Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model

<p>Abstract</p> <p>Background</p> <p>Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, <it>Aedes aegypti</it>, and the canine heartworm, <it>Dirofilaria immitis</it>.</p> <p>Results</p> <p>LAMP reactions amplifying the cytochrome oxidase subunit I gene demonstrated high sensitivity when a single purified <it>D. immitis </it>microfilaria was detected. Importantly, the robustness of the LAMP reaction was revealed upon identification of an infected mosquito carrying just a single parasite, a level easily overlooked using conventional microscopic analysis. Furthermore, successful detection of <it>D. immitis </it>in wild-caught mosquitoes demonstrated its applicability to field surveys.</p> <p>Conclusion</p> <p>Due to its simplicity, sensitivity, and reliability, LAMP is suggested as an appropriate diagnostic method for routine diagnosis of mosquito vectors carrying filarial parasites. This method can be applied to the survey of not only canine filariasis but also lymphatic filariasis, another major public health problem. Therefore, this method offers great promise as a useful diagnostic method for filarial parasite detection in endemic filariasis regions.</p

薬剤性過敏症症候群(DIHS)の皮疹部においてCD3陽性T細胞数に対するFoxP3陽性制御性T細胞数の割合は増加している

博士（医学）・甲第604号・平成25年11月27日© 2014 British Association of Dermatologists / The definitive version is available at http://onlinelibrary.wiley.com

Detection of G119S ace-1 R mutation in field-collected Anopheles gambiae mosquitoes using allele-specific loop-mediated isothermal amplification (AS-LAMP) method

Background Malaria vectors have developed resistance to the four families of insecticides available for public health purposes. For example, the kdr mutation is associated with organochlorines and pyrethroids resistance. It is of particular concern that organophosphate and carbamate resistance associated with the G119S ace-1 R mutation has recently increased in West Africa in extent and frequency, and is now spreading through the Anopheles gambiae malaria vector population. There is an urgent need to improve resistance management using existing insecticides and new tools to quickly assess resistance level for rapid decision-making. Methods DNA extracted from field-collected mosquitoes was used to develop the method. Specific primers were designed manually to match the mutation region and an additional mismatched nucleotide in the penultimate position to increase specificity. Other primers used are common to both wild and mutant types. The allele specific (AS)-LAMP method was compared to the PCR restriction fragment length polymorphism (PCR-RFLP) and real-time PCR (RT-PCR) methods using the genomic DNA of 104 field-collected mosquitoes. Results The primers designed for LAMP were able to distinguish between the wild type (ace-1 S ) and mutated type allele (ace-1 R ). Detection time was 50 min for the wild type homozygous and 64 min for the heterozygous. No amplification of the resistant allele took place within the 75-min test period when using the wild type primers. For the ace-1 R resistant type, detection time was 51 min for the resistant homozygous and 55 min for the heterozygous. No amplification of the wild type allele took place within the 75-min test period when using the resistant type primers. Gel electrophoresis of LAMP products confirmed that amplification was primer-DNA specific, i.e., primers could only amplify their target specific DNA. AS-LAMP, PCR-RFLP, and RT-PCR showed no significant difference in the sensitivity and specificity of their ace-1 R detection ability. Conclusions The AS-LAMP method could detect the ace-1 R mutation within 60 min, which is faster than conventional PCR-RFLP. This method may be used to quickly detect the ace-1 R mutation for rapid decision-making, even in less well-equipped laboratories

BUROSUMAB IN TUMOR-INDUCED OSTEOMALACIA

Patients with tumor-induced osteomalacia (TIO), an acquired paraneoplastic condition characterized by osteomalacia due to hypophosphatemia, exhibit a similar clinical picture to those with X-linked hypophosphatemic rickets/osteomalacia (XLH). The human monoclonal anti-fibroblast growth factor 23 (FGF23) antibody burosumab (KRN23) increases serum phosphate and improves bone turnover, fracture healing, pain, and physical function in XLH patients by inhibiting circulating FGF23; thus, burosumab is expected to be an effective treatment for TIO. We report here an interim analysis of a multicenter, open-label, intraindividual dose-adjustment study of burosumab (0.3 to 2.0 mg/kg every 4 weeks) in Japanese and Korean TIO patients. The primary endpoint was the fasting serum phosphate level at each visit. Key secondary endpoints were changes over time in bone biomarkers, pharmacodynamic markers, bone histomorphometric parameters, motor function, and patient-reported outcomes. Safety was assessed based on treatment-emergent adverse events (TEAEs). Thirteen patients received burosumab treatment, of whom 4 underwent bone biopsy. The mean dose after week 112 was approximately 1.0 mg/kg. After the first burosumab administration, mean serum phosphate levels increased and remained above the lower limit of normal and in the normal range from weeks 14 to 112. Bone biomarkers initially increased, reaching maximum values at week 16 or 24, and then gradually decreased. After burosumab treatment, patients were able to walk further (evaluated by the 6-minute walk test), reported decreased pain levels, and showed a tendency toward healing of baseline fractures and pseudofractures. Two patients discontinued, one each due to disease progression and consent withdrawal. Burosumab was generally well tolerated, with no treatment-related TEAEs of grade ≥3 and no treatment-related serious AEs. In conclusion, the interim results of this first study of burosumab to treat TIO patients indicate that this drug has the potential to provide clinical benefit for patients with unresectable tumors. The full study results are eagerly anticipated

血清TARC/CCL17値は薬剤性過敏症症候群(DIHS) の早期診断および病勢の指標となりうる。

BACKGROUND:Drug-induced hypersensitivity syndrome (DIHS)/drug rash with eosinophilia and systemic symptoms (DRESS) is a serious acute drug reaction with fever, cutaneous eruption, lymphadenopathy, and several visceral dysfunctions. Eosinophilia is a common hematological abnormality in DIHS/DRESS suggesting that the Th2-type immune response is involved. Thymus and activation-regulated chemokine (TARC/CCL17) is a family of CC chemokines known to play an important role in Th2-mediated immune-inflammatory processes. OBJECTIVE:We investigated the pathogenic role of TARC in patients with DIHS. METHODS:Sera were obtained from 8 patients with DIHS, 7 patients with Stevens-Johnson syndrome/Toxic epidermal necrolysis (SJS/TEN), and 14 patients with drug-induced maculopapular exanthema (MPE). Serum TARC levels were measured by ELISA. TARC levels were then compared with clinical symptoms and various hematological parameters. In addition, a biopsy was taken from the lesional skin of patients with DIHS and stained with anti-TARC Ab and anti-CD11c Ab. RESULTS:Serum TARC levels in patients with DIHS were significantly higher than those in patients with SJS/TEN and MPE during the acute phase. Serum TARC levels in DIHS patients correlated with skin eruptions, serum sIL-2R levels, eosinophil counts, and serum IL-5 levels. Immunohistochemical staining revealed that TARC was mainly expressed on CD11c+ dermal dendritic cells in patients with DIHS. CONCLUSION:Serum TARC levels may be associated with the initial presentation of DIHS as well as disease activity during the course. Thus, they could be useful as an indicator for early diagnosis and assessment of disease activity in DIHS. CD11c+ dendritic cells may be the main source of TARC in patients with DIHS.博士（医学）・甲第597号・平成25年3月15日Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved

Pooled blood volume measured by final flat-panel detector computed tomography predicts outcome after endovascular thrombectomy for acute ischemic stroke

Background: Pooled blood volume (PBV), measured in real-time in the angiography room using an angiography system, correlates with cerebral blood volume (CBV). We examined the usefulness of PBV in endovascular thrombectomy (EVT) for acute ischemic stroke (AIS). Methods: EVT for AIS in the anterior circulation (internal carotid artery (ICA) and middle cerebral artery (MCA)) was performed in 31 cases (13 males, 18 females, average age 75.7 years). PBV was acquired using a biplane flat-panel detector (FD) angiographic system. Then, we measured the average PBV value in the M1-6 regions similar to the Alberta Stroke Program Early CT score (ASPECTS) before and after EVT. We investigated factors associated with favorable outcome at 90 days after EVT. Results: There were 13 patients (41.9%) in the good outcome group (mRS (modified Rankin Scale) ≦2) and 18 patients (58.1%) in the poor outcome group (mRS>2). In univariate analysis, NIHSS (National Institutes of Health Stroke Scale) (odds ratio [OR] 0.74, 95% CI 0.57–0.87, p < 0.0001) and post PBV value (odds ratio [OR] 1.13, 95% CI 1.03–1.29, p = 0.0086) were significantly associated with good outcome. The good outcome group had significantly higher post-thrombectomy PBV value (3.69 ± 0.32 ml/100 g versus 2.78 ± 0.93 ml/100 g, P = 0.002) compared to that of the poor outcome group. The relationship between pre-thrombectomy PBV value and outcome at 90 days was not significant. Conclusions: Post-operative PBV value measured by FD-CT (computed tomography) correlated with 90-day outcome after EVT for AIS. FD-CT-PBV would be one of the good predictors of clinical outcome

Short- and Long-term Results of Modified Frey's Procedure in Patients with Chronic Pancreatitis: A Retrospective Japanese Single-Center Study

BACKGROUND: The study aim was to determine the short- and long-term results of surgical drainage procedure for chronic pancreatitis at a single center in Japan.METHODS: The records of 28 consecutive patients were retrospectively reviewed. All patients underwent surgery at Kobe University Hospital between June 1999 and April 2013. Long-term follow-up was performed in all patients for a median period of 77 months.RESULTS: The 26 men (93%) and 2 women (7%) had a mean age of 47 years. The etiology of pancreatitis was chronic alcohol abuse in 24 patients (86%). The major indication for surgery was persistent symptoms (97%). Modified Frey's procedure in 21 patients, lateral pancreaticojejunostomy (LPJ) in 6 patients, LPJ and distal pancreatectomy in one patient, were performed. There was no postoperative mortality. Postoperative morbidity occurred in 6 patients (21%). The percentage of pain-free patients after surgery was 97%, and further acute exacerbation was prevented in 97%. Two patients (6%) required subsequent surgery for infectious pancreatic cyst and intraabdominal abscess. Of the patients that completed follow-up, 13 (46%) had diabetes mellitus, including 5 patients (19%) with new-onset diabetes, and 6 patients (19%) developed pancreatic exocrine insufficiency.CONCLUSIONS: Modified Frey's procedure is safe, feasible, and effective to manage chronic pancreatitis. The technique prevents further exacerbations and maintains appropriate pancreatic endocrine and exocrine function