. These were used to iteratively refine the device. Conclusions. The active engagement of end users in the real-world testing of our nutritional assessment device allowed us to refine the innovation with special attention paid to the needs of dietitians. The result is a device the majority of dietitians found easy to use, efficient, convenient, and preferable to alternative measurement options.

Evaluating Mid-Upper Arm Circumference Z-Score as a Determinant of Nutrition Status.

BACKGROUND: Mid-upper arm circumference (MUAC) z-score, has recently been listed as an independent indicator for pediatric malnutrition. This investigation examined the relationship between MUAC z-score and the z-scores for conventional indicators (ie, weight-for-length and body mass index) to expand the available evidence for nutrition classification z-score threshold ranges in U.S. practice settings. METHODS: This was a single-center study of children through 18 years of age seen between October 2015 and September 2016. Height and weight were obtained on intake. MUAC was measured at midpoint of the humerus, between the acromion and olecranon. Age-specific and gender-specific z-score values were calculated using published λ, µ, and σ values derived from Centers for Disease Control and Prevention reference data. Nutrition status was determined from biochemical data; prior history; anthropometrics; weight gain velocity; weight loss, if present; and nutrient intake. RESULTS: 5,004 children (7.5 ± 5.7 years, 53% boys) were evaluated. As expected, MUAC z-scores were significantly correlated with body mass index (r = 0.789, P < .01) and weight-for-length (r = 0.638, P < .01) z-scores. There was a large degree of overlap in z-scores for all indicators between nutrition status groups; however, MUAC z-scores spanned a narrower range of values such that mean MUAC z-scores are lower in children classified as overweight/obese and higher in children who were severely malnourished than the corresponding body mass index or weight-for-length z-scores. CONCLUSION: These data are the first to suggest that the z-score ranges used to define various stages of malnutrition may not be the same for all indicators.

Rhodococcus fascians infection after haematopoietic cell transplantation: not just a plant pathogen?

INTRODUCTION: Rhodococcus spp. have been implicated in a variety of infections in immunocompromised and immunocompetent hosts. Rhodococcus equi is responsible for the majority of reported cases, but Rhodococcus erythropolis, Rhodococcusgordoniae and Rhodococcusruber infections have been described. There are no prior reports of human infection with Rhodococcus fascians. CASE PRESENTATION: We describe the unexpected finding of R. fascians in liver lesions incidentally noted at autopsy in an immunosuppressed patient status after bone-marrow transplant for acute lymphoblastic leukaemia who died of unrelated causes (septic shock due to Clostridium difficile colitis). At autopsy, an otherwise unremarkable liver contained several dozen well-demarcated sclerotic-appearing lesions measuring 0.1-0.3 cm in size. The absence of other bacterial or fungal DNA in the setting of histologically visible organisms argues against its presence as a contaminant and raises the consideration that R. fascians represents a human pathogen for the immunocompromised. CONCLUSION: Whether it represents the sole infectious agent responsible for the miliary lesions or a partially treated co-infection is impossible to determine, but our finding continues to reinforce the importance of molecular techniques in associating organisms with sites of infection and optimizing treatment of infectious diseases.

Incidental identification of Strongyloides stercoralis infection by broad-range 28S rDNA gene sequencing in a patient with a hematolymphoid malignancy.

Strongyloides stercoralis is an important human parasite, especially in rural areas and developing countries. Infected immunosuppressed patients are at risk for hyperinfection, with severe clinical consequences. Here we describe the incidental detection and diagnosis of an unexpected S. stercoralis infection by methods designed to detect fungal 28S ribosomal DNA.

The human cystatin M/E gene (CST6): exclusion candidate gene for harlequin ichthyosis.

Cystatin M/E is a recently discovered cysteine proteinase inhibitor whose expression is largely confined to cutaneous epithelia. In human skin it is expressed in sweat glands, hair follicles, and stratum granulosum of the epidermis where it presumably acts as a substrate for transglutaminase. Very recently we reported that a null mutation in the mouse cystatin M/E gene (Cst6) causes the murine ichq phenotype, which is characterized by abnormalities in cornification and desquamation, demonstrating an essential role for cystatin M/E in the final stages of epidermal differentiation. We here obtained the complete sequence of the human cystatin M/E gene (CST6), which provides a tool to investigate CST6 as a candidate gene in skin diseases characterized by abnormal cornification. The involvement of CST6 in harlequin ichthyosis in humans was evaluated by sequencing the entire coding region and intron-exon boundaries for mutations in 11 sporadic harlequin ichthyosis patients. No CST6 mutations were detected in this group, which comprised type 1 and type 2 harlequin ichthyosis patients. Disturbed transcription/translation due to mutations in regulatory and noncoding regions of cystatin M/E was unlikely because cystatin M/E protein expression was observed in all patients examined, as assessed by immunohistochemistry. Although our results indicate that CST6 is not a major gene contributing to type 1 and 2 harlequin ichthyosis, these data may facilitate further analysis of the role of cystatin M/E in normal human skin and other genetic disorders of cornification.

Diagnosis of five spinocerebellar ataxia disorders by multiplex amplification and capillary electrophoresis.

The autosomal-dominant spinocerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders with variable expression and phenotypic overlap. An accurate diagnosis relies on detection of a mutation in a specific causative gene, which is typically an abnormal number of CAG trinucleotide repeats. To streamline testing in a clinical setting, we converted our current panel of tests for the spinocerebellar ataxias (SCA) types SCA1, SCA2, SCA3, SCA6, and SCA7 from five independent amplification reactions analyzed by polyacrylamide gel electrophoresis (PAGE) to a single multiplex amplification reaction analyzed by capillary electrophoresis (CE). Multiplex amplification was facilitated by the use of chimeric primers; different lengths and fluorochromes distinguished the amplicons. During CE with commercially available molecular weight standards, the SCA amplicons migrated faster than predicted, thereby underestimating their length compared to that determined previously by PAGE. This was observed to varying degrees for each of the five loci, with the greatest size differential occurring in amplicons with greater (CAG)(n). To determine accurate amplicon length, and therefore an accurate number of CAG repeats, a size correction formula was calculated for each locus. This multiplex semi-automated assay has been reliable during 1 year of use in a clinical setting during which 57 samples were tested and five positive samples were detected.

Payment for obesity services: examples and recommendations for stage 3 comprehensive multidisciplinary intervention programs for children and adolescents.

OBJECTIVE: The Reimbursement and Payment Subcommittee of the National Association of Children's Hospitals and Related Institutions FOCUS on a Fitter Future group sought to guide medical providers, patients, and payers to better serve obese children and adolescents to enable optimum health. Recommendations are provided for the essential components of a stage 3 comprehensive multidisciplinary intervention program as defined by the 2007 Expert Committee recommendations. In addition, suggestions are offered for a stepwise approach to implement these recommendations. METHODS: In 2009, key informant interviews were conducted with 15 children's hospitals participating in FOCUS on a Fitter Future and 1 nonparticipating hospital. Interview transcripts identified 5 financially sustainable stage 3 programs, each funded differently. RESULTS: The stage 3 programs interviewed ranged from being nascent to 21 years old (27%, <2 years; 47%, 2-6 years; 27%, >6 years). All of them had multidisciplinary teams that delivered services through 1 of 3 institutional structures: 60% freestanding; 7% specialty; and 33% hospital within a hospital. One-third of them had 1 to 2 funding sources, and 67% had ≥ 3 sources. CONCLUSIONS: The stage 3 programs in this review shared some common strategies for achieving financial stability. All of them followed key strategies of the chronic care model, the details of which led to the following recommendation: stage 3 programs should include a health care team with a medical provider, registered dietitian, physical activity specialist, mental health specialist, and coordinator who, as a team, provide service to overweight and obese children at no less than moderate intensity (26-75 hours).

Primary hepatic Mycobacterium tuberculosis complex infection with terminal dissemination in a pig-tailed macaque (Macaca nemestrina).

An adult, female, pig-tailed macaque (Macaca nemestrina) of Indonesian origin presented with profound weight loss, anemia (PCV, 29%; normal, 36% to 45%), hypoalbuminemia (1.0 g/dL; normal, 3.5 to 5.2 g/dL), elevated alkaline phosphatase (1990 U/L; normal, 26 to 98 U/L), and an elevated erythrocyte sedimentation rate (75 mm/h; normal, less than 20 mm/h). Abdominal ultrasonography demonstrated an enlarged liver with hyperechoic areas. Euthanasia was performed. Grossly, the liver had multifocal, effacing, white masses throughout and was enlarged with rounded edges. There were 2, small nodules in the right lung lobes. Histologically, the hepatic masses were densely fibrous-encapsulated granulomas with vast central necrosis. The lung nodules also were maturing granulomas, and one kidney and one atrium had small, early granulomas. Fite acid-fast stains of liver and lung revealed very few acid-fast bacilli. PCR analysis of paraffin-embedded liver identified Mycobacterium tuberculosis complex. Culture of the liver was negative twice. This macaque had 16 negative intradermal tuberculin skin tests over the course of 6 y. We hypothesize that the animal arrived with a latent hepatic or enteric infection that later recrudesced and disseminated. Primary hepatic mycobacteriosis is not a typical presentation of tuberculosis in macaques. Negative tuberculin skin tests can be seen with latent infections and extrapulmonary tuberculosis such as Pott disease. This case underscores the problems associated with current surveillance procedures and the risks associated with latent mycobacterial infections in macaques.

Plexiform-like neurofibromas develop in the mouse by intraneural xenograft of an NF1 tumor-derived Schwann cell line.

Plexiform neurofibromas are peripheral nerve sheath tumors that arise frequently in neurofibromatosis type 1 (NF1) and have a risk of malignant progression. Past efforts to establish xenograft models for neurofibroma involved the implantation of tumor fragments or heterogeneous primary cultures, which rarely achieved significant tumor growth. We report a practical and reproducible animal model of plexiform-like neurofibroma by xenograft of an immortal human NF1 tumor-derived Schwann cell line into the peripheral nerve of scid mice. The S100 and p75 positive sNF94.3 cell line was shown to possess a normal karyotype and have apparent full-length neurofibromin by Western blot. These cells were shown to have a constitutional NF1 microdeletion and elevated Ras-GTP activity, however, suggesting loss of normal neurofibromin function. Localized intraneural injection of the cell line sNF94.3 produced consistent and slow growing tumors that infiltrated and disrupted the host nerve. The xenograft tumors resembled plexiform neurofibromas with a low rate of proliferation, abundant extracellular matrix (hypocellularity), basal laminae, high vascularity, and mast cell infiltration. The histologic features of the developed tumors were particularly consistent with those of human plexiform neurofibroma as well. Intraneural xenograft of sNF94.3 cells enables the precise initiation of intraneural, plexiform-like tumors and provides a highly reproducible model for the study of plexiform neurofibroma tumorigenesis. This model facilitates testing of potential therapeutic interventions, including angiogenesis inhibitors, in a relevant cellular environment.

Interstitial uniparental isodisomy at clustered breakpoint intervals is a frequent mechanism of NF1 inactivation in myeloid malignancies.

To identify the mechanism of loss of heterozygosity (LOH) and potential modifier gene(s), we investigated the molecular basis of somatic NF1 inactivation in myeloid malignancies from 10 children with neurofibromatosis type 1. Loci across a minimal 50-Mb region of primarily the long arm of chromosome 17 showed LOH in 8 cases, whereas a less than 9-Mb region of loci flanking NF1 had LOH in the remaining 2 cases. Two complementary techniques, quantitative polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), were used to determine whether the copy number at loci that showed LOH was 1 or 2 (ie, deleted or isodisomic). The 2 cases with LOH limited to less than 9 Mb were intrachromosomal deletions. Among the 8 leukemias with 50-Mb LOH segments, 4 had partial uniparental isodisomy and 4 had interstitial uniparental isodisomy. These isodisomic cases showed clustering of the centromeric and telomeric LOH breakpoints. This suggests that the cases with interstitial uniparental isodisomy arose in a leukemia-initiating cell by double-homologous recombination events at intervals of preferred mitotic recombination. Homozygous inactivation of NF1 favored outgrowth of the leukemia-initiating cell. Our studies demonstrate that LOH analyses of loci distributed along the chromosomal length along with copy-number analysis can reveal novel mechanisms of LOH that may potentially identify regions harboring "cryptic" tumor suppressor or modifier genes whose inactivation contributes to tumorigenesis.

ABCA12 is the major harlequin ichthyosis gene.

Harlequin ichthyosis (HI) is the most severe form of autosomal-recessive, congenital ichthyosis. Affected infants have markedly impaired barrier function and are more susceptible to infection. Abnormalities in the localization of epidermal lipids as well as abnormal lamellar granule formation are features of HI skin. Previously, we and others have shown that mutations in the ABCA12 gene encoding an adenosine triphosphate-binding cassette (ABC) transporter underlie the skin disease HI. In this study, we have sequenced the ABCA12 gene in an additional 14 patients and show that all contain mutations, with the majority being either nonsense substitution or frameshift mutations. Eleven HI patients had bi-allelic ABCA12 mutations, whereas in the remaining three HI patients in this study, ABCA12 mutations were detected on only one allele by sequencing. In addition, the one patient from the previous study where no sequence mutations were detected was screened for heterozygous deletions. A combination of oligonucleotide arrays, multiplex PCR analysis and single-nucleotide polymorphism genotyping revealed a heterozygous intragenic deletion in exon 8. These mutation data establish ABCA12 as the major HI gene.

Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor.

The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII-ORF4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium-dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII-ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway-specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.