Rapid detection of the most common high-risk human papillomaviruses by loop-mediated isothermal amplification

Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for development of cervical cancer. At present, polymerase chain reaction (PCR)-based methods, the most widely molecular tools used for HPV detection, are time-consuming and require expensive instruments. In this study, loop-mediated isothermal amplification (LAMP) was established for detection of HPV types 16, 18, 45 and 58 which are frequently found in Thailand. The optimal condition for detection of these high risk HPVs was 63°C for 60min. Since a white magnesium pyrophosphate precipitate is a characteristic by product of the LAMP reaction which can be visualized directly by the naked eye, the entire assay time of LAMP is 1h compared to 6-8h of for a nested PCR detection. The detection limit of LAMP assay was shown to be equivalent to nested PCR that could amplify 10(2) copies of HPV-18 and 10(3) copies of HPV 16, 45 and 58, as determined by either turbidity detection or agarose gel electrophoresis. No cross-reaction was observed, indicating that LAMP assay has high type-specificity. The assay showed successful detection of HPV in 56 clinical specimens. Using nested PCR as the gold standard, the sensitivity, specificity, negative predictive values and positive predictive values of LAMP assay were 100%. In conclusion, LAMP assay is a high efficiency, low cost diagnostic tool, useful for rapid, accurate, direct detection of HPV for clinical diagnosis.     

Verification of complete bisulfite modification using Calponin-specific primer sets

ObjectivesTo verify complete bisulfite modification.Design and methodsBisulfite modified DNA was screened by PCR with Calponin-specific primer sets prior to detecting methylation status of 14-3-3σ by methylation-specific PCR.ResultsFalse positive methylation was obtained from incomplete bisulfite modification. The lower detection limit of wild type Calponin-specific primer is approximately 0.375 ng of unmodified DNA.ConclusionsVerification of complete bisulfite modification by Calponin-specific primer sets can reduce misinterpretation of methylation.     

Primary hepatic clear cell myomelanocytic tumor. Case report and review of the literature

A case of hepatic clear cell myomelanocytic tumor in a 31-year-old woman presenting clinically with abdominal pain is reported. Histopathologic examination showed a lesion characterized by a population of large epithelioid cells with clear or eosinophilic granular cytoplasm, rich in glycogen. Immunohistochemically, the tumor cells were positive for HMB-45, Melan-A and muscle-specific actin, but negative for epithelial markers, desmin, S-100 protein, and neuroendocrine markers. Ultrastructurally, the tumor cells had abundant glycogen, well-developed rough endoplasmic reticulum, microtubules and aberrant melanosomes. Clinical and pathologic features with a brief review of the relevant literature for hepatic CCMMT as a variant of perivascular epithelioid cell tumor (PEComa) are discussed.     

Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis

Pythiosis is an emerging and life-threatening infectious disease of humans and animals living in tropical and subtropical countries and is caused by the fungus-like organism Pythium insidiosum. Antifungals are ineffective against this pathogen. Most patients undergo surgical removal of the infected organ, and many die from advanced infections. Early and accurate diagnosis leads to prompt management and promotes better prognosis for affected patients. Immunohistochemical assays (IHCs) have been developed using rabbit antibodies raised against P. insidiosum crude extract, i.e., culture filtrate antigen (CFA), for the histodiagnosis of pythiosis, but cross-reactivity with pathogenic fungi compromises the diagnostic performance of the IHC. Therefore, there is a need to improve detection specificity. Recently, the elicitin protein, ELI025, was identified in P. insidiosum, but it was not identified in other human pathogens, including true fungi. The ELI025-encoding gene was successfully cloned and expressed as a recombinant protein in Escherichia coli. This study aims to develop a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) and to compare its performance with the previously reported anti-CFA-based IHC. Thirty-eight P. insidiosum histological sections stained positive by anti-ELI-based and anti-CFA-based IHCs indicating 100% detection sensitivity for the two assays. The anti-ELI antibody stained negative for all 49 negative-control sections indicating 100% detection specificity. In contrast, the anti-CFA antibody stained positive for one of the 49 negative controls (a slide prepared from Fusarium-infected tissue) indicating 98% detection specificity. In conclusion, the anti-ELI based IHC is sensitive and specific for the histodiagnosis of pythiosis and is an improvement over the anti-CFA-based assay.     

The unique characteristics of Thai Leber hereditary optic neuropathy: analysis of 30 G11778A pedigrees

Leber hereditary optic neuropathy (LHON) is characterized by acute or subacute bilateral visual loss, and affects mostly young males. The most common mitochondrial DNA mutation responsible for LHON worldwide is G11778A. Despite different genetic backgrounds, which are believed to influence the disease expression, most features of LHON are quite common in different populations. However, there seem to be a few ethnic-specific differences. Analyses of our 30 G11778A LHON pedigrees in Thailand showed some characteristics different from those of Caucasians and Japanese. In particular, our pedigrees showed a lower male to female ratio of affected persons (2.6:1) and much higher prevalence of G11778A blood heteroplasmy (37% of the pedigrees contained at least one heteroplasmic G11778A individual). Heteroplasmicity seemed to influence disease manifestation in our patients but did not appear to alter the onset of the disease. The estimated overall penetrance of our G11778A LHON population was 37% for males and 13% for females. When each of our large pedigrees were considered separately, disease penetration varied from 9 to 45% between the pedigrees, and also varied between different branches of the same large pedigree. Survival analysis showed that the secondary LHON mutations G3316A and C3497T had a synergistic deleterious effect with the G11778A mutation, accelerating the onset of the disease in our patients.     

Creation of an integrated outcome database for pediatric anesthesia

Outcome analysis is essential to health care quality improvement efforts. Pediatric anesthesia faces unique challenges in analyzing outcomes. Anesthesia most often involves a one‐time point of care interaction where work flow precludes detailed feedback to care givers. In addition, pediatric outcome evaluations must take into account patients' age, development, and underlying illnesses when attempting to establish benchmarks. The deployment of electronic medical records, including preoperative, operative, and postoperative data, offers an opportunity for creating datasets large and inclusive enough to overcome these potential confounders. At our institution, perioperative data exist in five distinct environments. In this study, we describe a method to integrate these datasets into a single web‐based relational database that provides researchers and clinicians with regular anesthesia outcome data that can be reviewed on a daily, weekly, or monthly basis. Because of its complexity, the project also entailed the creation of a ‘dashboard,’ allowing tracking of data trends and rapid feedback of measured metrics to promote and sustain improvements. We present the first use of such a database and dashboard for pediatric anesthesia professionals as well as successfully demonstrating its capabilities to perform as described above.     

Alteration of noncollagenous bone matrix proteins in distal renal tubular acidosis

Our previous report on bone histomorphometry in patients with distal renal tubular acidosis (dRTA) revealed decreased bone formation rate (BFR) when compared to healthy subjects. The abnormality improved significantly after alkaline therapy. The modest increase in osteoblastic surface, after correction of metabolic acidosis, could not explain the striking improvement in bone formation, suggesting additional influence of metabolic acidosis on osteoblast function and/or bone matrix mineralization. Osteoblasts and, to a lesser extent, osteoclasts synthesize and secrete bone matrix including type I collagen and various noncollagenous proteins (NCPs). Substantial evidence suggested diverse functions of NCPs related to bone formation, resorption, and mineralization. Metabolic acidosis, through its effect on bone cells, may result in an alteration in the production of NCPs. Our study examined bone histomorphometry with detailed analysis on the mineralization parameters and NCPs expression within the bone matrix of patients with dRTA before and after treatment with alkaline. Seven dRTA patients underwent bone biopsy at their initial diagnosis and again 12 months after alkaline therapy. Bone mineral density (BMD) and bone histomorphometry were obtained at baseline and after the treatment. The expression of NCPs was examined by immunohistochemistry, quantitated by digital image analysis, and reported as a percentage of area of positive staining or mineralized trabecular bone area. Alkaline therapy normalized the low serum phosphate and PTH during acidosis. The reduction in BMD at baseline improved significantly by the treatment. Bone histomorphometry demonstrated the increase in osteoid surface and volume without significant alteration after acidosis correction. In comparison to the normal subjects, osteoid thickness was slightly but insignificantly elevated. Osteoblast and osteoclast populations and their activities were suppressed. The reduction in mineral apposition rate and adjusted apposition rate were observed in conjunction with the prolongation of mineralization lag time. Alkaline therapy improved the mineralization parameters considerably. In addition to the increase in BFR relative osteoblast number after acidosis correction, osteocalcin expression in the bone matrix increased significantly from 16.7% to 22.3%. Six of seven patients had decreased osteopontin expression. In conclusion, the abnormal bone remodeling in dRTA is characterized by low turnover bone disease with some degree of defective mineralization. Alteration of NCPs expression suggested the effect of metabolic acidosis on bone cells. Alkaline therapy increased bone mass through the restoration of bone mineral balance and, perhaps, improved osteoblast function.