Mycoplasma genitalium Endocarditis in Prosthetic Aortic Valve.

We report a case of Mycoplasma genitalium endocarditis in a prosthetic heart valve of a woman who sought care in Switzerland for acute aortic valve dysfunction 3 years after valve replacement. This unusual manifestation of infection with this bacterium was diagnosed using broad-range PCR despite suspicion of a mechanical disinsertion.

Characterization of therapy-related acute myeloid leukemia: increasing incidence and prognostic implications.

Studies of therapy-related AML (t-AML) are usually performed in selected cohorts and reliable incidence rates are lacking. In this study, we characterized, defined the incidence over time and studied prognostic implications in all t-AML patients diagnosed in Sweden between 1997 and 2015. Data were retrieved from nationwide population-based registries. In total, 6,779 AML patients were included in the study, of whom 686 (10%) had t-AML. The median age for t-AML was 71 years and 392 (57%) patients were females. During the study period, the incidence of t-AML almost doubled with a yearly increase in t-AML of 4.5% (95% confidence interval: 2.8%-6.2%), which contributed significantly to the general increase in AML incidence over the study period. t-AML solidly constituted over 10% of all AML cases during the later period of the study. Primary diagnoses with the largest increase in incidence and decrease in mortality rate during the study period (i.e., breast and prostate cancer) contributed significantly to the increased incidence of t-AML. In multivariable analysis, t-AML was associated with poorer outcome in cytogenetically intermediate- and adverse-risk cases but t-AML had no significant impact on outcome in favorable-risk AML, including core binding leukemias, acute promyelocytic leukemia and AML with mutated NPM1 without FLT3-ITD. We conclude that there is a strong increase in incidence in t-AML over time and that t-AML constitutes a successively larger proportion of the AML cases. Furthermore, we conclude that t-AML confers a poor prognosis in cytogenetically intermediate- and adverse-risk, but not in favorable-risk AML.

Metagenomics for the microbiological diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia (HAP/VAP) in intensive care unit (ICU): a proof-of-concept study.

BACKGROUND: Hospital-acquired and ventilator-associated-pneumonia (HAP/VAP) are one of the most prevalent health-care associated infections in the intensive care unit (ICU). Culture-independent methods were therefore developed to provide faster route to diagnosis and treatment. Among these, metagenomic next-generation sequencing (mNGS) has shown considerable promise. METHODS: This proof-of-concept study describes the technical feasibility and evaluates the clinical validity of the mNGS for the detection and characterization of the etiologic agents causing hospital-acquired and ventilator-associated pneumonia. We performed a prospective study of all patients with HAP/VAP hospitalized in our intensive care unit for whom a bronchoalveolar lavage (BAL) was performed between July 2017 and November 2018. We compared BAL fluid culture and mNGS results of these patients. RESULTS: A total of 32 BAL fluids were fully analyzed. Of these, 22 (69%) were positive by culture and all pathogens identified were also reported by mNGS. Among the culture-positive BAL samples, additional bacterial species were revealed by mNGS for 12 patients, raising the issue of their pathogenic role (colonization versus coinfection). Among BALF with culture-negative test, 5 were positive in mNGS test. CONCLUSIONS: This study revealed concordant results for pneumonia panel pathogens between mNGS and culture-positive tests and identified additional pathogens potentially implicated in pneumonia without etiologic diagnosis by culture. mNGS has emerged as a promising methodology for infectious disease diagnoses to support conventional methods. Prospective studies with real-time mNGS are warranted to examine the impact on antimicrobial decision-making and clinical outcome.

A risk score based on real-world data to predict early death in acute promyelocytic leukemia.

With increasingly effective treatments, early death (ED) has become the predominant reason for therapeutic failure in patients with acute promyelocytic leukemia (APL). To better prevent ED, patients with high-risk of ED must be identified. Our aim was to develop a score that predicts the risk of ED in a real-life setting. We used APL patients in the populationbased Swedish AML Registry (n=301) and a Portuguese hospital-based registry (n=129) as training and validation cohorts, respectively. The cohorts were comparable with respect to age (median, 54 and 53 years) and ED rate (19.6% and 18.6%). The score was developed by logistic regression analyses, risk-per-quantile assessment and scoring based on ridge regression coefficients from multivariable penalized logistic regression analysis. White blood cell count, platelet count and age were selected by this approach as the most significant variables for predicting ED. The score identified low-, high- and very high-risk patients with ED risks of 4.8%, 20.2% and 50.9% respectively in the training cohort and with 6.7%, 25.0% and 36.0% as corresponding values for the validation cohort. The score identified an increased risk of ED already at sub-normal and normal white blood cell counts and, consequently, it was better at predicting ED risk than the Sanz score (AUROC 0.77 vs. 0.64). In summary, we here present an externally validated and population-based risk score to predict ED risk in a real-world setting, identifying patients with the most urgent need of aggressive ED prevention. The results also suggest that increased vigilance for ED is already necessary at sub-normal/normal white blood cell counts.

Central venous catheter-related complications in hematologic patients: An observational study.

BACKGROUND: The frequency of central venous catheter (CVC)-related complications in hematologic patients has previously been studied but some uncertainty remains. Therefore, this observational cohort study was designed primarily to investigate mechanical and infectious complications related to CVC insertion in hematologic patients and secondarily to identify factors associated with these complications. METHODS: Documented data on CVC insertions in all adult hematologic patients who received a CVC from 2013 to 2019 at a University Hospital in Sweden were retrospectively collected. RESULTS: A total of 589 CVC insertions in 387 patients were included. The prevalence of moderate and severe mechanical complications, predominantly comprising grades 2-4 bleeding, was 11%. Preprocedural coagulopathy, number of needle passes, and arterial puncture were all independently associated with grades 2-4 bleeding. The incidence of suspected catheter-related infections (sCRI) was 3.7/1000 catheter days. Higher body mass index and male gender were independently associated with sCRI. CONCLUSIONS: Patients with hematologic malignancies have a high risk of both grades 2-4 bleeding and sCRI after CVC insertion. This underlines the importance of optimizing the conditions at the insertion and also of daily inspections, evaluation of future needs, and extra precautions to avoid sCRI in these susceptible patients.

Clinical and genomic characterization of patients diagnosed with the provisional entity acute myeloid leukemia with BCR-ABL1, a Swedish population-based study.

Acute myeloid leukemia (AML) with t(9;22)(q34;q11), also known as AML with BCR-ABL1, is a rare, provisional entity in the WHO 2016 classification and is considered a high-risk disease according to the European LeukemiaNet 2017 risk stratification. We here present a retrospective, population-based study of this disease entity from the Swedish Acute Leukemia Registry. By strict clinical inclusion criteria we aimed to identify genetic markers further distinguishing AML with t(9;22) as a separate entity. Twenty-five patients were identified and next-generation sequencing using a 54-gene panel was performed in 21 cases. Interestingly, no mutations were found in NPM1, FLT3, or DNMT3A, three frequently mutated genes in AML. Instead, RUNX1 was the most commonly mutated gene, with aberrations present in 38% of the cases compared to around 10% in de novo AML. Additional mutations were identified in genes involved in RNA splicing (SRSF2, SF3B1) and chromatin regulation (ASXL1, STAG2, BCOR, BCORL1). Less frequently, mutations were found in IDH2, NRAS, TET2, and TP53. The mutational landscape exhibited a similar pattern as recently described in patients with chronic myeloid leukemia (CML) in myeloid blast crisis (BC). Despite the concomitant presence of BCR-ABL1 and RUNX1 mutations in our cohort, both features of high-risk AML, the RUNX1-mutated cases showed a superior overall survival compared to RUNX1 wildtype cases. Our results suggest that the molecular characteristics of AML with t(9;22)/BCR-ABL1 and CML in myeloid BC are similar and do not support a distinction of the two disease entities based on their underlying molecular alterations.

Mycobacterium chelonae Infection Identified by Metagenomic Next-Generation Sequencing as the Probable Cause of Acute Contained Rupture of a Biological Composite Graft-A Case Report.

We present the case of a 72-year-old female patient with acute contained rupture of a biological composite graft, 21 months after replacement of the aortic valve and the ascending aorta due to an aortic dissection. Auramine-rhodamine staining of intraoperative biopsies showed acid-fast bacilli, but classical culture and molecular methods failed to identify any organism. Metagenomic analysis indicated infection with Mycobacterium chelonae, which was confirmed by target-specific qPCR. The complexity of the sample required a customized bioinformatics pipeline, including cleaning steps to remove sequences of human, bovine ad pig origin. Our study underlines the importance of multiple testing to increase the likelihood of pathogen identification in highly complex samples.

Septic shock caused by Capnocytophaga canis after a cat scratch.

Capnocytophaga canis is an uncommon cause of septic shock. Only three cases have been previously reported in the literature. In this article, we describe the case of a 70-year-old male admitted to the intensive care unit for septic shock of unknown origin. On day 2, one anaerobic bottle out of the two sets taken at admission turned positive with Gram-negative bacilli. The pathogen was identified by 16S rRNA gene as C. canis. The strain was characterized and compared with other clinical isolates of Capnocytophaga spp.

Safety and Effectiveness of Vedolizumab in Patients with Steroid-Refractory Gastrointestinal Acute Graft-versus-Host Disease: A Retrospective Record Review.

Allogeneic hematopoietic cell transplantation (allo-HCT) can be curative in patients with hematologic malignancies but carries a significant risk of graft-versus-host disease (GVHD). There are no standard treatments for steroid-refractory (SR) gastrointestinal (GI) acute GVHD (aGVHD). This multicenter, international, retrospective medical record review aimed to evaluate the off-label use of vedolizumab, a gut-selective immunomodulator, for treating SR GI aGVHD. Data were collected from patients' medical records; criteria for extraction included no more than 1 allo-HCT and at least 1 dose of vedolizumab as treatment for SR GI aGVHD (ie, stage 1 to 4 GI aGVHD following ≥1 previous treatment regimen(s) containing ≥1 mg/kg methylprednisolone or equivalent). Descriptive analyses of response rate, overall survival (OS), and serious adverse effects (SAEs) were performed. Twenty-nine patients were identified from 7 sites who had received 1 to 10 doses of vedolizumab 300 mg i.v. (median 3 doses) as treatment for SR GI aGVHD. The overall response rate at 6 to 10 weeks after vedolizumab initiation was 64%, and OS at 6 months was 54%. There were 29 SAEs, including 12 infections; 3 SAEs were considered possibly related to vedolizumab, 2 of which were infections. Thirteen SAEs were fatal, 1 of which was possibly vedolizumab-related. There were 8 nonserious infections and 1 serious infection with confirmed GI origin in 8 patients; there was no apparent pattern in the timing of these infections relative to the initiation of vedolizumab treatment. Further data on the efficacy and safety of vedolizumab in this setting from prospective trials are needed.

Listeria monocytogenes infectious periaortitis: a case report from the infectious disease standpoint.

BACKGROUND: Endograft infection is a rare but extremely dangerous complication of aortic repair (25-100% of mortality). We describe here the first case of Listeria monocytogenes abdominal periaortitis associated with a vascular graft. We also discuss the differential diagnosis of periaortitis and provide a literature review of L. monocytogenes infectious aortitis. CASE PRESENTATION: Nine months after endovascular treatment of an abdominal aortic aneurysm (abdominal stent graft), a 76-year-old man was admitted for severe abdominal pain radiating to the back. Laboratory tests were normal apart from elevated C-reactive protein (CRP). Injected abdominal computed tomography (CT) showed infiltration of the fat tissues around the aortic endoprosthesis and aneurysmal sac expansion; positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro- D-glucose integrated with computed tomography (18F-FDG PET/CT) showed a hypermetabolic mass in contact with the endoprosthesis. Blood cultures were negative. At surgical revision, an infra-renal peri-aortic abscess was evident; post-operative antibiotic therapy with ciprofloxacin and doxycycline was started. Cultures of intraoperative samples were positive for L. monocytogenes. Results were further confirmed by a broad-range polymerase chain reaction (PCR) and next-generation sequencing. Antibiotic treatment was switched to intravenous amoxicillin for 6 weeks. Evolution was uneventful with decrease of inflammatory parameters and regression of the abscess. CONCLUSION: An etiologic bacterial diagnosis before starting antibiotic therapy is paramount; nevertheless, culture-independent methods may provide a microbiological diagnosis in those cases where antimicrobials are empirically used and when cultures remain negative.

Effects of the gut-liver axis on ischaemia-mediated hepatocellular carcinoma recurrence in the mouse liver.

BACKGROUND & AIMS: There is growing evidence that liver graft ischemia-reperfusion (I/R) is a risk factor for hepatocellular carcinoma (HCC) recurrence, but the mechanisms involved are unclear. Herein, we tested the hypothesis that mesenteric congestion resulting from portal blood flow interruption induces endotoxin-mediated Toll-like receptor 4 (Tlr4) engagement, resulting in elevated liver cancer burden. We also assessed the role of remote ischemic preconditioning (RIPC) in this context. METHODS: C57Bl/6j mice were exposed to standardized models of liver I/R injury and RIPC, induced by occluding the hepatic and femoral blood vessels. HCC was induced by injecting RIL-175 cells into the portal vein. We further evaluated the impact of the gut-liver axis (lipopolysaccharide (LPS)-Tlr4 pathway) in this context by studying mice with enhanced (lipopolysaccharide infusion) or defective (Tlr4-/- mice, gut sterilization, and Tlr4 antagonist) Tlr4 responses. RESULTS: Portal triad clamping provoked upstream mesenteric venous engorgement and increased bacterial translocation, resulting in aggravated tumor burden. RIPC prevented this mechanism by preserving intestinal integrity and reducing bacterial translocation, thereby mitigating HCC recurrence. These observations were linked to the LPS-Tlr4 pathway, as supported by the high and low tumor burden displayed by mice with enhanced or defective Tlr4 responses, respectively. CONCLUSIONS: Modulation of the gut-liver axis and the LPS-Tlr4 response by RIPC, gut sterilization, and Tlr4 antagonism represents a potential therapeutic target to prevent I/R lesions, and to alleviate HCC recurrence after liver transplantation and resection. LAY SUMMARY: Cancer recurrence can occur after liver resection or liver transplantation for hepatocellular carcinoma (HCC). This study suggests that intestinal venous congestion, which often occurs during liver surgery, favors the translocation of gut-derived bacterial products in the portal vein, thereby facilitating cancer recurrence by enhancing the signaling of Toll-like receptor 4 in the liver. Using a mouse model of HCC recurrence, we show that strategies that (i) reduce bacterial translocation (by gut decontamination, or by protecting the intestine from venous ischemia damage) or (ii) inhibit Tlr4 signaling in the liver, could reduce cancer recurrence.

Relatively favorable outcome after allogeneic stem cell transplantation for BCR-ABL1-positive AML: A survey from the acute leukemia working party of the European Society for blood and marrow transplantation (EBMT).

The aim of the study was to assess the role of allogeneic stem cell transplantation (SCT) in patients diagnosed with BCR-ABL1-positive acute myeloid leukemia (AML). Fifty-seven patients (median age, 48 years, range: 19-67) with BCR-ABL1 positive AML undergoing SCT were identified. The majority of the patients (70%) received a TKI before the transplant. At SCT 48 patients were in CR (45 in CR1), while 9 patients were transplanted in a more advanced stage of the disease. MRD was negative (BCR-ABL1/ABL < 104 ) at time of SCT in 36.1% (14/40). After SCT, 16 (61.5%) out of 26 patients with MRD positive at transplantation reached MRD negativity. After a median follow-up of 6.3 years (0.7-14.2), NRM, RI, LFS, OS, and GRFS at 5 years were 18.1%, 37%, 44.2%, 53.8%, and 32.1%, respectively. The cumulative incidence of acute GvHD grade II-IV was 16.4%, incidence of chronic GvHD 24.9%, and of extensive cGvHD 21.4%, respectively. In patients who received SCT in CR1, 5-yr NRM, RI, LFS, OS, and GRFS were 15.9%, 36.4%, 46.5%, 59.4%, and 34.9%, respectively. Univariate analysis showed that age (<50 vs. ≥50 years) was associated with RI (5-yr: 22.7 vs. 50%), LFS (5-yr: 61.9 vs. 31.8%), and GRFS (5-yr: 52.4 vs. 18.2%), whereas MRD-negative status before SCT was associated with an improved GRFS (38.9 vs. 16.7%). We conclude that the outcome of patients <50 years of age with BCR-ABL1-positive AML receiving allogeneic SCT in CR is relatively favorable, possibly reflecting the beneficial effect of the use of TKI.

[Microbiota and malnutrition: an overview]. / Microbiote et dénutrition : état des lieux.

Currently, there is an increased interest in the role of gut microbiota in health issues. Evidence shows that an imbalance of gut microbiota or dysbiosis is involved in the mechanisms of weight changes. This review aims at summarizing the present knowledge between gut microbiota and malnutrition. Intestinal bacterial diversity and richness are altered in malnourished people compared to healthy people. The first studies on the modulation of the gut microbiota by probiotics, prebiotics, symbiotics, fecal transplantation and antibiotics for weight gain are encouraging. However, further studies are needed to develop and implement effective treatment for malnutrition.

L'analyse du microbiote intestinal suscite un intérêt grandissant. Il est maintenant établi qu'un déséquilibre du microbiote intestinal ou dysbiose est impliqué dans les mécanismes de variations pondérales. Cet article vise à faire l'état des lieux des connaissances actuelles entre microbiote intestinal et dénutrition. Les personnes dénutries ont une diversité et une abondance bactérienne intestinale altérées en comparaison des personnes en bonne santé. Les premières études concernant la modulation du microbiote intestinal par les probiotiques, les prébiotiques, les symbiotiques, la transplantation fécale et les antibiotiques dans un but de prise pondérale sont encourageantes. Cependant, de nombreuses études sont encore nécessaires avant toute implication thérapeutique.

Targeting Integrin α4ß7 in Steroid-Refractory Intestinal Graft-versus-Host Disease.

Steroid refractory acute graft-versus-host-disease of the gut is a serious complication associated with high mortality after allogeneic stem cell transplantation. Treatment options are limited and not predictably effective. We describe the treatment of steroid-refractory acute graft-versus-host-disease with vedolizumab, an antibody directed against integrin α4ß7, in 6 patients. All patients responded, and 4 of 6 patients are alive with a median follow-up of 10 months.

Linear decline of corrected platelet count increment within 24 hours after platelet transfusion in haematological patients: A prospective observational study.

OBJECTIVES: The aim of this study was to prospectively explore the detailed longitudinal development of platelet increments in patients with chemotherapy-induced bone marrow aplasia during the first 24 hours after platelet transfusion. METHODS: Patients admitted to the Haematology department during 7 months, and fulfilled inclusion criteria were divided into 4 groups: Group 1, patients with acute leukaemia; Group 2, patients after autologous stem cell transplantation (SCT); Group 3, patients after allogeneic SCT; and Group 4, patients given platelet transfusion prior to intervention. We used frequent blood sampling within 24 hours after platelet transfusion to investigate the kinetics of platelet counts following transfusion. RESULTS AND CONCLUSIONS: Fifty-four platelet transfusion occasions in patients with chemotherapy-induced bone marrow aplasia were included. The decrease in corrected count increment (CCI) 1-24 hours after platelet transfusions in all groups could be described as linear functions. For patients in the aggregated Groups 1-3, the decline was 2.0% ± 0.6% (mean ± standard deviation) per hour. For patients in Group 4, the decline of CCI was 2.8% ± 1.2% per hour. We found no differences between the groups, either in the rate of platelet elimination from the bloodstream or in the mean CCI, in the first 24 hours post-transfusion.

Incidence and prognostic significance of isolated trisomies in adult acute myeloid leukemia: A population-based study from the Swedish AML registry.

OBJECTIVES AND METHODS: To ascertain the incidence/clinical implications of isolated autosomal trisomies in adult acute myeloid leukemia (AML), all such cases were retrieved from the Swedish AML Registry. RESULTS: Of the 3179 cytogenetically informative AMLs diagnosed January 1997-May 2015, 246 (7.7%) had isolated trisomies. The frequency increased by age (2.4% at age 18-60 years vs. 23% at >60 years; P<.0001); the median age was 69 years. The five most common were +8 (4.0%), +13 (0.9%), +11 (0.8%), +21 (0.7%), and +4 (0.5%). Age and gender, types of AML and treatment, and complete remission and early death rates did not differ between the single trisomy and the intermediate risk (IR) groups or among cases with isolated gains of chromosomes 4, 8, 11, 13, or 21. The overall survival (OS) was similar in the single trisomy (median 1.6 years) and IR groups (1.7 years; P=.251). The OS differed among the most frequent isolated trisomies; the median OS was 2.5 years for +4, 1.9 years for +21, 1.5 years for +8, 1.1 years for +11, and 0.8 years for +13 (P=.013). CONCLUSION: AML with single trisomies, with the exception of +13, should be grouped as IR.

Detection of Bacterial Pathogens from Broncho-Alveolar Lavage by Next-Generation Sequencing.

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus, Corynebacterium jeikeium and Rothia dentocariosa, the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

[Intestinal microbiota: towards therapeutic applications]. / Microbiote intestinal :vers des applications thérapeutiques.

Due to technological advances in DNA sequencing, the number of microbiota studies has increased exponentially in recent years. The intestinal microbiota, characterized by high diversity and bacterial richness, received particular interest. The studies showed that the modifications of the intestinal microbiota derive from and lead to different mechanisms and explain various pathologies, including those outside the digestive system.The aim of this short article is to comment on some recent findings on the human microbiota in a clinical context. We attempt to identify the strengths of current research as well as the trends that emerge from an abundant literature of a rather heterogeneous quality.

Favorisé par les avancées technologiques du séquençage de l'ADN, le nombre d'études sur le microbiote a augmenté de manière exponentielle ces dernières années. Le microbiote intestinal, caractérisé par sa diversité et sa richesse bactérienne, a suscité un intérêt tout particulier. Il en ressort que les conséquences associées aux modifications du microbiote intestinal peuvent provenir de mécanismes très différents et expliquer ainsi une variété de pathologies s'étendant bien au-delà du système digestif. Le but de ce bref article est de repositionner les découvertes récentes sur le microbiote humain dans un contexte clinique. Il nous paraît judicieux de tenter d'identifier les points forts de la recherche actuelle ainsi que les tendances qui se dessinent au travers d'une littérature foisonnante et de qualité assez hétérogène.

Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR.

BACKGROUND: Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators. RESULTS: For in silico removal of reagent contaminants, a pipeline was used which combines the relative abundance of operational taxonomic units (OTUs) in V3-4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene. Serially diluted cultures of Escherichia coli and Staphylococcus aureus were used for 16S rDNA profiling, and DNA from each of these species was used as a qPCR standard. OTUs assigned to Escherichia or Staphylococcus were virtually unaffected by the decontamination procedure, whereas OTUs from Pseudomonas, which is a major reagent contaminant, were completely or nearly completely removed. The decontamination procedure also attenuated the trend of increase in OTU richness in serially diluted cultures. CONCLUSIONS: Removal of contaminant sequences derived from reagents based on use of qPCR data may improve taxonomic representation in samples with low DNA concentration. Using the described pipeline, OTUs derived from cross-contamination of negative extraction controls were not recognized as contaminants and not removed from the sample dataset.