Detection of Chitinase 3-Like 1 in Symptomatic Primary Care Patient Faecal Samples is Not a Reliable Biomarker of Colonic Lesions.

BACKGROUND: The current gold standard non-invasive test for detecting pre-cancerous changes is the faecal immunochemical test (FIT). However, this test can lack sensitivity and specificity and testing for another biomarker may address these limitations. Chitinase 3-like 1 (CHI3L1) is emerging as a potential biomarker of inflammation-associated carcinogenic changes in epithelial cells.  In this study CHI3L1 levels were analysed in patients and controls to determine their ability to improve detection of early CRC either alone or in combination with a FIT. METHODS: CHI3L1 levels were measured by ELISA in serum and stool samples from cohorts of CRC and healthy donors as well as stool samples from a cohort of symptomatic primary care patients. Faecal haemoglobin was also analysed in the same primary care samples using FIT. RESULTS: CHI3L1 levels were a good discriminatory marker of CRC, with no significant difference between levels detected in the stool and serum samples.  ROC curves that determined the optimal cut-point however identified that stool samples gave higher sensitivity (83% versus 69%) and specificity (89% versus 74%) than matched serum samples. Faecal CHI3L1 levels in the primary care patients were not significantly different (p=0.193) from those detected in the healthy controls. ROC curve analysis confirmed that faecal CHI3L1 levels had limited ability to discriminate between patients who did or didn't have evidence of lesions (AUC=0.52, p=0.74). Similarly, CHI3L1 levels did not reliably identify those symptomatic primary care patients who subsequently presented with early-stage disease (polyps and adenomas) or CRC. The discriminatory power of FIT was not increased by incorporating the CHI3L1 results in this setting. CONCLUSION: There was no evidence that measurement of faecal CHI3L1 has the potential to increase diagnostic accuracy, either alone or in combination with a FIT, in symptomatic primary care patients.

Could Microplastics Be a Driver for Early Onset Colorectal Cancer?

Introduction: The incidence of colorectal cancer in those under 50 years of age (early onset colorectal cancer (EOCRC)) is increasing throughout the world. This has predominantly been an increase in distal colonic and rectal cancers, which are biologically similar to late onset colorectal cancer (LOCRC) but with higher rates of mucinous or signet ring histology, or poorly differentiated cancers. The epidemiology of this change suggests that it is a cohort effect since 1960, and is most likely driven by an environmental cause. We explore the possible role of microplastics as a driver for this change. Review: The development of sporadic colorectal cancer is likely facilitated by the interaction of gut bacteria and the intestinal wall. Normally, a complex layer of luminal mucus provides colonocytes with a level of protection from the effects of these bacteria and their toxins. Plastics were first developed in the early 1900s. After 1945 they became more widely used, with a resultant dramatic increase in plastic pollution and their breakdown to microplastics. Microplastics (MPs) are consumed by humans from an early age and in increasingly large quantities. As MPs pass through the gastrointestinal tract they interact with the normal physiological mechanism of the body, particularly in the colon and rectum, where they may interact with the protective colonic mucus layer. We describe several possible mechanisms of how microplastics may disrupt this mucus layer, thus reducing its protective effect and increasing the likelihood of colorectal cancer. Conclusions: The epidemiology of increase in EOCRC suggests an environmental driver. This increase in EOCRC matches the time sequence in which we could expect to see an effect of rapid increase of MPs in the environment and, as such, we have explored possible mechanisms for this effect. We suggest that it is possible that the MPs damage the barrier integrity of the colonic mucus layer, thus reducing its protective effect. MPs in CRC pathogenesis warrants further investigation. Future directions: Further clarification needs to be sought regarding the interaction between MPs, gut microbiota and the mucus layer. This will need to be modelled in long-term animal studies to better understand how chronic consumption of environmentally-acquired MPs may contribute to an increased risk of colorectal carcinogenesis.

An epigenetic signature of advanced colorectal cancer metastasis.

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. The majority of CRC deaths are caused by tumor metastasis, even following treatment. There is strong evidence for epigenetic changes, such as DNA methylation, accompanying CRC metastasis and poorer patient survival. Earlier detection and a better understanding of molecular drivers for CRC metastasis are of critical clinical importance. Here, we identify a signature of advanced CRC metastasis by performing whole genome-scale DNA methylation and full transcriptome analyses of paired primary cancers and liver metastases from CRC patients. We observed striking methylation differences between primary and metastatic pairs. A subset of loci showed coordinated methylation-expression changes, suggesting these are potentially epigenetic drivers that control the expression of critical genes in the metastatic cascade. The identification of CRC epigenomic markers of metastasis has the potential to enable better outcome prediction and lead to the discovery of new therapeutic targets.

Distinct changes in the colonic microbiome associated with acute diverticulitis.

AIM: The pathogenesis of acute diverticulitis (AD) remains incompletely understood, despite it being one of the most common gastrointestinal conditions worldwide. The aim of this study was to investigate the role of the colonic microbiome in the pathogenesis of AD. METHOD: A prospective case-control study was performed, comparing the microbiome of AD patients with that of controls, using 16S rRNA sequencing of rectal swab samples. RESULTS: The microbiome of individuals with AD showed lower diversity than that of controls. There were significant compositional differences observed, with a lower abundance of commensal bacterial families and genera such as Lachnospiraceae, Ruminococcus and Faecalibacterium in AD patients compared with controls, and there was an increase in several genera with known pathogenic roles including Fusobacteria, Prevotella and Paraprevotella. CONCLUSION: This is the largest study to date to examine the microbiota of AD patients, and adds evidence to the proposed hypothesis that alterations in the colonic microbiome play a role in the pathogenesis of AD.

Detection of Fusobacterium nucleatum DNA in primary care patient stool samples does not predict progression of colorectal neoplasia.

BACKGROUND: Carriage of certain bacterial species may represent potential biomarkers of colorectal cancer (CRC). Prominent among these is Fusobacterium nucleatum. We explored the association of F. nucleatum DNA in stool samples with the presence of colonic neoplastic lesions in a cohort of primary care patients, and compared our findings with those from an unrelated cohort of colonoscopy patients followed clinically over time. METHODS: Carriage rates of F. nucleatum in stool samples were assessed in 185 patients referred for a faecal immunochemical test (FIT) by their general practitioners (GPs). Comparisons were made with stool samples from 57 patients diagnosed with CRC and 57 age-matched healthy controls, and with tissue samples taken at colonoscopy from 150 patients with a decade of subsequent clinical follow-up. FINDINGS: F. nucleatum DNA was found at a high rate (47.0%) in stool samples from primary care patients, and more often in stool samples from CRC patients (47.4%) than in healthy controls (7.0%), (P = 7.66E-7). No association was found between carriage of F. nucleatum and FIT positivity (P = 0.588). While evidence of stool-associated F. nucleatum DNA was significantly more likely to indicate a lesion in those primary care patients progressed to colonoscopy (P = 0.023), this finding did not extend to the progression of neoplastic lesions in the 150 patients with a decade of follow up. CONCLUSION: The finding of F. nucleatum DNA at similar rates in stool samples from patients diagnosed with CRC and in primary care patients with pre-cancerous lesions supports growing awareness that the presence of these bacteria may be a biomarker for increased risk of disease. However, molecular evidence of F. nucleatum did not predict progression of colonic lesions, which may lessen the utility of this bacterium as a biomarker for increased risk of disease.

Biomarkers to Detect Early-Stage Colorectal Cancer.

Colorectal cancer is a leading cause of mortality worldwide. The high incidence and the acceleration of incidence in younger people reinforces the need for better techniques of early detection. The use of noninvasive biomarkers has potential to more accurately inform how patients are prioritised for clinical investigation, which, in turn, may ultimately translate into improved survival for those subsequently found to have curable-stage CRC. This review surveys a wide range of CRC biomarkers that may (alone or in combination) identify symptomatic patients presenting in primary care who should be progressed for clinical investigation.

Acute Diverticulitis in Young Patients: A Review of the Changing Epidemiology and Etiology.

Acute diverticulitis is one of the leading gastrointestinal causes for hospitalization. The incidence of acute diverticulitis has been increasing in recent years, especially in patients under 50 years old. Historically, acute diverticulitis in younger patients was felt to represent a separate entity, being more virulent and associated with a higher rate of recurrence. Accordingly, young patients were often managed differently to older counterparts. Our understanding of the natural history of this condition has evolved, and current clinical practice guidelines suggest age should not alter management. The purpose of this review is to evaluate the changing epidemiology of acute diverticulitis, consider potential explanations for the observed increased incidence in younger patients, as well as review the natural history of acute diverticulitis in the younger population.

The Epidemiology and Etiology of Right-Sided Colonic Diverticulosis: A Review.

Diverticulosis of the colon is a common condition in Western countries and most patients will remain asymptomatic, but some will present with symptoms of acute diverticulitis or bleeding. Our understanding of diverticulosis is evolving but is mostly derived from diverticulosis affecting the left-sided colon. In contrast, right-sided colonic diverticulosis (RCD) is more commonly seen in Asian countries but is much less common overall. Based on the marked differences in epidemiology, it is commonly thought that these are 2 distinct disease processes. A review of the literature describing the epidemiology and etiology of RCD was performed, with a comparison to the current understanding of left-sided diverticulosis. RCD is becoming increasingly common. The epidemiology of RCD shows it to be a mostly acquired condition, and not congenital as previously thought. Many factors in the etiology of RCD are similar to that seen in left-sided diverticulosis, with a few variations. It is therefore likely that most cases of RCD represent the same disease process that is seen in the left colon.

The prevalence of right-sided colonic diverticulosis in a New Zealand population.

BACKGROUND: Once considered to be a congenital condition, the epidemiology of right-sided colonic diverticulosis (RCD) is evolving. Acute diverticulitis (AD) is a complication of RCD which is frequently misdiagnosed as appendicitis, resulting in unnecessary surgery, as there is strong evidence supporting medical management for right-sided AD. In general, the incidence of AD correlates with the prevalence of RCD, which shows marked geographic variation. Few data reporting RCD prevalence come from Western countries, so the aim of this study is to define the prevalence of RCD in a New Zealand population. METHODS: Independent review of the imaging from 1000 consecutive patients undergoing a computed tomography Kidney/Ureter/Bladder scan for suspected urolithiasis at Christchurch Hospital between January and November 2017 was undertaken, to determine the presence or absence, and distribution of colonic diverticulosis. Patients were excluded if they had a history of colonic resection, known IBD, or were less than 18-years old. RESULTS: Thirty-one patients were excluded, leaving 969 eligible patients. Overall, 95 patients (9.8%) had RCD identified. The prevalence of RCD increased significantly with advancing age, being present in 2.3% of those aged 18-29, increasing to 20.3% in those greater than 70-years old (p < 0.001). CONCLUSION: The prevalence of RCD in a New Zealand population is relatively high and increases significantly with age. This adds support to the role of cross-sectional imaging in the evaluation of suspected appendicitis, to exclude right-sided AD. The association with advancing age supports RCD being an acquired condition rather than a congenital condition as was previously thought.

Quality of life preferences in colorectal cancer patients aged 80 and over.

BACKGROUND: Management of patients with colorectal cancer (CRC) is about not only survival, but also quality of life (QoL). What patients want is important but is not well researched or understood for elderly patients where it is very relevant. This study aimed to measure and compare what patients with CRC aged 80 and over and surgeons consider important in terms of survivorship after surgery for CRC. METHODS: Patients aged 80 and over who were having surgery for CRC were recruited and interviewed using closed and open questions about their expectations of surgery and various QoL dimensions. These were assessed preoperatively and 3 months post-operatively. Surgeons ranked the same QoL dimensions of patients by questionnaire. RESULTS: Nineteen patients (median age 87.5, range 80-95, eight males and 11 females) were recruited. Patients rated items relating to health, mobility and independence (n = 23) as top three items most often followed by people outside self (n = 13). Surgeons underestimated importance in 17 domains with the biggest discrepancy being in 'avoiding a stoma' (4.11 versus 2.3, P < 0.01). CONCLUSION: With patients over 80 years having surgery for CRC, there is a lack of concordance between what surgeons think is important and what patients think is important. Despite this, CRC patients aged 80 and older are almost always satisfied with the outcome of surgery. Surgeons should ensure that they understand patients' expectations and that they are aligned with likely outcomes of surgery.

Clinical predictors of response to chemoradiotherapy for rectal cancer as an aid to organ preservation.

AIM: Clinical predictors of pathological response to chemoradiotherapy for rectal cancer can influence patient management including selection for organ preservation. This study aimed to identify clinical predictors at a tertiary referral hospital. METHODS: A retrospective review of clinical records was undertaken after identifying all patients with stage 1-3 rectal cancer treated with long course chemoradiotherapy and total mesorectal excision from 2013 to 2018. Clinicopathological factors were recorded and multivariate analysis performed to identify predictors of pathological complete response (pCR) and good response (AJCC TRG 0-1). RESULTS: A total of 470 patients with rectal cancer were identified of which 164 met the inclusion criteria for the study. The pCR rate was 14.6% and good response (TRG 0-1) rate 43.7%. On univariate analysis, lower T stage, older age, node negative status, anterior tumour position and shorter tumour length on magnetic resonance imaging (MRI) were associated with good response (TRG 0-1). On univariate analysis cN stage, carcinoembryonic antigen <5 and shorter tumour length on MRI were associated with pCR. On binary logistic regression shorter length on MRI and lower clinical nodal stage were predictive of pCR and lower body mass index, anterior tumour position and higher haemoglobin were predictive of good response (TRG 0-1). CONCLUSION: Anterior tumour position is newly identified as an independent predictor of good response (TRG 0-1) to nCRT for rectal cancer and this should be explored in future studies. Higher haemoglobin and lower body mass index were also independent predictors of good response (TRG 0-1) and optimisation of these factors should be considered when using neoadjuvant chemoradiotherapy for rectal cancer.

Pre-operative frailty is predictive of adverse post-operative outcomes in colorectal cancer patients.

BACKGROUND: An increasing number of elderly patients are presenting for elective surgery. Pre-operative risk assessment in this population is inexact due to the complex interplay between age, comorbidity and functional status. Frailty assessment may provide a surrogate measure of a patient's physiological reserve and aid operative decision-making. The aim of this study is to determine the association between pre-operative frailty, as assessed using the Edmonton Frail Scale, and post-operative outcomes in elderly patients undergoing elective colorectal cancer surgery. METHODS: A prospective analysis of 86 patients over the age of 65 undergoing elective colorectal cancer surgery at a tertiary centre between October 2017 and October 2018 was performed. Frailty assessment was conducted pre-operatively using the Edmonton Frail Scale. Primary outcomes included length of stay and post-operative complication rates. Multivariable logistic regression analyses were used to determine the influence of frailty on post-operative outcomes including mortality, prolonged hospital admission, complication rates and quality of life. RESULTS: Of 86 patients, 12 (14.0%) were identified as frail. Frailty was associated with a significantly increased median length of stay (20 days versus 6 days, incidence rate ratio 2.83, P < 0.01) and a significantly increased risk of major post-operative complications (50.0% versus 6.7%, odds ratio 13.8, P < 0.01). Frailty was not associated with a significant reduction in quality of life scores at 30 and 90 days post-operatively. CONCLUSION: Frailty is associated with adverse post-operative outcomes in elderly patients undergoing elective colorectal cancer surgery. Frailty assessment is an important component of pre-operative risk assessment and may identify targets for pre-operative optimisation.

Surgical outcomes following colorectal cancer resections in patients aged 80 years and over: results from the Australia and New Zealand Binational Colorectal Cancer Audit.

AIM: The primary aim was to compare the 30-day morbidity and mortality in patients aged ≥80 years undergoing surgery for colorectal cancer with those aged <80 years. The secondary aim was to identify independent outcome predictors. METHOD: This was a retrospective study of patients undergoing surgery for colorectal cancer between January 2007 and February 2018. Patients were divided into those <80 years and those ≥80 years at the time of surgery. Data had been collected prospectively by the Australasian Binational Colorectal Cancer Audit and included patient demographics, site and stage of tumour, comorbidity, operative details, American Society of Anesthesiologists score (ASA), pathological staging, 30-day mortality and morbidity (medical and surgical). Univariate and multivariate analyses were used to identify predictors of 30-day morbidity and mortality. RESULTS: During the study period, 4600 out of 20 463 (22.5%) patients were ≥80 years. They had a greater 30-day mortality after both colonic (97/2975 [3.3%] vs. 66/7010 [0.9%], P < 0.001) and rectal resections (50/1625 [3.1%] vs. 36/9006 [0.4%], P < 0.001) compared with younger patients. They also had an increased length of stay (colon cancer, 9 vs. 7 days; rectal cancer, 10 vs. 8 days; P < 0.001) and medical complications (colon cancer, 23.5% vs. 12.7%; rectal cancer, 25.2% vs. 11.2%; P < 0.001). Surgical complications were equivalent. Age ≥80 years was not an independent predictor of 30-day morbidity or mortality. Patients ≥80 years who were ASA 2/3 and had rectal cancer seemed to fare worse in terms of 30-day mortality (ASA 2, 22%, 95% CI 9%-36%, P < 0.001; ASA 3, 11%, 95% CI 4%-19%, P< 0.001). CONCLUSIONS: Postoperative morbidity and mortality are significantly greater in patients ≥80 years undergoing colorectal cancer surgery. Any recommendation for surgery in this age group should take into account patient comorbidity and not be based on age alone.

Predicting pathological response to chemoradiotherapy for rectal cancer: a systematic review.

Introduction: Pathological complete response (pCR) rates of approximately 20% following neoadjuvant long-course chemoradiotherapy for rectal cancer have given rise to non-operative or watch-and-wait (W&W) management. To improve outcomes there has been significant research into predictors of response. The goal is to optimize selection for W&W, avoid chemoradiotherapy in those who won't benefit and improve treatment to maximize the clinical complete response (cCR) rate and the number of patients who can be considered for W&W.Areas covered: A systematic review of articles published 2008-2018 and indexed in PubMed, Embase or Medline was performed to identify predictors of pathological response (including pCR and recognized tumor regression grades) to fluoropyrimidine-based chemoradiotherapy in patients who underwent total mesorectal excision for rectal cancer. Evidence for clinical, biomarker and radiological predictors is discussed as well as potential future directions.Expert opinion: Our current ability to predict the response to chemoradiotherapy for rectal cancer is very limited. cCR of 40% has been achieved with total neoadjuvant therapy. If neoadjuvant treatment for rectal cancer continues to improve it is possible that the treatment for rectal cancer may eventually parallel that of anal squamous cell carcinoma, with surgery reserved for the minority of patients who don't respond to chemoradiotherapy.

APC Mutations Are Not Confined to Hotspot Regions in Early-Onset Colorectal Cancer.

While overall colorectal cancer (CRC) cases have been declining worldwide there has been an increase in the incidence of the disease among patients under 50 years of age. Mutation of the APC gene is a common early event in CRC but is reported at lower rates in early-onset colorectal cancer (EOCRC) than in older patients. Here we investigate the APC mutation status of a cohort of EOCRC patients in New Zealand using a novel sequencing approach targeting regions of the gene encompassing the vast majority of known APC mutations. Using this strategy we find a higher rate (72%) of APC mutation than previously reported in EOCRC with mutations being spread throughout the gene rather than clustered in hotspots as seen with sporadic mutations in older patients. The rate of mutations falling within hotspots was similar to those previously seen in EOCRC and as such our study has implications for sequencing strategies for EOCRC patients. Overall there were low rates of both loss of heterozygosity and microsatellite instability whereas a relatively high rate (40%) of APC promoter methylation was found, possibly reflecting increasing exposure of young people to pro-oncogenic lifestyle factors.

The association between low pre-operative step count and adverse post-operative outcomes in older patients undergoing colorectal cancer surgery.

BACKGROUND: Multiple tools exist estimating perioperative risk. With an ageing surgical demographic, frailty is becoming an increasingly important concept in perioperative medicine due to its association with adverse post-operative outcomes. Reduced physical activity is a hallmark of frailty, and we postulate that a low pre-operative step count may be an objective measure of frailty. This study aimed to determine the association between low pre-operative step count and post-operative outcomes in patients undergoing elective colorectal cancer surgery. METHODS: A prospective analysis of 85 older patients undergoing major elective colorectal surgery was performed at a tertiary centre between October 2017 and October 2018. Patients aged 65 years and over who met inclusion criteria were provided with an activity tracker to wear for 14 days prior to planned surgery. Their median daily step count was measured and a cut-off of < 2500 steps/day was used to define a reduced step count. Primary outcomes included length of stay and 30-day post-operative complication rate. Multivariable logistic regression analyses were used to analyze the influence of low pre-operative step count and other preoperative variables, on post-operative outcomes including mortality, prolonged hospital admission, and complication rates. RESULTS: Of 85 patients, 17 (20%) were identified as having a low pre-operative step count. A low pre-operative step count was associated with a significantly increased length of stay (14 vs. 6 days, IRR 2.09, 95% CI 1.55-2.83, p ≤ 0.01) and rate of major post-operative complications (29.4% vs. 8.8%, OR 3.34, 95% CI 1.03-14.3, p = 0.04). It was also associated with significantly increased rates of discharge to care facilities (p < 0.01) and requiring support on discharge (p = 0.03). CONCLUSION: Low pre-operative step count (< 2500 steps/day) is predictive of an increased risk of post-operative morbidity in patients undergoing elective colorectal surgery. Accurate preoperative identification may allow for treatment modification and tailored perioperative care. The possibility of using a wearable activity tracker as a simple but powerful pre-habilitation tool is raised as an important avenue for future study. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ACTRN12618000045213).

Predictors of overall survival following extended radical resections for locally advanced and recurrent pelvic malignancies.

PURPOSE: In an era of personalised medicine, there is an overwhelming effort for predicting patients who will benefit from extended radical resections for locally advanced pelvic malignancy. However, there is paucity of data on the effect of comorbidities and postoperative complications on long-term overall survival (OS). The aim of this study was to define predictors of 1-year and 5-year OS. METHODS: Data were collected from prospective databases at two high-volume institutions specialising in beyond TME surgery for locally advanced and recurrent pelvic malignancies between 1990 and 2015. The primary outcome measures were 1-year and 5-year OS. RESULTS: A total of 646 consecutive extended radical resections were performed between 1990 and 2015. The majority were female patients (371, 57.4%) and the median age was 63 years (range 19-89 years). One-year OS, primary rectal adenocarcinoma had the best survival while recurrent colon cancer had the worse survival (p = 0.047). The 5-year OS between primary and recurrent cancers were 64.7% and 53%, respectively (p = 0.004). Poor independent prognostic markers for 5-year OS were increasing ASA score, cardiovascular disease, recurrent cancers, ovarian cancers, pulmonary embolus and acute respiratory distress syndrome. A positive survival benefit was demonstrated with preoperative radiotherapy (HR 0.55; 95% CI 0.4-0.75, p < 0.001). CONCLUSION: Patient comorbidities and specific complications can influence long-term survival following extended radical resections. This study highlights important predictors, enabling clinicians to better inform patients of the potential short- and long-term outcomes in the management of locally advanced and recurrent pelvic malignancy.

MinION Sequencing of colorectal cancer tumour microbiomes-A comparison with amplicon-based and RNA-Sequencing.

BACKGROUND: Recent evidence suggests a role for the gut microbiome in the development and progression of many diseases and many studies have been carried out to analyse the microbiome using a variety of methods. In this study, we compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples. METHODS: DNA and RNA were extracted from 11 colorectal tumour samples. 16S rRNA amplicon sequencing and MinION sequencing was carried out using genomic DNA, and RNA-Sequencing for meta-transcriptomic analysis. Non-human MinION and RNA-Sequencing reads, and 16S rRNA amplicon sequencing reads were taxonomically classified using a database built from available RefSeq bacterial and archaeal genomes and a k-mer based algorithm in Kraken2. Concordance between the three platforms at different taxonomic levels was tested on a per-sample basis using Spearman's rank correlation. RESULTS: The average number of reads per sample using RNA-Sequencing was greater than 129 times that generated using MinION sequencing. However, the average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing. Taxonomic assignment using 16S sequencing was less reliable beyond the genus level, and both RNA-Sequencing and MinION sequencing could detect greater numbers of phyla and genera in the same samples, compared to 16S sequencing. Bacterial species associated with colorectal cancer, Fusobacterium nucleatum, Parvimonas micra, Bacteroides fragilis and Porphyromonas gingivalis, were detectable using MinION, RNA-Sequencing and 16S rRNA amplicon sequencing data. CONCLUSIONS: Long-read sequences generated using MinION sequencing can compensate for low numbers of reads for bacterial classification. MinION sequencing can discriminate between bacterial strains and plasmids and shows potential as a cost-effective tool for rapid microbiome sequencing in a clinical setting.