Development of a LAMP assay for rapid and sensitive detection and differentiation of Mycobacterium avium subsp. avium and subsp. hominissuis.

Mycobacterium avium causes atypical mycobacterial infection in humans and animals worldwide. M. avium comprises the subspecies avium (MAA), hominissuis (MAH), silvaticum (MAS) and paratuberculosis (MAP). The M. avium complex (MAC), comprising M. avium and M. intracellulare, causes opportunistic infections of humans. M. avium subsp. avium (MAA) mainly causes avian tuberculosis while subsp. hominissuis (MAH) mainly infects pig. Distinguishing between these two subspecies is essential to the effective control of these atypical mycobacterial infections and minimization of the resulting economic loss. For this purpose, we developed a loop-mediated isothermal amplification (LAMP) assay that rapidly and sensitively detects and differentiates MAA and MAH. This MAA-LAMP assay targeting IS901 correctly detected four MAA isolates but did not detect 27 MAH and 19 non-MAA/non-MAH mycobacterial isolates. The MAAH-LAMP assay targeting IS1245 detected four MAA and 27 MAH isolates but not the other 19 mycobacterial isolates. We believe that implementation of this LAMP assay will significantly improve public health and safety. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycobacterium avium, which is pathogenic for humans and animals, represents a continuing threat to public health and safety and to food production. Therefore, improved methods are urgently required to readily and efficiently identify M. avium subspecies. Compared with conventional PCR methods, the LAMP assay herein developed more rapidly detects and better distinguishes between two major M. avium subspecies that cause disease of pig. Importantly, this highly accurate and sensitive LAMP assay detects mycobacterial DNAs using real-time fluorescence or the unaided eye with a colour-change dye, making it ideal for translation to the clinic and slaughterhouse.

Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting.

OBJECTIVES: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses. METHODS: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test. RESULTS: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5). CONCLUSIONS: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.

Molecular epidemiological survey and phylogenetic analysis of bovine influenza D virus in Japan.

The influenza D virus, a new member of the Orthomyxoviridae family, is predominantly found in cattle. Although viral pathology and clinical disease in cattle appear mild, this virus plays an important role as a trigger of bovine respiratory disease (BRD). BRD is a costly illness worldwide. Thus, epidemiological surveys of the influenza D virus are necessary. Here, we conducted a molecular epidemiological survey for the influenza D virus in healthy and respiratory-diseased cattle in Japan. We found that 2.1% (8/377) of the cattle were infected with influenza D. The cattle with and without respiratory symptoms had approximately equal amounts of the virus. A full-genome sequence analysis revealed that the influenza D virus that was isolated in Japan formed an individual cluster that was distinct from the strains found in other countries. These results suggest that this virus might have evolved uniquely in Japan over a long period of time and that the viral pathology of Japanese strains might be different from the strains found in other countries. Continuous surveillance is required to determine the importance of this virus and to characterize its evolution.

Chimeric Allografts Induced by Short-Term Treatment With Stem Cell Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: II, Study in Miniature Swine.

Transplantation is now lifesaving therapy for patients with end-stage organ failure but requires lifelong immunosuppression with resultant morbidity. Current immunosuppressive strategies inhibit T cell activation and prevent donor-recipient engagement. Therefore, it is not surprising that few host cells are demonstrated in donor grafts. However, our recent small animal studies found large numbers of recipient stem cells present after transplantation and pharmacological mobilization, resulting in a chimeric, repopulated organ. We now confirm these findings in a well-characterized large animal preclinical model. Here, we show that AMD3100 and FK506 mobilization of endogenous stem cells immediately post kidney transplantation combined with repeat therapy at 1, 2, and 3 months led to drug-free long-term survival in maximally immunologically mismatched swine. Three long-term recipients have stable chimeric transplants, preserved antidonor skin graft responses, and normal serum creatinine levels despite withdrawal of all medication for 3 years.

Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats.

Transplant tolerance allowing the elimination of lifelong immunosuppression has been the goal of research for 60 years. The induction of mixed chimerism has shown promise and has been extended successfully to large animals and to the clinic; however, it remains cumbersome and requires heavy early immunosuppression. In this study, we reported that four injections of AMD3100, a CXCR4 antagonist, plus eight injections of low-dose FK506 (0.05 mg/kg per day) in the first week after kidney transplantation extended survival, but death from renal failure occurred at 30-90 days. Repeating the same course of AMD3100 and FK506 at 1, 2 and 3 mo after transplant resulted in 92% allograft acceptance (n = 12) at 7 mo, normal kidney function and histology with no further treatment. Transplant acceptance was associated with the influx of host stem cells, resulting in a hybrid kidney and a modulated host immune response. Confirmation of these results could initiate a paradigm shift in posttransplant therapy.

Fourier transform microwave and millimeter-wave spectroscopy of bromoiodomethane, CH2BrI.

Bromoiodomethane, CH2BrI, is a molecule of natural origin emitted in significant amount into the marine boundary layer. It can easily be decomposed by solar radiation, releasing Br and I atoms in the troposphere, which in turn impacts the atmospheric chemistry. Spectroscopy is an invaluable tool to monitor species present in the atmosphere. Since no high-resolution spectroscopic studies are available for this dihalomethane, we have investigated the rotational spectra of the two bromine isotopologues of CH2BrI in its vibrational ground state in the microwave and millimeter-wave regions. Transitions of b-type have been recorded by Fourier transform microwave spectroscopy below 25 GHz while both a- and b-type spectral lines have been measured below 230 GHz. Observed transitions correspond to energy levels with J ≤ 132 and Ka ≤ 14. Molecular constants including those describing the nuclear quadrupole coupling tensors for (79)Br, (81)Br, and (127)I were accurately determined from the least-squares analysis of a total of 1873 distinct transition frequencies (of which 943 belong to the CH2(79)BrI isotopologue). An experimental (r0) structure of the title species has been derived from the two sets of rotational constants.

Mobilization of host stem cells enables long-term liver transplant acceptance in a strongly rejecting rat strain combination.

Careful examination of liver, kidney and heart transplants in human recipients has revealed small numbers of host bone marrow derived stem cells in the graft. If the limited recipient repopulation of a donor graft that is currently observed could be facilitated, it is possible that conversion to a predominantly host phenotype would permit long-term graft function without immunosuppression. We proposed to "engineer" repopulation after transplant in a strain combination (dark agouti [DA] to Lewis green fluorescent protein+[LEW GFP+]) which rejects liver grafts strongly, a model that more closely resembles the situation in humans. Treatment on days 0, 1, 2, 3 and 7 after transplantation with low-dose (0.1 mg/kg) tacrolimus (T) designed to blunt rejection combined with plerixafor (P) to mobilize host stem cells resulted in greater than 180 days graft survival with extensive albeit spotty conversion of a small (50%) DA graft to the recipient LEW GFP+ genotype. Subsequent skin grafting revealed donor-specific graft prolongation. The T plus P treatment resulted in higher levels of Lin-Thy1+CD34+CD133+ stem cells and Foxp3+ regulatory T cells in the blood and liver at day 7. Thus, pharmacological mobilization of host stem cells sustains liver allografts by two mechanisms: repopulation of injured donor cells and regulation of the immune response.

Risk factors and predictors for surgical site infection after hepatic resection.

Strict control of blood glucose levels with insulin in a surgical intensive care unit reduces postoperative morbidity and mortality. The aim of this study was to identify risk factors and the predictors for the prevention of surgical site infection (SSI) in a consecutive series of hepatectomised cases in a single institution. The association between SSI and various clinical parameters was investigated in 152 patients who underwent hepatic resection at Kochi Medical School from January 2000 through March 2007. The incidence of SSI in these patients was 14.5%. Multivariate analysis identified four independent parameters correlating with the occurrence of SSI: (i) body mass index >23.6 kg/m(2); (ii) estimated blood volume loss >810 mL; (iii) presence of postoperative bile leakage of organ/space SSI; and (iv) use of the sliding scale method for postoperative glucose control. There was no observed SSI after liver resection in the group whose postoperative blood glucose levels were controlled by an artificial pancreas. This study reveals that lack of postoperative glycaemic control is associated with a significantly higher incidence of postoperative infectious complications and longer hospitalisation. Obesity and the level of intraoperative estimated blood loss and bile leakage after hepatic resection are also risk factors with predictive value for SSI. Artificial pancreas is a safe and beneficial device to perform postoperative strict glycaemic control without hypoglycaemia for patients who undergo hepatic resection for liver diseases.

Accuracy and reliability of continuous blood glucose monitor in post-surgical patients.

BACKGROUND: The STG-22 is the only continuous blood glucose monitoring system currently available. The aim of this study is to determine the accuracy and reliability of the STG-22 for continuously monitoring blood glucose level in post-surgical patients. METHODS: Fifty patients scheduled for routine surgery were studied in surgical intensive care unit (ICU) of a university hospital. After admission to the ICU, the STG-22 was connected to the patients. An attending physician obtained blood samples from a radial arterial catheter. Blood glucose level was measured using the ABL800FLEX immediately after blood collection at 0, 4, 8, and 16 h post-admission to the ICU (total of 200 blood glucose values). RESULTS: The correlation coefficient (R2) was 0.96. In the Clarke error grid, 100% of the paired measurements were in the clinically acceptable zone A and B. The Bland and Altman analysis showed that bias+/-limits of agreement (percent error) were 0.04(0.7)+/-0.35(6.3) mmol (mg/dl) (7%), -0.11(-2)+/-1.22(22) (15%) and -0.33(-6)+/-1.28(23) (10%) in hypoglycemia (<70(3.89) mmol (mg/dl), normoglycemia (3.89(70)-10(180) mmol (mg/dl), and hyperglycemia (>10(180) mmol (mg/dl), respectively. CONCLUSIONS: The STG-22 can be used for measuring blood glucose level continuously and measurement results are consistent with intermittent measurement (percentage error within 15%). Therefore, the STG-22 is a useful device for monitoring in blood glucose level in the ICU for 16 h.

Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects.

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.

Bilateral ischemic necrosis of the tongue due to disseminated intravascular coagulation.

Ischemic necrosis of the tongue is a rare condition because the tongue has a rich blood supply. Temporal arteritis appears to be the most frequent cause of tongue necrosis. The case is presented of an 82-year-old man who developed bilateral ischemic necrosis of the tongue. The necrosis was considered to be a consequence of thrombosis of the bilateral lingual arteries due to disseminated intravascular coagulation. To the authors' knowledge, this is the first reported case of necrosis of the tongue secondary to disseminated intravascular coagulation.

Hyperfine resolved spectrum of the bromomethyl radical, CH2Br, by Fourier transform microwave spectroscopy.

Pure rotational spectra of the bromomethyl radical, CH(2)Br, were measured by using a Fourier transform microwave (FT-MW) spectrometer in order to fully resolve hyperfine structures arising from both the bromine and hydrogen nuclei. We detected a total of 124 lines for the (79)Br and (81)Br isotopomers, including K(a)=0 (ortho species) and K(a)=1 (para species). No hyperfine splitting due to the hydrogen nuclei was observed for the para species, directly confirming the planarity of the radical. We conducted a global analysis of our present FT-MW results and previous measurements in the millimeter-wave region and obtained an exhaustive list of molecular constants. The sign of the Fermi constant of the bromine nucleus was unambiguously determined to be positive, which is opposite to that found in previous work in the millimeter-wave region and in electron spin resonance experiment on this radical. The present study permitted a systematic comparison to be made of the hyperfine coupling constants of both the halogen and hydrogen nuclei for CH(2)X-type compounds, where X=F, Cl, and Br.

Usefulness of multi-detector row computed tomography for accurate preoperative assessment of pancreatic adenocarcinoma: report of a case.

A 74-year-old female underwent surgical treatment for adenocarcinoma of the pancreatic head. Preoperative multi-detector row computed tomography (MD-CT) demonstrated tumor invasion into the accessory right colic vein and the branch of the middle colic artery (MCA), which was not detected by digital subtraction angiography. MD-CT showed anatomical variants in the left hepatic artery arising from the left gastric artery, and the right posterior hepatic artery arising from the superior mesenteric artery. Three-dimensional reconstruction CT generated a clear picture of the anatomy of the region concerned, which is essential for a safe operation. The MD-CT findings were highly consistent with the intraoperative findings. We have demonstrated that MD-CT is an important and highly accurate modality for pancreatic surgery.

Laparoscopic incisional hernia repair. A new mesh fixation method without stapling.

BACKGROUND: Recent advances in laparoscopic surgery have made various abdominal surgeries possible. To avoid wound infection, mesh repair of abdominal incisional hernias is performed laparoscopically. Here we present a new procedure to fix mesh to the abdominal wall. SURGICAL TECHNIQUE: Four anchoring sutures are made using a suture-grasping device; the additional transabdominal sutures are then made with a modified double-needle device. Additional circumferential fixation with tacks is not necessary. CONCLUSIONS: This new mesh fixation method involves simple suturing techniques and is less time consuming than the conventional procedure.

Laparoscopic lymph node dissection around the inferior mesenteric artery for cancer in the lower sigmoid colon and rectum: is D3 lymph node dissection with preservation of the left colic artery feasible?

BACKGROUND: When we perform laparoscopic lymph node dissection around the inferior mesenteric artery (IMA), we preserve the left colic artery (LCA) to maintain the blood supply to the proximal sigmoid colon. In this study, we present our laparoscopic D2 and D3 lymph node (LN) dissection technique and evaluate its applicability and safety. METHODS: We performed LN dissection on 23 rectal and lower sigmoid colon cancer cases from April 2002 to December 2004. For D3 LN dissection, the incision to the mesosigmoid extends to just before the root of the IMA, which is exposed with an ultrasonic cutting and coagulating surgical device to avoid bleeding. Then, the arterial wall is exposed with a dissecting electrocautery spatula down to the LCA, at least 2 cm of which is exposed. Adipose tissue surrounding the IMA and inferior mesenteric vein is dissected. For D2 LN dissection, we partially expose the IMA to confirm the location of the LCA. RESULTS: The mean times taken for D2 and D3 LN dissections were 36.2 and 68.2 min, respectively. Both procedures took longer in male patients. There was a trend for the procedure overall to take less time in female patients. However, D2 dissection took significantly longer in male than female patients (p < 0.05). In women, D3 dissection took significantly longer than D2 (p < 0.05), but this trend was not seen in men. Increased experience among surgeons with this procedure was associated with significantly faster LN dissections in men (p < 0.05), but not in women (p = 0.493). Pearson product moment analysis identified a relationship between body mass index (BMI) and the time taken for D2 LN dissection (r = 0.765), but not D3 LN dissection (r = 0.158). There was no treatment-related morbidity with this technique. CONCLUSIONS: This method was safe and feasible for all patients in this series, but takes longer to perform in male patients.

CT imaging of intrabiliary growth of colorectal liver metastases: a comparison of pathological findings of resected specimens.

The objective of this study was to assess the usefulness of CT in the pre-operative evaluation of macroscopic intrabiliary tumour growth of colorectal liver metastases. 25 metastatic nodules of 18 patients who underwent an initial hepatectomy for colorectal liver metastasis were retrospectively evaluated. The CT appearance and pathological findings of the resected specimens were correlated. A number of unusual peritumoral features associated with intrabiliary tumour growth were detected by pre-operative CT. These were classified into three patterns: (1) thickened portal tract; (2) intrahepatic bile duct dilatation; and (3) a wedge-shaped area with enhancement. In 8 (32%) of the 25 nodules the portal tract was depicted as thicker than usual and these features were found proximal to the tumour in three instances, distal to the tumour in four instances, and both proximal and distal in one instance. All of the three intrabiliary tumours larger than 30 mm resulted in thickening of the portal tract. Intrahepatic bile duct dilatation was detected in association with 10 (40%) of 25 nodules. Bile duct dilatation was observed in more than one segment when intrabiliary tumour reached the hepatic hilus from the tumour. The presence of bile duct dilatation was not related to either the size of the tumour or the extent of intrabiliary tumour growth. An abnormally high density wedge-shaped area on contrast enhanced CT was another feature indicating intrabiliary tumour growth and was seen in association with four nodules. Such areas were seen in the liver parenchyma distal to the tumour on three occasions, or encompassing the tumour on one accasion. This wedge-shaped area appeared as a well demarcated dark red-brown region in the cut surface of the resected specimen. CT was useful for detecting the presence of intrabiliary tumour growth with these three patterns of radiological findings in patients with liver metastases from colorectal cancer

A new staging system for mass-forming intrahepatic cholangiocarcinoma: analysis of preoperative and postoperative variables.

BACKGROUND: The objective of this study was to analyze the clinicopathologic variables and the postoperative outcome in patients with mass-forming intrahepatic cholangiocarcinoma (ICC) to identify important factors for predicting postresection prognosis. Although it has been reported that mass-forming ICC has a different etiology and biologic features compared with hepatocellular carcinoma (HCC), patients with ICC have been dealt with clinicopathologically in the same manner as patients with HCC. METHODS: Sixty patients who underwent hepatectomy for mass-forming ICC with curative intent between 1981 and 1999 were studied. Fourteen preoperative clinical and diagnostic parameters and 12 postoperative surgicopathologic parameters were analyzed. RESULTS: The rate of operative mortality in this patient cohort was 5%, and the overall 1-year, 3-year, and 5-year survival rates were 68%, 35%, and 29%, respectively, with a median survival of 19.6 months. A multivariate analysis revealed that independent negative prognostic factors were 1) hepatic regional lymph node metastasis, 2) multiple tumor presentation, 3) symptomatic tumor, and 4) the presence of vascular invasion. Using these factors, a new staging system was devised: Stage I disease was defined as a solitary tumor without vascular invasion, Stage II disease was defined as a solitary tumor with vascular invasion, Stage IIIA disease was defined as multiple tumors with or without vascular invasion, Stage IIIB disease was defined as any tumor with regional lymph node metastasis, and Stage IV disease was defined as any tumor with distant metastases. The Kaplan-Meier estimated 3-year survival rate and the median survival for each subgroup were 74% for patients with Stage I disease (median survival is the time when the cumulative survival rate of some patients' group declined to 50%; thus, the median survival could not be calculated in patients with Stage I disease because survival was 74% at the latest follow-up), 48% and 26.2 months for patients with Stage II disease, 18% and 16.8 months for patients with Stage IIIA disease, and 7% and 11.2 months for patients with Stage IIIB disease, respectively (P < 0.0001). None of the patients met the criteria for Stage IV disease. CONCLUSIONS: The current results support the use of a new staging system for patients with ICC that is simple and predicts well the differences in survival after patients undergo hepatic resection.

Seroprevalence of antibodies against spotted fever group rickettsia among dogs and humans in Okinawa, Japan.

We evaluated serum antibodies against Rickettsia japonica in 517 dogs (430 stray dogs and 87 pet dogs) and 164 humans in Okinawa, Japan, by indirect immunofluorescence assay. The seropositive rate in stray dogs was significantly higher than that in pet dogs (30.7 versus 4.6%, P<0.01). This high prevalence rate is attributed to the understandably frequent environmental exposure of stray dogs to tick infestation. Human samples obtained from Okinawa and Sapporo also showed a significant difference in seropositive antibody percentages (45.1 and 12.0%, respectively, P<0.01). This result suggests that there has been pre-exposure to spotted fever group rickettsia in humans in Okinawa.