Standards for clinical trials for treating TB.

BACKGROUND: The value, speed of completion and robustness of the evidence generated by TB treatment trials could be improved by implementing standards for best practice.METHODS: A global panel of experts participated in a Delphi process, using a 7-point Likert scale to score and revise draft standards until consensus was reached.RESULTS: Eleven standards were defined: Standard 1, high quality data on TB regimens are essential to inform clinical and programmatic management; Standard 2, the research questions addressed by TB trials should be relevant to affected communities, who should be included in all trial stages; Standard 3, trials should make every effort to be as inclusive as possible; Standard 4, the most efficient trial designs should be considered to improve the evidence base as quickly and cost effectively as possible, without compromising quality; Standard 5, trial governance should be in line with accepted good clinical practice; Standard 6, trials should investigate and report strategies that promote optimal engagement in care; Standard 7, where possible, TB trials should include pharmacokinetic and pharmacodynamic components; Standard 8, outcomes should include frequency of disease recurrence and post-treatment sequelae; Standard 9, TB trials should aim to harmonise key outcomes and data structures across studies; Standard 10, TB trials should include biobanking; Standard 11, treatment trials should invest in capacity strengthening of local trial and TB programme staff.CONCLUSION: These standards should improve the efficiency and effectiveness of evidence generation, as well as the translation of research into policy and practice.

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles.

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.

Association Between Minimum Inhibitory Concentration, Beta-lactamase Genes and Mortality for Patients Treated With Piperacillin/Tazobactam or Meropenem From the MERINO Study.

INTRODUCTION: This study aims to assess the association of piperacillin/tazobactam and meropenem minimum inhibitory concentration (MIC) and beta-lactam resistance genes with mortality in the MERINO trial. METHODS: Blood culture isolates from enrolled patients were tested by broth microdilution and whole genome sequencing at a central laboratory. Multivariate logistic regression was performed to account for confounders. Absolute risk increase for 30-day mortality between treatment groups was calculated for the primary analysis (PA) and the microbiologic assessable (MA) populations. RESULTS: In total, 320 isolates from 379 enrolled patients were available with susceptibility to piperacillin/tazobactam 94% and meropenem 100%. The piperacillin/tazobactam nonsusceptible breakpoint (MIC >16 mg/L) best predicted 30-day mortality after accounting for confounders (odds ratio 14.9, 95% confidence interval [CI] 2.8-87.2). The absolute risk increase for 30-day mortality for patients treated with piperacillin/tazobactam compared with meropenem was 9% (95% CI 3%-15%) and 8% (95% CI 2%-15%) for the original PA population and the post hoc MA populations, which reduced to 5% (95% CI -1% to 10%) after excluding strains with piperacillin/tazobactam MIC values >16 mg/L. Isolates coharboring extended spectrum ß-lactamase (ESBL) and OXA-1 genes were associated with elevated piperacillin/tazobactam MICs and the highest risk increase in 30-day mortality of 14% (95% CI 2%-28%). CONCLUSIONS: After excluding nonsusceptible strains, the 30-day mortality difference from the MERINO trial was less pronounced for piperacillin/tazobactam. Poor reliability in susceptibility testing performance for piperacillin/tazobactam and the high prevalence of OXA coharboring ESBLs suggests that meropenem remains the preferred choice for definitive treatment of ceftriaxone nonsusceptible Escherichia coli and Klebsiella.

Clinical evaluation of serological IgG antibody response on the Abbott Architect for established SARS-CoV-2 infection.

OBJECTIVE: This study aimed to evaluate the diagnostic performance of the Abbott Architect SARS-CoV-2 IgG assay in COVID-19 patients. METHODS: Residual sera from 177 symptomatic SARS-CoV-2-positive patients and 163 non-COVID-19 patients were tested for antibody with the Abbott SARS-CoV-2 IgG assay (Abbott Diagnostics, Chicago, USA). Clinical records for COVID-19 patients were reviewed to determine the time from onset of clinical illness to testing. RESULTS: Specificity of the assay was 100.0% (95%CI: 97.1-100.0%). The clinical sensitivity of the assay varied depending on time from onset of symptoms, increasing with longer periods from the onset of clinical illness. The clinical sensitivity at ≤6 days was 8.6% (7/81; 95%CI: 3.8-17.5%), at 7-13 days 43.6% (17/39; 95%CI: 28.2-60.2%), at 14-20 days 84.0% (21/25; 95%CI: 63.1-94.7%), and at ≥21 days 84.4% (27/32; 95%CI: 66.5-94.1%). Clinical sensitivity was higher in the ≥14-day group compared to <14 days. There were no differences between the 14-20-day and ≥21-days groups; the combined clinical sensitivity for these groups (≥14 days) was 84.2% (49/57; 71.6-92.1%). CONCLUSION: The Abbott SARS-CoV-2 IgG test has high specificity. Clinical sensitivity was limited in the early stages of disease but improved from 14 days after the onset of clinical symptoms.

A model for decentralised grey wastewater treatment system in Singapore public housing.

Global concerns over the sustainable use of natural resources provided the impetus for research into water reclamation from wastewater within the Singapore context. The objective of the research is to study and develop a water infrastructure system as an integral element of architecture and the urbanscape, thereby reducing the need for the large area requirements associated with centralised treatment plants. The decentralised plants were considered so as to break up the large contiguous plot of land otherwise needed, into smaller integrated fragments, which can be incorporated within the housing scheme. This liberated more usable space on the ground plane of the urban housing master plan, enabling water-edge and waterscape relationships within both the private and public domains of varying scale.

Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization.

PURPOSE: Breast cancer is thought to develop from noninvasive precursor lesions, although the earliest steps of neoplastic transformation are still undefined. Usual ductal hyperplasia (UDH) is considered to represent a benign proliferation of ductal epithelial cells, whereas atypical ductal hyperplasia (ADH) may represent the first clonal neoplastic expansion of these cells. The aim of this study was to examine genetic alterations in UDH and ADH and to determine the relationship between these lesions in the same breast biopsy. EXPERIMENTAL DESIGN: Comparative genomic hybridization analysis was used to define copy number alterations in DNA extracted from archival sections of 18 patients. Nine patients showed ADH with adjacent UDH, and nine showed pure UDH. None showed evidence of invasive cancer or ductal carcinoma in situ. RESULTS: Five of the nine ADH lesions showed chromosome copy number alterations. 16q loss (five cases) and 17p loss (two cases) were the most frequent changes. The associated UDH lesions in these five patients also showed copy number alterations, always a subset of the changes present in the paired ADH. In one other patient, the UDH showed eight chromosomal alterations, whereas the paired ADH showed no changes. Only one of nine cases with pure UDH showed comparative genomic hybridization abnormalities. CONCLUSIONS: These data support the likelihood that UDH is a precursor of ADH, at least in some cases representing neoplastic growth. The frequencies of 16q and 17p losses suggest that alterations of candidate genes located in these chromosomal regions may play a role early in breast carcinogenesis.

Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences.

BACKGROUND: Ductal carcinoma in situ (DCIS) recurs in the same breast following breast-conserving surgery in 5%-25% of patients, with the rate influenced by the presence or absence of involved surgical margins, tumor size and nuclear grade, and whether or not radiation therapy was performed. A recurrent lesion arising soon after excision of an initial DCIS may reflect residual disease, whereas in situ tumors arising after longer periods are sometimes considered to be second independent events. The purpose of this study was to determine the clonal relationship between initial DCIS lesions and their recurrences. METHODS: Comparative genomic hybridization (CGH) was used to compare chromosomal alterations in 18 initial DCIS lesions (presenting in the absence of invasive disease) and in their subsequent ipsilateral DCIS recurrences (detected from 16 months to 9.3 years later). RESULTS: Of the 18 tumor pairs, 17 showed a high concordance in their chromosomal alterations (median = 81%; range = 65%-100%), while one case showed no agreement between the paired samples (having two and 20 alterations, respectively). Morphologic characterization of the DCIS pairs showed clear similarities. The mean number of CGH changes was greater in the recurrent tumors than in the initial lesions (10.7 versus 8.8; P =.019). The most common changes in both the initial and the recurrent in situ lesions were gains involving chromosome 17q and losses involving chromosomes 8p and 17p. The degree of concordance was independent of the time interval before recurrence and of the presence of positive surgical margins. CONCLUSIONS: In this study, DCIS recurrences were clonally related to their primary lesions in most cases. This finding is consistent with treatment paradigms requiring wide surgical margins and/or postoperative radiation therapy.

Tumor angiogenesis as a prognostic assay for invasive ductal breast carcinoma.

BACKGROUND: Tumor angiogenesis, as assayed by microvessel density, has been proposed as an independent prognostic marker for clinical outcome in breast cancer patients. PURPOSE: The present study evaluated the microvessel density assay and assessed its utility, alone and together with the evaluation of other tumor characteristics, in predicting outcome in patients with invasive ductal carcinomas. METHODS: In a blinded design, cases of invasive ductal carcinoma were selected from a registry containing the records of and tumor specimens from 386 breast cancer patients treated at the Massachusetts General Hospital from 1977 through 1982. After the exclusion of ineligible patients and inadequate specimens, 220 patients were included in the study; their median time of follow-up was 11.5 years. Half of these patients (n = 110) were positive for axillary lymph node metastases. Histologic sections of the tumors were stained immunocytochemically for factor VIII, a coagulation protein expressed by blood vessel endothelium, and for p53 protein. Independently, two analysts counted microvessels in three microscope fields selected from separate vascular regions of the tumor. Variability in microvessel scores between analysts and among different fields of the same tumor was summarized by the coefficient of variation. The kappa statistic tested for agreement between the analysts while correcting for chance agreement. The effects of tumor characteristics on metastasis-free survival and overall survival were tested univariately by the Harrington-Fleming rank test procedure. The effect of multiple factors on survival was tested under a Cox multivariate proportional hazards model. RESULTS: Microvessel count showed considerable variability between the two analysts and among regions within each tumor, with an overall concordance for tumor classification of 73%. Univariate analysis revealed no association between microvessel count and any other tumor or patient characteristic. Multivariate analysis indicated, for these patients, that nodal status and p53 staining predicted metastasis-free survival and that nodal status, estrogen receptor (ER) status and tumor grade predicted overall survival. CONCLUSIONS: Microvessel count showed much variation among different regions of each tumor. It did not predict metastasis-free survival or overall survival. Nodal status was the most powerful criterion to stratify these patients with invasive ductal carcinoma of the breast into different survival groups. Only ER status, tumor grade, and p53 staining had additional prognostic utility for these patients after they had been stratified by nodal status.

Cell proliferation in hyperplastic and in situ carcinoma lesions of the breast estimated by in vivo labeling with bromodeoxyuridine.

The proliferative activity of normal, benign proliferative, and carcinoma in situ (CIS) breast lesions was studied by in vivo labeling with 5-bromodeoxyuridine (BrdU) in 35 patients with concurrent breast carcinoma. The BrdU-labeled cells were identified on histologic sections by an anti-BrdU monoclonal antibody and an immunoperoxidase reaction. The percentage of BrdU-labeled cells in nonatypical hyperplasia (NAH) was higher (1.15 +/- 1.14%) than normal epithelial cells (0.67 +/- 0.56%, p = 0.066, borderline significance). This difference was very significant in postmenopausal women and disappeared in premenopausal women. No significant difference was found in the fraction of proliferating cells between NAH and atypical hyperplasia (AH): 1.15 +/- 1.14% for NAH versus 1.26 +/- 1.19% for AH. In CIS and in invasive carcinoma (ICA), a significant increase in the percent of BrdU-labeled cells was observed when compared to the normal epithelial cells or NAH (p < 0.001). No significant difference was found in the values of BrdU-labeled cells between CIS and ICA (p = 0.29). The percent of BrdU-labeled cells in benign breast lesions, including fibroadenoma, papilloma, and sclerosing adenosis, did not differ from those of the normal epithelial cells. The menopausal status of the patients did not affect the proliferative activity in NAH or CIS. No correlation was found in the fraction of BrdU-labeled cells between the normal and hyperplastic epithelial cells (r2 = 0.012) or between NAH and CIS (r2 = 0.406) or between CIS and ICA (r2 = 0.429).

Effects of short-term culture on benign and malignant human breast epithelium analyzed by image analysis.

The ability to culture malignant breast cells is crucial to the success of many scientific studies. However, short-term cultures of breast cancers are composed predominantly of DNA diploid cells, leading to doubt regarding the presence of bonafide cancer cells in these cultures. Morphology by conventional light microscopy is not helpful since cultured benign cells take on features usually associated with malignancy. In this study we examined by image analysis both benign and malignant breast cancer cells before and after culture. Consistent changes in both benign and malignant cells were found following culturing. There also were consistent differences distinguishing the cultures derived from benign and malignant specimens. While the features used were not sufficiently powerful to identify individual cells or cultures as benign or malignant, our results strongly suggest that malignant breast cells do indeed grow in short-term cultures.

Correlation of Pneumocystis carinii cyst density with mortality in patients with acquired immunodeficiency syndrome and pneumocystis pneumonia.

Fifteen percent to 20% of patients with the acquired immunodeficiency syndrome and pneumocystis pneumonia do poorly despite early intervention. It is not known what distinguishes those who die, despite early intervention and aggressive therapy, from those who readily respond to therapy. We used image analysis to determine the relative abundance of cysts within aggregates of Pneumocystis carinii found in induced sputa (21 patients) and bronchoalveolar lavage fluid (14 patients) from 35 patients with pneumocystis pneumonia. We calculated a cyst density (number of cysts per area of aggregate) for each aggregate and a mean cyst density for all of the aggregates on the smear. Six patients died within 2 weeks of diagnosis; four of these six patients who had autopsies all had residual P carinii. The mean cyst density for those who died was 9.7 +/- 3.9 (range, 5 to 15 x 10(-3)). The 29 patients who survived beyond 2 weeks had a mean cyst density of 18.4 +/- 8.7 (range, 5 to 35 x 10(-3); P = .01). Mean cyst density was not influenced by the number of aggregates present in the smear, the variation in cyst density among aggregates in a smear, or the episode of pneumocystis pneumonia. Cyst density determinations alone should not be used to predict outcome for individuals with P carinii pneumonia until further study is completed. Nevertheless, the current study suggests that a low cyst density specimen, which may indirectly indicate a greater proportion of trophozoites compared with a high cyst density specimen, may be associated with an unfavorable outcome in acquired immunodeficiency syndrome-associated pneumocystis pneumonia.

Breast cancer incidence in women with abnormal cytology in nipple aspirates of breast fluid.

This is a prospective study of breast cancer risk in relation to nipple aspirate fluid cytology in 2,701 volunteer white women from the San Francisco Bay Area first enrolled between 1973 and 1980. The women were not pregnant or lactating and were free of breast cancer within 6 months of entry into the study. The breast cancer status of this cohort was determined between June 1988 and April 1991. Follow-up was complete for 87% (n = 2,343) of the cohort, representing 29,961 person-years and an average of 12.7 years of follow-up. The overall breast cancer incidence was 4.4% (104 of 2,343) and rose with fluid cytology findings as follows: no fluid obtained, 2.6% (9 of 352); unsatisfactory specimen, 4.8% (15 of 315); normal cytology, 4.3% (56 of 1,291); epithelial hyperplasia, 5.5% (18 of 327); and atypical hyperplasia, 10.3% (6 of 58). Relative risks for breast cancer and their 95% confidence intervals were estimated by Cox regression, adjusting for age and year of entry. Compared with the relative risk for women who yielded no fluid, relative risks were: unsatisfactory specimen, relative risk (RR) = 1.4 (95% confidence interval (CI) 0.6-3.3); normal cytology, RR = 1.8 (95% CI 0.9-3.6); epithelial hyperplasia, RR = 2.5 (95% CI 1.1-5.5); and atypical hyperplasia, RR = 4.9 (95% CI 1.7-13.9). These findings were strongest for and were mainly confined to women aged 25-54 years. Women with atypical hyperplasia and a first-degree family history of breast cancer were six times more likely to develop breast cancer than were women with atypical hyperplasia but without a family history of breast cancer (95% CI 1.0-30.2). These findings provide strong support for our hypothesis that hyperplasia and atypical hyperplasia diagnosed in nipple aspirates of breast fluid are associated with an increased risk of breast cancer.

Proliferation of normal breast epithelial cells as shown by in vivo labeling with bromodeoxyuridine.

The proliferative activity of normal acinar and ductal breast epithelial cells was studied by in vivo labeling with 5-bromodeoxyuridine (BrdUrd) in 26 cases with concurrent breast carcinoma. The BrdUrd-labeled cells were recognized in histologic sections of paraffin-embedded tissue, using an anti-BrdUrd antibody and an immunoperoxidase reaction. The percentage of BrdUrd-labeled cells showed great variability for both acinar (0% to 2.66%; mean, 0.70%; standard deviation [SD], 0.80%) and ductal cells (0% to 1.99%; mean, 0.51%; SD, 0.57%). The fraction of proliferating epithelial cells declined with the age of the patients and was significantly higher in premenopausal women (1.16% +/- 0.85% for acinar and 0.94% +/- 0.60% for ductal cells) as compared with the postmenopausal women (0.27% +/- 0.46% for acinar and 0.17% +/- 0.22% for ductal cells), P less than 0.01 for acinar and P less than 0.001 for ductal cells, respectively. In some patients, great variability in distribution of proliferating acinar and ductal cells among different lobules and ducts was observed. No difference was found in the number of proliferating acinar and ductal cells situated near or far from their corresponding tumors. No correlation was seen between cell proliferation of normal acinar or ductal cells and cell proliferation of the respective tumors.