Comparison of molecular diagnosis with serum markers and synovial fluid analysis in patients with prosthetic joint infection.

AIMS: The aim of this study was to compare the results of 16S/28S rRNA sequencing with the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and synovial fluid analysis in the diagnosis of prosthetic joint infection (PJI). PATIENTS AND METHODS: Between September 2015 and August 2016, 214 consecutive patients were enrolled. In the study population, there were 25 patients with a PJI and 189 controls. Of the PJI patients, 14 (56%) were women, and the mean age at the time of diagnosis was 65 years (38 to 83). The ESR and CRP levels were measured, and synovial fluid specimens were collected prospectively. Synovial fluid was subjected to reverse transcription polymerase chain reaction (RT-PCR)/sequence analysis targeting the 16S/28S rRNA, and to conventional culture. Laboratory personnel who were blind to the clinical information performed all tests. The diagnosis of PJI was based on the criteria of the Musculoskeletal Infection Society. RESULTS: A total of 25 patients had a confirmed PJI. In 20 cases of monomicrobial PJI, the PCR products could be perfectly matched with the 16S/28S rRNA genes specific for different species of bacteria provided by sequence analysis. Of the five polymicrobial cases of PJI, 16S/28S rRNA PCR sequence analysis failed to identify the concordant bacteria species. In the 189 control patients, there was one false-positive RT-PCR result. The sensitivity and specificity of the molecular diagnosis method were 100% (95% confidence interval (CI) 85.7 to 100) and 99.5% (95% CI 97.1 to 99.9), respectively, whereas the positive and negative predictive values of PCR were 96.1% (95% CI 79.6 to 99.9) and 100% (95% CI 98.1 to 100), respectively. The PCR results were significantly better than serological diagnostic methods (p = 0.004 and p = 0.010 for ESR and CRP, respectively), the synovial fluid white blood cell (WBC) count (p = 0.036), and percentage of polymorphonuclear cells (PMN%) (p = 0.014). CONCLUSION: Stepwise RT-PCR and sequence analysis of the 16S/28S rRNA carried out under stringent laboratory conditions achieved highly sensitive and specific results for the differentiation between aseptic and septic joints undergoing arthroplasty. Sequence analysis successfully identified bacterial strains in monomicrobial infections but failed to identify molecular targets in polymicrobial infections. Further refinement of the protocols to identify the bacteria in polymicrobial infections is needed. Cite this article: Bone Joint J 2018;100-B:1345-51.

A regime of two intravenous injections of tranexamic acid reduces blood loss in minimally invasive total hip arthroplasty: a prospective randomised double-blind study.

Tranexamic acid (TXA), an inhibitor of fibrinolysis, reduces blood loss after total knee arthroplasty. However, its effect on minimally invasive total hip arthroplasty (THA) is not clear. We performed a prospective, randomised double-blind study to evaluate the effect of two intravenous injections of TXA on blood loss in patients undergoing minimally invasive THA. In total, 60 patients (35 women and 25 men with a mean age of 58.1 years; 17 to 84) who underwent unilateral minimally invasive uncemented THA were randomly divided into the study group (30 patients, 20 women and ten men with a mean age of 56.5 years; 17 to 79) that received two intravenous injections 1 g of TXA pre- and post-operatively (TXA group), and a placebo group (30 patients, 15 women and 15 men with a mean age of 59.5 years; 23 to 84). We compared the peri-operative blood loss of the two groups. Actual blood loss was calculated from the maximum reduction in the level of haemoglobin. All patients were followed clinically for the presence of venous thromboembolism. The TXA group had a lower mean intra-operative blood loss of 441 ml (150 to 800) versus 615 ml (50 to 1580) in the placebo (p = 0.044), lower mean post-operative blood loss (285 ml (120 to 570) versus 392 ml (126 to 660) (p = 0.002), lower mean total blood loss (1070 ml (688 to 1478) versus 1337 ml (495 to 2238) (p = 0.004) and lower requirement for transfusion (p = 0.021). No patients in either group had symptoms of venous thromboembolism or wound complications. This prospective, randomised controlled study showed that a regimen of two intravenous injections of 1 g TXA is effective for blood conservation after minimally invasive THA.

Amoxicillin resistance with beta-lactamase production in Helicobacter pylori.

BACKGROUND: Amoxicillin-resistant Helicobacter pylori with minimal inhibitory concentration (MIC) >or= 256 mg L(-1) was isolated from a gastritis patient. The aims were to investigate the mechanism of high-level amoxicillin resistance in H. pylori. MATERIALS AND METHODS: The beta-lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the beta-lactam antibiotic resistance enzyme TEM beta-lactamase was analysed by polymerase chain reaction (PCR), sequencing and dot-blot hybridization. Sequencing analysis of pbp1A gene was performed and amoxicillin-susceptible isolate was transformed with pbp1A PCR products from the resistant isolate. The expression of hefC efflux system was analysed using real-time quantitative PCR. RESULTS: Activity of beta-lactamase was detected. Sequence analysis showed that the PCR product derived from H. pylori 3778 was identical to the bla(TEM-1) (GenBank accession EU726527). Dot-blot hybridization confirmed the presence of beta-lactamase gene bla(TEM-1.) By transformation of PCR product of mutated pbp1A gene from H. pylori 3778 into amoxicillin-susceptible strain showed that substitutions in Thr(556)-->Ser, Lys(648)-->Gln, Arg(649)-->Lys and Arg(656)-->Pro contribute to low-level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0.75 mg L(-1). Over-expression of hefC was not found. CONCLUSIONS: High-level amoxicillin resistance is associated with beta-lactamase production in H. pylori. Low-level amoxicillin resistance is linked to a point mutation on pbp1A. Because H. pylori can exchange DNA through natural transformation, spreading of bla(TEM-1) amoxicillin resistance gene among H. pylori is a potential threat when treating H. pylori infection.

Interaction between cigarette, alcohol and betel nut use on esophageal cancer risk in Taiwan.

OBJECTIVE: In 2003 esophageal cancer was the sixth leading cause of death among men in Taiwan, but it is the fastest increasing (70%) alimentary tract cancer. The aim of this study was to investigate the impact of different habits of betel nut chewing on esophageal squamous cell carcinoma (SCC) and its interaction with cigarette use and alcohol consumption. MATERIALS AND METHODS: All 165 cases were pathologically proven esophageal SCC patients (all male, mean age = 56.0, range = 35-92 years) diagnosed by biopsy during gastroendoscopic examinations. The control group comprised 255 subjects (all male, mean age = 54.8, range = 40-92 years) selected from patients who had visited the Otolaryngology Outpatient or Inpatient Department of KMUH owing to a benign lesion over this field. All were interviewed to collect demographic and substance use information by a trained interviewer using a standardized questionnaire. RESULTS: Smoking (aOR = 5.4, 95% CI = 2.4-12.9, PAR = 72%), alcoholic beverage drinking (aOR = 17.6, 95% CI = 9.3-35.2, PAR = 76%) and low education level are independent risk factors for esophageal cancer. Although betel nut chewers only had a borderline significant higher risk than nonchewers (aOR = 1.7; 95% CI = 0.8-3.1), those who chewed with a piece of betel inflorescence (aOR = 4.2, 95% CI = 1.4-16.0) and swallow betel-quid juice (aOR = 3.3, 95% CI = 1.3-9.3) had a significant higher risk. Significant dose-response effects were found in daily quantity of drinking and smoking. There is a synergistic effect of these three substances on the development of esophageal cancer. CONCLUSION: Betel nut chewing plays a relevant role in the development of esophageal SCC but adds to the carcinogenetic effect of smoking and alcohol drinking. Direct mucosal contact of betel juice may contribute to its carcinogenesis.

Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors.

Endometrial stromal tumors are divided into three types: benign stromal nodules, endometrial stromal sarcomas, and undifferentiated endometrial sarcomas. A variety of cytogenetic abnormalities involving chromosome 7 have been reported in endometrial stromal sarcomas, including a recurrent t(7;17)(p15;q21). We have identified two zinc finger genes, which we have termed JAZF1 and JJAZ1, at the sites of the 7p15 and 17q21 breakpoints. Analyses of tumor RNA indicate that a JAZF1/JJAZ1 fusion is present in all types of endometrial stromal tumors; however, the fusion appears to be rarer among endometrial stromal sarcomas that would be considered high-grade according to certain classification schemes. These findings suggest that the less malignant endometrial stromal tumors may evolve toward more malignant types, but that some endometrial stromal sarcomas with relatively abundant mitotic activity may compose a biologically distinct group.

Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations.

We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.

Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1.

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.

Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Diffuse colonic mantle cell lymphoma in a patient with presumed ulcerative colitis: detection of a precursor monoclonal lymphoid population using polymerase chain reaction and immunohistochemistry.

Primary lymphoma of the colon, a rare and typically late complication of ulcerative colitis, exhibits high-grade morphology and behavior when it occurs. Recently, several reports of colonic lymphoma masquerading as ulcerative colitis have been described. These previous reports described inflammatory mucosal changes typical of ulcerative colitis as being present in superficial biopsies, leading to the initial diagnosis of ulcerative colitis; however, further workup resulted in a diagnosis of primary colonic lymphoma within several months in these cases, and all symptoms and mucosal changes resolved after treatment of the lymphoma. Herein we report a case of mantle cell lymphoma arising in the colon and rectum in a 71-year-old woman with a 4-year history of ulcerative colitis. Immunoglobulin heavy-chain gene rearrangements were detected using the polymerase chain reaction procedure in fixed tissue in the lymphoma as well as in a prior resection specimen that histologically appeared to show only changes of severe ulcerative colitis. This finding suggests that an indolent lymphoid proliferation may have been the underlying disease in this patient and raises questions about the role of colonic lymphoma in causing mucosal injury.

An early haematopoietic defect in mice lacking the transcription factor GATA-2.

Blood cell development relies on the expansion and maintenance of haematopoietic stem and progenitor cells in the embryo. By gene targeting in mouse embryonic stem cells, we demonstrate that the transcription factor GATA-2 plays a critical role in haematopoiesis, particularly of an adult type. We propose that GATA-2 regulates genes controlling growth factor responsiveness or the proliferative capacity of early haematopoietic cells.

Evidence that interaction of hepatocytes with the collecting (hepatic) veins triggers position-specific transcription of the glutamine synthetase and ornithine aminotransferase genes in the mouse liver.

We previously demonstrated that glutamine synthetase (GS) and ornithine aminotransferase (OAT) mRNAs are expressed in the mouse liver acinus preferentially in pericentral hepatocytes, that is, those immediately surrounding terminal central veins (A.L. Bennett, K.E. Paulson, R.E. Miller, and J.E. Darnell, Jr., J. Cell Biol. 105:1073-1085, 1987, and F.C. Kuo, W.L. Hwu, D. Valle, and J.E. Darnell, Jr., Proc. Natl. Acad. Sci. USA, in press). We now show that hepatocytes surrounding large collecting hepatic veins but not portal veins also express these two mRNAs. The pericentral hepatocytes are the most distal hepatocytes with respect to acinar blood flow, whereas this is not necessarily the case for hepatocytes next to the large collecting hepatic veins. This result implies that it is contact with some hepatic venous element which signals positional expression. In an effort to induce conditions that change relationships between hepatocytes and blood vessels, regenerating liver was studied. After surgical removal of two-thirds or more of the liver, there was no noticeable change in GS or OAT expression in the remaining liver tissue during regeneration. However, treatment with carbon tetrachloride (CCl4), which specifically kills pericentral hepatocytes, completely removed GS- and OAT-containing cells and promptly halted hepatic transcription of GS. Repair of CCl4 damage is associated with invasion of inflammatory and scavenging cells, which remove dead hepatocytes to allow regrowth. Only when hepatocytes resumed contact with pericentral veins were the pretreatment levels of OAT and GS mRNA and high levels of GS transcription restored.

Colocalization in pericentral hepatocytes in adult mice and similarity in developmental expression pattern of ornithine aminotransferase and glutamine synthetase mRNA.

In situ hybridization showed that the mRNA for ornithine aminotransferase (OAT; ornithine-oxo-acid aminotransferase; L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13) colocalized with glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] in pericentral hepatocytes of the adult mouse liver. In addition to an identical distribution in adult hepatocytes, OAT and GS have very similar expression patterns in fetal and neonatal liver. As was earlier described for GS, there is a low level of OAT mRNA in fetal cells and increasing pericentral levels in neonates that reach adult patterns within 2 weeks. These results suggest that the transcriptional regulation of the two genes is similar in the liver. However, there was a lack of colocalization of the mRNAs for the two enzymes in cells of the kidney, intestine, and brain, suggesting different regulatory decisions for the OAT and GS genes in the cells of these different tissues. The metabolic consequences of these localized expression patterns favor ammonia clearance from the blood by the liver and urea synthesis by the kidney.