In our hospital, we use the BACTEC^TM FX (Becton, Dickinson, and Company [BD]) automated blood culture system for bacterial detection. Blood samples are cultured in a set of three vials, each containing one of the following BD mediums: BACTEC^TM aerobic plus (Aero-plus), BACTEC^TM anaerobic plus (Anaero-plus), and BACTEC^TM anaerobic lytic (Anaero-lytic). The performances of these vials were compared by examining the rate and time of bacterial detection in samples. Of the 3,217 blood culture samples collected from April 2012 to March 2014, 2,435 samples with more than two sets of vials were analyzed, and 440 samples were positive for bacteria. Of the 306 samples collected before starting antibiotic therapy, the bacterial detection rate of the Anaero-lytic vial (84.3%, 258/306) was significantly higher than that of the Anaero-plus vial (77.1%, 236/306). The detection rate for the 134 samples collected during antibiotic therapy was not significantly different between the two kinds of anaerobic vials. Of the samples collected before the start of antibiotic therapy, the average detection time of the Anaero-lytic vials was significantly shorter than that of the Anaero-plus vials. No significant difference was found in the average detection time between the two kinds of anaerobic vials in samples collected during antibiotic therapy. We conclude that the use of the Anaero-lytic vials improved the rate and time of bacterial detection in blood cultures, especially for samples collected before the start of antibiotic therapy.

Pseudomonas aeruginosa is a common cause of chronic respiratory tract infection in patients with pulmonary disorders such as diffuse panbronchiolitis (DPB). Long-term, low-dose macrolide treatment has been reported to significantly increase the survival rate of patients with DPB. However, macrolides do not exert any antibacterial effect on P. aeruginosa. Macrolides have been reported to inhibit P. aeruginosa virulence factors; however, in previous studies, the effects of macrolides on P. aeruginosa via short-term exposure were evaluated. Hence, in this study, we aimed to determine the effects of long-term, low-dose macrolide (erythromycin and clarithromycin) treatment on P. aeruginosa over 2 years in vitro. The virulence factors of P. aeruginosa (total proteolytic activity, elastase activity, and pyocyanin production) and cytotoxicity to the human lung epithelial adenocarcinoma cell line A549 treated with the supernatant of macrolide-exposed P. aeruginosa were analyzed. Long periods of macrolide exposure decreased P. aeruginosa virulence factors and A549 cell cytotoxicity. Long-term, low-dose macrolide treatment temporally reduced the pathogenicity of P. aeruginosa by decreasing its virulence factors. The present findings may help modify the clinical course of DPB.

Evaluation of unbound bilirubin concentration and total bilirubin/albumin　ratio in infants at Gunma Children’s Medical CenterUnbound bilirubin (UB) concentrations were retrospectively investigated for infants admitted to our hospital between 1 January 2011 and 31 December 2015. Peak UB concentrations in 1926 infants were targeted. UB concentrations were mostly within the range of 0.40–0.59 μg/dL (704/1,926, 36.6%). The percentage of infants with 0.60 μg/dL or more was 22.2% (427/1,926), and that with 0.80 μg/dL or more was 3.8% (74/1,926). In terms of age (0–27 days), the age of peak UB concentration was three days after birth (494/1,926, 25.6%). When UB concentrations were compared among the four groups divided by birth weight, the UB concentration tended to be higher in the group with a high birth weight. We examined the relationship between UB concentration and the results of several biochemical tests. Among them, the bilirubin/serum albumin ratio showed the best correlation with UB concentration (r = 0.826, p < 0.01), when the indirect bilirubin/total bilirubin ratio was limited to more than 50%. Therefore, by using this ratio, a cut-off UB concentration of 0.60 μg/dL or more, which was one standard of phototherapy, and a UB concentration of 0.80 μg/dL or more, which was one standard of exchange transfusion, were determined. As a result, the cut-off UB concentration of 0.60 μg/dL or more was 3.5 mg/g (sensitivity, 93.1%; specificity, 77.6%), and the UB concentration of 0.80 μg/dL or more was 4.0 mg/g (sensitivity, 87.8%; specificity, 73.0%).

Available examination methods to detect the capsular-type pneumococci include the slide agglutination test and multiplex PCR method, which are used to identify pneumococcal serotypes. From December 2008 to September 2016, 59 strains of Streptococcus pneumoniae isolated from patients with invasive pneumococcal diseases (IPDs) at Aichi Medical University Hospital were subjected to slide agglutination and multiplex PCR methods. We found that we can reduce the amount of work needed for the multiplex PCR method by determining the capsule types using the slide agglutination method. When we found that the strain as non-aggregated by the slide agglutination method and not containing the antigen factor 6d, and when the 6A/6B/6C/6D-type band was also observed by the multiplex PCR method, we evaluated the strain as serotype 6C. The combination of the slide agglutination test and the multiplex PCR method might be useful for determining serotype 6C.

In our facility, we deal with spcimens immersed in a preservative liquid containing 50% alcohol, some of these specimens contain many blood components. In liquid-based cytology centrifugation, a macroscopically visible layer of nucleated cells, i.e., the buffy coat, is formed on the upper part of the sediment. This nucleated cell layer is the focus of cytological examination. However, after the addition of a preservative liquid to the specimens, the nucleated cell layer formed becomes unclear, and gross observation is difficult. It is necessary to confirm the validity of sampling because it is possible that a test result could be a false negative. Therefore, to find an effective sampling method, a cell block was prepared to investigate how the cells were distributed in the sediments of 15 specimens where the nucleated cell layer was unclear. As a result, we recognized that nucleated cells were mostly distributed in the lower part of the sediment of a specimen to which a preservative liquid was added, and malignant cell clusters tended to concentrate in the lower layer. In this study, we verified the effectiveness of taking samples from the lower part of the sediment for cytological examination of specimens to which a preservative liquid was added. We used the preservative liquid containing approximately 50% alcohol, but there are various types of preservatives, and we think that it is important to understand the fixation actions and hemolysis ability of preservatives used for specimens.

We usually prepare products of the same blood type for transfusion; however, in emergency cases, we prepare unmatched type O red blood cells. The necessity of emergency transfusions has been increasing recently. It is often difficult to identify the patient’s original blood type after the transfusion of type O red blood cells because the original patient’s blood and the transfused blood are mixed and mixed-field agglutination (mf) can be found. In the determination of the blood type of such patients, the transfused blood is found at the bottom after sample centrifugation because mature erythrocytes show increased weight with time. The sampling position may affect the judgement of “mf”. In this study, we investigated four causes that affect the typing of the blood, namely, the mixed type O cell concentration, the staff members’ ability, the differences in the reactivity of erythrocytes over time, and the reproducibilities of two different automated analyzers and the slide method. We found that a mixture of more than 2 units (about 10% of the circulating blood) of type O red blood cells can be examined even when the “mf” was found in any of these methods. Mixed-field agglutination might not be detected by the slide method when an inexperienced staff member examines or when mature erythrocytes were transfused. The samples with 10% type O red blood cells were judged as “mf” in 11 out of 15 tests, because the automated analyzer usually aspirates samples from the bottom. The sampling position is critical for the decision of the blood type when testing samples of mixed cells with different specific weights. When an unexpected reaction occurs, we should check the patient’s clinical background and try to test by another appropriate method.

In immune serum test items, the actual differences among devices and reagents have not been sufficiently elucidated owing to the matrix effect of samples for external accuracy control. In this study, we performed factor analyses of differences among reagents and evaluated the appropriateness of test samples. Devices from four equipment manufacturers were investigated using a prostate-specific antigen (PSA), ferritin, α-fetoprotein (AFP), and carcinoembryonic antigen (CEA) as the test items. For samples, 30 panel sera and 16 test samples were used. Regarding the total average as the standard, the measured values of the panel sera were evaluated by regression analysis. The measured values were noted to be proportional among the methods used for PSA, ferritin, and AFP, and no value showed deviation. However, the measured value of CEA differed among the methods, showing no consistent tendency. By setting the criterion to the 95% confidence interval determined by the regression analysis of the panel sera, the reactivity of the test samples was evaluated. Samples prepared with pooled sera met the evaluation criterion, suggesting the usefulness of the pooled serum as a test sample.

We investigated the appropriateness of samples for external accuracy control using a prostate-specific antigen (PSA), ferritin, α-fetoprotein (AFP), and carcinoembryonic antigen (CEA) as test items and found the usefulness of pooled serum in the first report. In this study, we evaluated devices and reagents from 13 manufacturers. Two series of PSA, ferritin, and AFP and four series of CEA of 5-fold serial dilutions of serum were prepared with sera from different patients. The CV of measured values of PSA was 8.6–11.9%, and none of the values showed deviation. The CV of measured values of ferritin was 13.5–19.5%, and differences among the origins of the samples were noted in two devices. In AFP, the value of one sample deviated in one device, and CV was 10.6%, but the CVs of the other samples were 4.5–7.2%. CEA was measured in serum samples prepared from patients with cancers of different organs. The mean CV of samples from colorectal cancer patients was 10.9%, and that of samples from breast cancer patients was 19.9%, showing a difference in reactivity between the origins of samples.

Utilizing a portable ultraviolet lamp (black light), we estimated the amount of 12 fluorescent substances in urine specimens. The lamp can radiate light of not only the main excitation wavelengths of 350 and 370 nm, but also light of wavelengths shorter than 300 nm (the ultraviolet region). The substances may be considered as fluorescent if they have excitation wavelengths lower than 300 nm on filter papers or thin layer chromatography (TLC) plates. Using the fluorescent spot test on filter papers and TLC plates, we have defined 12 fluorescent substances, namely, amino acids (tryptophan and histidine), flavine compounds (FMN and FAD), aromatic amino acid metabolites (gentisic acid, tryptamine, serotonin, 5-hydroxyindole acetic acid, skatole, and indole) and hemoglobin metabolites (coproporphyrin and urobilin) in urine specimens.

In cerebrospinal fluid (CSF) examinations, the use of cell collecting smears is recommended for accurate cell identification. Therefore, we devised a new method of cell collection using a general-purpose swing-type centrifuge for smear preparation, and the validity of this method was evaluated. In our method, a “chamber system” kit (Sakura Finetek) and a general-purpose centrifuge were utilized, and the cells were collected for smear preparation by centrifugation at 800 rpm and 3 min. In the case of a large centrifuge, we made an adapter to fix the chamber system to the metal basket of the centrifuge. In the evaluations, we used a WBC suspension as a sample, and the number of cells, the cell fraction, and the number of destroyed cells identified on the smear slides by our method were compared with those obtained using an automatic smear method. As a result, the number of smeared cells was found to be higher in our method using a large centrifuge. However, there were no significant differences in the number of destroyed cells or in the ratio of mononuclear cells to polymorphonuclear cells between the two methods. Regarding the number of cells most suitable for such smears, the optimum total number of cells ranged from 10,000 to 50,000. In addition, excellent smear images were obtained even with actual CSF specimens. Our new method is easy to perform without using any expensive equipment, and it is most suitable for CSF testing, which requires rapid sample preparation.

The results of the basic examination of AQT as a target reagent by immunochromatography were mostly good. For N = 50, the rate of agreement is 88.0%, and it is considered that the introduction of AQT to routine examination is possible in all six mismatched cases, which depend on individual differences in visual observation. In addition, the rate of agreement with the blood culture was 75.0%, and only a small number of samples is required, but the positivity rate of the blood cultures tended to be high if the PCT level was high. Because we can measure whole blood samples immediately without preprocessing a specimen, which is a characteristic of AQT, a quick result report is possible. In addition, without individual differences occurring in comparison with the immunochromatography method, which requires complicated visual judgment and quantitative measurement, human errors, such as input mistakes, can be prevented because AQT can be applied online.

It is important to determine the optimum conditions for the argentation reaction to obtain the best results of Grocott staining. Since methenamine-silver nitrate solution often causes unstable staining, chromic acid ammoniacal-silver nitrate solution is appropriate for fungal staining. However, the optimum temperature and duration of argentation reaction have not been sufficiently investigated. In this study, we examined various staining durations for both argentation reaction and gold chloride solution using a dissolver or water bath in the method using chromic acid ammoniacal-silver nitrate solution. The fungi stained here were Aspergillus, Cryptococcus, and Pneumocystis jirovecii. It was confirmed that the duration of the argentation reaction should be 5–10 min to obtain good results when a dissolver is used. Fungal staining color could be well regulated by modulating the duration of the gold chloride solution reaction, and appropriate staining was achieved when the staining duration was 1–5 min for all types of fungi. The chromic acid ammoniacal-silver nitrate method using a dissolver is considered to provide stable staining results with less difference among performers when the staining duration with the gold chloride solution is adjusted for each fungus.

In recent years, the number of infections caused by macrolide-resistant Mycoplasma pneumoniae has been increasing, particularly in children, and has been the focus of interest in the field of internal medicine. In this study, we compared the performance of GENECUBE with that of immunochromatography in M. pneumoniae identification, and studied the rate and clinical effects of macrolide-resistant gene mutations. The samples used in this study were obtained from patients who were suspected of having mycoplasma pneumonia at our Department of Pediatrics and were examined for M. pneumoniae using GENECUBE. The sensitivity of the immunochromatography method was 14.7%. The macrolide-resistant gene mutation retention rate was 20.6%, which was lower than that previously reported. The reasons for the low rate were considered to be the region and medical facility function. Furthermore, the presence or absence of macrolide resistance gene mutation determines the type of antibacterial drugs to be administered. It is considered that further studies of the method of GENECUBE operation and the test results are necessary. Our findings showed that GENECUBE can rapidly detect not only M. pneumoniae but also the presence or absence of macrolide-resistant gene mutations. This method may be useful for the rapid diagnosis of M. pneumoniae infections.

Daratumumab (DARA) is a human monoclonal IgG antibody preparation against CD38, which was developed to treat multiple myelomas (MMs). CD38 is expressed not only on myeloma cells but also on red blood cells; hence, it may cause a false positive result of the indirect antiglobulin test (IAT) in patients administered with DARA. In this study, we encountered cases with positive IAT results, which were assumed to be caused by DARA, and investigated the duration of DARA’s interference with IAT as well as a method of avoiding DARA’s interference by the dithiothreitol (DTT) processing of red blood cells (RBCs). Our subjects included 4 MM patients using DARA. In the DTT processing of RBCs, 100 μL of 3–5% RBC suspension was washed 4 times with PBS. Then, 400 μL of 0.2 mol/L DTT was added to the suspension, and the suspension was warmed at 37°C for 30 min and then washed 4 times with PBS. For the duration of DARA’s interference with IAT, a positive IAT result was observed on day 3 after starting DARA administration. One patient that we investigated became negative on day 137 after the completion of DARA administration. The positive IAT reaction induced by DARA became negative with the DTT processing of RBCs. The processing did not deactivate blood group antigens other than K, and examination for irregular antibody expression was possible, suggesting that it could be useful as a pre-blood transfusion process for patients using DARA.

In this study, we evaluated the performance of a new method of measuring neutrophil gelatinase-associated lipocalin (NGAL), the “ARCHITECT urine NGAL assay”. Satisfactory results in terms of precision and linearity were obtained. There was good long-term stability for 14 weeks. The sensitivity using the 2SD method was 0.42 ng/mL. There was no interference from substances such as l-ascorbic acid, d-glucose, NaCl, urea, and hemoglobin. Strong correlations were observed between the ARCHITECT urine NGAL assay and ELISA (r = 0.990, y = 0.855x + 13.025). In summary, our results suggest that the newly developed ARCHITECT urine NGAL assay using the ARCHITECT analyzer is useful for routine examinations.

We report a case of chronic otitis media caused by the Mycobacterium abscessus complex. The patient was a 72-year-old female, whose chief complaints were otalgia and otorrhea. The patient had been prescribed with ofloxacin (OFLX) otic solution and garenoxacin (GRNX) by her previous doctor and was subsequently admitted to our hospital, because her symptoms had not improved. A Gram stain of an otorrheal sample showed Gram-positive bacilli. On the basis of our suspicion of an acid-fast bacillus, we conducted Ziehl–Neelsen staining, which revealed acid-fast bacilli (Gaffky 4). The acid-fast bacilli showed rapid growth after 48 h of culture. Identification by DNA-DNA hybridization revealed the presence of M. abscessus. Combined treatment with clarithromycin (CAM) and GRNX diminished the patient’s lesion. Treatment was continued for one month, after which the patient’s symptoms were resolved. In summary, we have isolated a fast-growing M. abscessus complex in the otorrhea of a patient with chronic otitis media that was successfully treated with CAM and GRNX.

We report on a case of rupture of the urinary bladder diagnosed on the basis of the presence of mesothelial cells in urine sediments. An eighty-year-old woman was admitted to our hospital with the complaint of back pain, and end-stage renal disease was suspected from her medical history and laboratory data. Massive proteinuria was detected in the urine test. RBCs, WBCs and casts were very few in the urinary sediments. In addition, oval cells were seen scattered or in small clusters, and cell-to-cell apposition was recognized at the points where cells are joined. Cells had nucleoli in mononuclei and multinuclei, but no hyperchromatin was seen in the nuclei. Therefore, we identified them as mesothelial cells from these findings. Five days after admission, ascites accumulated prominently, and serum BUN and CRE levels further increased. However, massive amounts of urine were excreted with urinary catheter use, and pooling ascites disappeared. No rupture sites were identified by contrast-enhanced CT, but were confirmed by cystoscopy. A few days later, her kidney function returned to normal with the use of an indwelling balloon catheter in the urinary bladder. This can be considered as an instructive case in terms of her medical history, that is, although rupture sites were not identified by contrast-enhanced CT, rupture of the urinary bladder was diagnosed on the basis of the findings in urine sediments.

Analysis of urinary creatinine level is frequently used for the evaluation of general clinical conditions, which is especially useful for data corrections in urinalysis or the evaluation of renal function. A man in his sixties with advanced bladder cancer presented with an extremely low urinary creatinine level. Confirmatory tests ruled out technical errors and revealed that Prebotella loescheii and Anaerococcus tetradius degraded creatinine in the urine. Creatinine degradation depended on the amount of bacteria and temperature. Occasionally, urinary creatinine levels were also very low, which demonstrated that creatinine was degraded in his bladder. In addition, the urinary creatinine level decreased with increasing amount (10⁶–10⁷/μL) of the two bacteria. Therefore, attention should be paid to the potential negative error of urinary creatinine in bacteria-contaminated urine.

Fabry disease is a lysosomal storage disorder caused by a deficiency in α-galactosidase A. It is an X-linked inherited disease that develops mostly in men having only one X chromosome, but it may also develop in women. For men with the classical-type Fabry disease, symptoms such as pain, angiokeratoma, and hypohidrosis of the limbs appear from childhood. Patients with the late-onset type show symptoms mainly in the heart or kidney later in adulthood. The classical type is characterized by low enzyme activity, whereas the late-onset type shows slightly low enzyme activity. There are also women who are asymptomatic, whereas others show severe symptoms. In the sediments of urine from patients with Fabry disease, mulberry bodies and mulberry cells appear. It is characterized by the appearance of an eddy form. Here, we report 2 cases of the disease characterized by the appearance of mulberry bodies that did not present an eddy-formed structure.

We report a case of uncomplicated cystitis caused by CO₂-dependent Escherichia coli small colony variants (SCVs). The initial E. coli isolate came from a midstream urine specimen containing large numbers of Gram-negative rod-shaped organisms that failed to grow on both Drigalski agar and sheep blood agar incubated in ambient air. The isolate grew when the urine was cultured overnight on GAM agar under anaerobic conditions and on chocolate agar under 5% CO₂ atmosphere. Further investigation revealed that the isolate grew on both Drigalski agar and sheep blood agar under 5% CO₂ atmosphere. Initially, we failed to identify the isolate or determine its antimicrobial susceptibilities using the MicroScan WalkAway System because the isolate did not grow in the system. However, the isolate was subsequently identified as E. coli on the basis of its morphological, cultural, and biochemical properties using a commercially available kit, rapid ID 32 E API. The identification of this isolate was confirmed by sequencing the 16S rRNA gene of the organism and MALDI-TOF MS. Cefdinir or fosfomycin was administered, and the patient’s condition improved. CO₂-dependent SCVs should be suspected when samples show positive Gram staining and negative culture test results under ambient air.