Accurate subspecies-level identification of clinically significant Mycobacterium avium and Mycobacterium intracellulare by whole-genome sequencing.

Whole genome sequencing (WGS) of Mycobacterium avium complex (MAC) isolates in the clinical laboratory setting allows for rapid and reliable subspecies identification of a closely related complex of human pathogens. We developed a bioinformatics pipeline for accurate subspecies identification and tested 74 clinical MAC isolates from various anatomical sites. We demonstrate that reliable subspecies level identification of these common and clinically significant MAC isolates, including M. avium subsp. hominissuis (most dominant in causing lower respiratory tract infections in our cohort), M. avium subsp. avium, M. intracellulare subsp. intracellulare, and M. intracellulare subsp. chimaera, can be achieved by analysis of only two marker genes (rpoB and groEL/hsp65). We then explored the relationship between these subspecies and anatomical site of infection. Further, we conducted an in silico analysis and showed our algorithm also performed well for M. avium subsp. paratuberculosis but failed to consistently identify M. avium subsp. silvaticum and M. intracellulare subsp. yongonense, likely due to a lack of available reference genome sequences; all the 3 subspecies were not found in our clinical isolates and rarely reported to cause human infections. Accurate MAC subspecies identification may provide the tool and opportunity for better understanding of the disease-subspecies dynamics in MAC infections.

Reinfection or relapse? A case study of whole genome sequencing guided genomic characterization of Mycobacterium abscessus chronic infection in a cystic fibrosis patient.

A 7-year-old cystic fibrosis patient with increased cough, new pulmonary infiltrate, and declining pulmonary function was diagnosed with clarithromycin resistant Mycobacterium abscessus infection. Treatment was initiated with clofazimine, linezolid and cefoxitin; she responded well to therapy and achieved microbiological clearance after completion of 12-month treatment. One year later, she had re-emergence of worsening symptoms and her sputum culture again grew clarithromycin resistant M. abscessus. Using a laboratory developed whole genome sequencing (WGS) test, the bacterium was determined to be the same strain with the same resistance mechanisms, indicating a relapse. This was deemed a critical element of clinical information, as the isolation of a genetically distinct organism would have indicated a new infection and would have served as evidence that a 12-month regimen was likely sufficient to achieve eradication. The confirmation of a relapse prompted the prolongation of the therapy plan to a goal of 24 months. Reinfection and relapse are great challenges in patients with cystic fibrosis who may acquire new strain of M. abscessus from the environment, may harbor multiple subpopulations of bacteria, or may have persistent infections but intermittent bacteria shedding that could not be eradicated. WGS has emerged as a powerful molecular tool to accurately differentiate re-infection from relapse thus solving this conundrum.

Clinical and Laboratory Predictors of Shiga Toxin-Producing Escherichia coli Infection in Children With Bloody Diarrhea.

OBJECTIVES: Children with acute bloody diarrhea are at risk of being infected with Shiga toxin-producing Escherichia coli (STEC) and of progression to hemolytic uremic syndrome. Our objective was to identify clinical and laboratory factors associated with STEC infection in children who present with acute bloody diarrhea. METHODS: We performed a prospective cohort study of consecutive children younger than 18 years who presented with acute (<2-week duration) bloody diarrhea between August 1, 2013, and August 1, 2014. We excluded patients with a chronic gastrointestinal illness and/or an obvious noninfectious source of bloody stool. We obtained a standardized history and study laboratory tests, performed physical examinations, and recorded patient outcomes. RESULTS: Of the 135 eligible patients, 108 were enrolled; 27 declined consent. The median patient age was 3 years, and 56% were male. Ten (9%) patients tested positive for STEC (E coli O157:H7, n = 8; E coli O111, n = 1; E coli O103, n = 1), and 62 had negative stool culture results. Children infected with STEC were older (8.5 vs 3 years, respectively) (P < .001) and more likely to have abdominal tenderness (83% vs 17%, respectively) than those in the other groups. D-Dimer concentrations had a 70% sensitivity and 55% specificity for differentiating children with STEC from those with another cause of bloody diarrhea and 75% sensitivity and 70% specificity in differentiating children with a bacterial etiology from those with negative stool culture results. CONCLUSION: Clinical assessment and laboratory data cannot reliably exclude the possibility that children with bloody diarrhea have an STEC infection and are at consequent risk of developing hemolytic uremic syndrome. Abnormal D-dimer concentrations (>0.5 µg/mL) were insufficiently sensitive and specific for distinguishing patients with STEC from those with another bacterial cause of bloody diarrhea. However, this marker might be useful in identifying children whose bloody diarrhea is caused by a bacterial enteric pathogen.

Pattern of Pediatric Oncology Cases in the Western Region of Nepal

Childhood cancers form a rare and heterogeneous group which fortunately have a higher cure rate than adult cancers. A few non-profit organizations in Nepal have extended support to help patients suffering from cancer, but their main focus has been on adults. The objective of this study was to establish the pattern of childhood cancers in the Western region of Nepal. We reviewed cases receiving external radiotherapy with both palliative and curative intent between 28th September 2010 and 30th September 2015 at the Department of Radiotherapy and Oncology, Manipal Teaching Hospital affiliated with Manipal College of Medical Sciences, Pokhara, Nepal. Of the total of 1217 cases, 2.71% involved children. The gender distribution showed a male preponderance (M:F= 1.35:1). The patients' mean age was 11.4 years (range 2 - 14 years). Considering the caste, Brahmins and Gurungs constituted 33.0% and 21.2%, respectively, of children with cancer.

Epidermal growth factor preserves myelin and promotes astrogliosis after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) leads to reduced myelination and astrogliosis of the white matter in premature infants. No therapeutic strategy exists to minimize white matter injury in survivors with IVH. Epidermal growth factor (EGF) enhances myelination, astrogliosis, and neurologic recovery in animal models of white matter injury. Here, we hypothesized that recombinant human (rh) EGF treatment would enhance oligodendrocyte precursor cell (OPC) maturation, myelination, and neurological recovery in preterm rabbits with IVH. In addition, rhEGF would promote astrogliosis by inducing astroglial progenitor proliferation and GFAP transcription. We tested these hypotheses in a preterm rabbit model of IVH and evaluated autopsy samples from human preterm infants. We found that EGF and EGFR expression were more abundant in the ganglionic eminence relative to the cortical plate and white matter of human infants and that the development of IVH reduced EGF levels, but not EGFR expression. Accordingly, rhEGF treatment promoted proliferation and maturation of OPCs, preserved myelin in the white matter, and enhanced neurological recovery in rabbits with IVH. rhEGF treatment inhibited Notch signaling, which conceivably contributed to OPC maturation. rhEGF treatment contributed to astrogliosis by increasing astroglial proliferation and upregulating GFAP as well as Sox9 expression. Hence, IVH results in a decline in EGF expression; and rhEGF treatment preserves myelin, restores neurological recovery, and exacerbates astrogliosis by inducing proliferation of astrocytes and enhancing transcription of GFAP and Sox9 in pups with IVH. rhEGF treatment might improve the neurological outcome of premature infants with IVH. GLIA 2016;64:1987-2004.

Awareness and assessment of risk factors for lung cancer in residents of Pokhara Valley, Nepal.

OBJECTIVE: The objective of this study was to evaluate the awareness and assessment of lung cancer risk factors with respect to sociodemographic factors among residents of Pokhara Valley, Nepal. MATERIALS AND METHODS: A cross sectional study was carried out in 240 residents between 01 September 2009 and 31 March 2010 using a structured questionnaire containing details of lung cancer risk factors viz., smoking, environmental pollution, insecticide exposure, hereditary factors, protective diet and socio demographic details. Descriptive statistics and testing of hypothesis were used for the analysis using EPI INFO and SPSS 16 software. RESULTS: In the 240 subjects, the mean age was 33.4 ± SD 11.4 years, with a slight male preponderance in gender distribution (57.5% males vs. 42.5% females). 32.5% out of the study population were smokers (43.5% of males and 17.6% of females). Relationships could be established between gender and smoking (p=0.001, odds ratio=3.58), stoppage or restriction of tobacco use (p=0.001), smoking by mother during subjects' childhood as a motivation to develop smoking habit (p= 0.001), tobacco use as a cause of cancer (p=0.001), cancer as the most dreaded disease (p=0.009). Positive relationships were found between educational level and risk factors viz. smoking by mother during subjects' childhood (p= 0.03), wood or coal exposure causing lung cancer (p=0.0001), protection from lung cancer by consumption of green and yellow vegetables (p=0.0001) and insecticide exposure as a cause of lung cancer (p=0.0001). No strong relationship could be established between gender and outdoor pollution (p=0.721), insecticide exposure (p=0.219), protective diet (p=0.979) and hereditary factors (p=0.273). CONCLUSION: Awareness of lung cancer by tobacco use and other risk factors varied with socioeconomic status amongst residents of Pokhara. Despite their awareness of smoking as a risk factor for lung cancer, most of them still continue to smoke. Government and NGOs should gear up a population based counselling programme in this community.