[The Plasma (1→3)-ß-D-glucan Assay, a Japanese Contribution to the Diagnosis of Invasive Fungal Infection].

In 1985, a (1→3)-ß-D-glucan assay based on the discovery of a (1→3)-ß-D-glucan-sensitive coagulation factor in the horseshoe crab amebocytes was developed in Japan, as a test aid in the diagnosis of invasive fungal infections. After many clinical trials, it has now been incorporated in practice guidelines both in Japan and abroad for the diagnosis and management of invasive fungal infection, and is now available worldwide. In Japan, however, two commercial kits that differ widely in sensitivity to (1→3)-ß-D-glucan are used; namely, the colorimetric and the turbidimetric assays. Thus, attention must be paid when comparing measurement values between the two tests. In most cases, one pg/ml of the latter corresponds to about 7 pg/ml of the former. This raises the cutoff value of the turbidimetric assay far higher than that of the colorimetric assay, raising concern about missing many true cases of invasive fungal infections. The detection limit of the turbidimetric method is equivalent to as high as 60 pg/ml if converted to the colorimetric measurement. Thus, early diagnosis will be difficult to make based on detection of a rise in the ß-D-glucan level in the lower range of concentration. The kits used overseas also differ in sensitivity; thus, global standardization or harmonization will be required to establish inter-assay data comparability and to enhance the usefulness of the tests. In the future, genetic engineering may provide an alternative source of the coagulation pathway enzymes other than the horseshoe crabs, in accordance with the global trends in animal protection, and bring about new opportunities for standardization.

[Validation of a New (1 → 3)-ß-D-Glucan Test Kit, Fungitec G Test MKâ ¡ " Nissui "].

Using 415 residual blood samples subjected to (1→3) -ß-D-glucan assay, we studied the correlation of measurement results between Fungitec G Test MKII "Nissui" (Nissui Pharmaceutical Co., Ltd., Tokyo) and its predecessor, Fungitec G Test MK (Seikagaku Corporation, Tokyo), which is now out of production. Their major difference is that MK II uses reagents derived from blood cells of Limulus polyphemus, the American horseshoe crab, whereas MK used those of Tachypleus tridentatus, an Asian horseshoe crab. Passing-Bablok analysis showed a linear regression with nearly one-to-one correspondence (slope=1.065, intercept=-0.287) between the two test kits over the regular range of measurements (4.0 pg/ml -500 pg/ml ). This was also true when data were subdivided and analyzed in the low to medium (≦150 pg/ml ) and in the low range (≦50 pg/ml ). There were several cases, however, that showed a wide discrepancy between the pair of measurements. This discrepancy is believed to be due in part to the difference between Limulus and Tachypleus in their reactivity to ß-glucans with diverse side chains. Despite of this, the even distribution on either side of the regression line of those samples that are associated with deep fungal infection and the abrupt disappearance of such samples below 20 pg/ml attest that MK II "Nissui" is an acceptable successor of MK.

Extrahepatic bile duct measurement by using transabdominal ultrasound in Japanese adults: multi-center prospective study.

BACKGROUND: In adults, less than 10-mm bile duct has idiomatically been recognized as "non-dilated bile duct" though there was no obvious evidence. The aim of this study was to prospectively examine the maximum inner diameter of extrahepatic bile duct (MDEBD) in consecutive adults. METHODS: Transabdominal ultrasound (US) was performed to measure the MDEBD of 8840 cases (4420 male) in five institutions. The frequency of ultrasound probe ranged from 3.5 to 5 MHz. RESULTS: The mean diameter of MDEBD was 4.5 ± 1.4 mm (range 1-14 mm). The relationship between the MDEBD and age was shown as follows: MDEBD = 2.83 + 0.03 × age. Multiple regression analysis was analyzed between 6 groups and significant α level is 0.008 in this analysis. In all age groups but 20s and 30s, there was statistically significant MDEBD among each age group (p < 0.0001). Mean, mode value and median MDEBD is increasing according to the age as follows: 20s: 3.9 ± 1.0 mm, 30s: 3.9 ± 1.2 mm, 40s: 4.3 ± 1.2 mm, 50s: 4.6 ± 1.3 mm, 60s: 4.9 ± 1.4 mm, >70s: 5.3 ± 1.6 mm. CONCLUSION: The present study revealed that MDEBD positively correlates with age. Therefore, when we examine the presence of dilation of the bile duct, our calculating formula appears to be suitable for accurate evaluation.

Rapid typing of influenza viruses using super high-speed quantitative real-time PCR.

The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18s/cycle; 40 cycles in less than 20min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(-1) plaque-forming unit/reaction for viruses in culture supernatants during 20min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.

Serial changes of elevated serum IgG4 levels in IgG4-related systemic disease.

OBJECTIVE: Autoimmune pancreatitis (AIP) and Mikulicz's disease have recently been recognized as pancreatic or salivary gland lesions of IgG4-related systemic disease. These are frequently associated with elevated serum IgG4 levels. This study aimed to clarify clinical implications of serial changes of elevated serum IgG4 levels in IgG4-related systemic diseases. METHODS: Serial changes of elevated serum IgG4 levels were examined in patients with IgG4-related systemic diseases. Patients Serial changes of elevated serum IgG4 levels were examined in 44 patients: AIP (n=24), Mikulicz's disease (n=8), pancreatic cancer (n=5), bile duct cancer (n=1), sclerosing cholangitis (n=1), hypereosinophilic syndrome (n=1), chronic thyroiditis (n=1), hypophysitis (n=1), idiopathic pancreatitis (n=1), and Behcet's disease (n=1). RESULTS: The serum IgG4 levels decreased in all patients with AIP and Mikulicz's disease after steroid therapy. The serum IgG4 levels were normalized in 46% of AIP patients and 38% of Mikulicz's disease patients. The serum IgG4 levels were not normalized at remission in 3 of 4 relapsed AIP patients, and re-elevation of serum IgG4 levels was detected in all relapsed patients. Elevated serum IgG4 levels decreased in 3 patients with pancreatic cancer after resection or chemotherapy, and decreased in patients with hypereosinophilic syndrome, sclerosing cholangitis, and hypophysitis after steroid therapy. CONCLUSION: Measurement of serial serum IgG4 levels is useful to determine the disease activity of IgG4-related systemic diseases.

[IgG4-related sclerosing disease].

Autoimmune pancreatitis (AIP) is a peculiar type of pancreatitis of presumed autoimmune etiology. AIP is diagnosed currently based on characteristic radiological findings (irregular narrowing of the main pancreatic duct and enlargement of the pancreas) in combination with serological findings (elevation of serum IgG, and IgG4, and presence of autoantibodies), clinical findings (elderly male preponderance, occasional extrapancreatic lesions, and favorable response to steroid therapy), and histopathological findings (dense infiltration of IgG4-positive plasma cells and T lymphocytes with fibrosis and obliterative phlebitis in the pancreas). Based on histological and immunohistochemical examination of various organs of AIP patients, a novel clinicopathological entity of "IgG4-related sclerosing disease" has been proposed. This is a systemic disease that is characterized by extensive IgG4-positive plasma cell and T lymphocyte infiltration of various organs. Clinical manifestations are apparent in organs such as the pancreas, bile duct, gallbladder, salivary or lacrimal gland, and retroperitoneum, where tissue fibrosis with obliterative phlebitis is pathologically induced. AIP is not simply a form of pancreatitis but rather a pancreatic lesion reflecting an IgG4-related sclerosing disease. This disease includes autoimmune pancreatitis, IgG4-related sclerosing cholangitis, IgG4-related cholecystitis, IgG4-related sialadenitis, IgG4-related retroperitoneal fibrosis. In some cases, only 1 or 2 organs are clinically involved, while in others 3 or 4 organs are affected. Serum IgG4 levels and immunostaining with anti-IgG4 antibody are useful in establishing the diagnosis. Since malignant tumors are frequently suspected on initial presentation, IgG4-related sclerosing disease should be considered in the differential diagnosis to avoid unnecessary surgery.

Serum IgG4 concentrations and IgG4-related sclerosing disease.

BACKGROUND: Based on histological and immunohistochemical examinations of various organs of patients with autoimmune pancreatitis (AIP), a new clinicopathological entity, IgG4-related systemic disease, was proposed. This study aimed to clarify clinical utility of serum IgG4 levels in differentiating AIP from other pancreatobiliary diseases, clinical utility of serum IgG4 levels in differentiating Mikulicz's disease from other salivary gland disorders, as well as in identifying other IgG4-related diseases. METHODS: Serum IgG4 levels were measured in 468 patients. RESULTS: The median serum IgG4 level was significantly greater in AIP (301.5mg/dl) than in other pancreatobiliary diseases (p<0.01). Using the cutoff value of 119 mg/dl that was determined on the basis of this study's ROC curve data, the sensitivity and specificity to distinguish AIP from pancreatic cancer were 82.1% and 94.8%, respectively. The median serum IgG4 level was significantly greater in Mikulicz's disease (357.0mg/dl) than in other salivary gland diseases (p<0.01). Of 75 patients with elevated serum IgG4 levels, 15 had diseases other than pancreatobiliary and salivary gland diseases. CONCLUSIONS: Serum IgG4 levels were useful for diagnosing AIP and Mikulicz's disease. Some diseases with serum IgG4 level elevations may be lesions of IgG4-related systemic disease without manifestations of AIP and Mikulicz's disease.

Reappraisal of the serum (1-->3)-beta-D-glucan assay for the diagnosis of invasive fungal infections--a study based on autopsy cases from 6 years.

BACKGROUND: The prevalence of invasive fungal infection is increasing. An effective diagnostic test is required to identify and treat them successfully. METHODS: All autopsy records at our hospital for the period from January 2000 through December 2005 [corrected] were reviewed for cases of invasive fungal infection. The diagnostic efficacy of a serum (1-->3)-beta-D-glucan (beta-glucan) assay was examined using only those cases in which patients had been tested for fungal infection within 2 weeks before death. RESULTS: Of 456 autopsies, 54 (11.8%) involved cases of invasive fungal infection. Leukemias were the most frequent underlying disease (in 52% of cases of invasive fungal infection), and Aspergillus species was the most frequent pathogen detected (in 70%). Of the 54 patients with invasive fungal infection, 41 had beta-glucan testing performed within 2 weeks before death, as did 63 patients without invasive fungal infection; 48 of 54 patients with invasive fungal infection had a blood culture performed. The sensitivity and specificity of the beta-glucan test for the detection of invasive fungal infection were 95.1% and 85.7%, respectively, with a cutoff value of 30 pg/mL; 85.4% and 95.2%, respectively, with a cutoff value of 60 pg/mL; and 78.0% and 98.4%, respectively, with a cutoff value of 80 pg/mL. The sensitivity of blood culture testing was 8.3%. With a prevalence of 11.8%, the positive and negative predictive values for the beta-glucan test were 47.1% and 99.2%, respectively, with a cutoff of 30 pg/mL; 70.4% and 98.0%, respectively, with a cutoff of 60 pg/mL; and 86.7% and 97.1%, respectively, with a cutoff of 80 pg/mL. During the 6-year period studied, of 21 patients with fungus-positive blood cultures that were preceded or followed by a beta-glucan test within 2 weeks, 4 had negative beta-glucan test results (beta-glucan level, <30 pg/mL), and 17 had positive results (beta-glucan level, >60 pg/mL); the concordance between culture results and beta-glucan test results was 81.0%. Contrary to the general belief, 5 of 6 cases of cryptococcemia were associated with high serum beta-glucan levels. CONCLUSION: The beta-glucan test is an effective diagnostic tool for invasive fungal infection.

[Detection of acute HIV infection by serum HIV-1 RNA quantification].

To assess the merit of serum HIV-1 RNA quantification in detecting acute HIV infection, we reviewed the results of HIV-1 RNA assay and antibody tests in all patients who received those tests at our hospital from August 1999 to December 2004. Of 3530 such patients, five were sero-negative and PCR-positive with more than 105 copies/ml at initial examination. Four of them had HAART and followed a favorable course; they were later confirmed to be sero-positive by Western blotting. The other one, positive only by CLEIA, was lost to follow-up. This reconfirms the importance of using PCR in the initial assessment of HIV infection in high-risk patients.

[Evaluation of an improved pretreatment method for the measurement of (1-->3)-beta-D-glucan in blood samples].

Measurement of blood (1-->3)-beta-D-glucan is useful for early diagnosis and follow-up of the therapeutic process of deep seated mycoses. The Fungitec G test MK (Seikagaku Corp., Tokyo) kit using alkaline-pretreatment followed by chromogenic kinetic assay has been widely used in Japan because of its high sensitivity and easy handling of a large number of samples. Discrepancy in the levels of (1-->3)-beta-D-glucan and/or in the quantitative judgement, however, has been pointed out between this kit and other commercial kits. One of the reasons for this discrepancy has been reported to be non-specific reactions caused by substances other than beta-glucan. In this study, we have improved the alkaline pretreatment reagent by changing the concentration of KOH and salts, resulting in a marked reduction of the non-specific reaction. Recovery of standard beta-glucan added to plasma or serum after the improved pretreatment was 80 to 120%, and no amidolytic activity was detected either in plasma or in serum. By the improved pretreatment, the incidence of non-specific reactions, i.e., those that exceed the quantitation limit (3.9 pg/mL), were markedly decreased from 139 to 16 out of 200 plasma samples and from 106 to 22 out of 170 serum samples. The incidence of strong non-specific reactions, i.e., those that exceed the cut-off level (20 pg/mL), were also decreased from seven to one with plasma and seven to zero with serum samples. Correlation between corrected beta-glucan measurements by the current pretreatment and non-corrected ones by the improved pretreatment was quite good. The improved method is thus expected to decrease the frequency of non-specific false-positive reactions, with the high sensitivity of Fungitec G test MK.

Environmental sound recognition after unilateral subcortical lesions.

Nonverbal environmental sound recognition was investigated in 24 subjects with unilateral subcortical lesions and 20 age-matched normal controls. All patients incurred putaminal hemorrhage at least three months before examination, and had a cystic lesion under the insula on CT or MRI at the time of evaluation. A mild impairment was found in association with extensive damage to the lateral and ventral portions of the putamen and the adjacent white matter in either the right or left hemisphere. Degree of impairment and type of error did not differ significantly between the sides of lesions. These observations and a review of literature suggest that the impairment of environmental sound recognition may arise with a unilateral subcortical lesion disrupting the geniculo-auditory association cortex projection fibers.