Detection of Respiratory Viruses in SARS-CoV-2-Negative Specimens from January to March 2020 in Fukuoka, Japan.

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first confirmed in Japan on January 15, 2020. The Fukuoka Institute of Health and Environmental Sciences conducted testing using polymerase chain reaction (PCR) for SARS-CoV-2 from January 31 to March 4, 2020. Samples (n = 119) were collected from 81 patients suspected of having SARS-CoV-2 infection, presenting with fever, cough, fatigue, pneumonia, and other symptoms; all the samples tested during that period were negative. To identify the pathogens responsible for these symptoms, we conducted multiplex PCR. Respiratory viruses, human metapneumovirus (hMPV) was detected in 10 patients (12%), human rhinovirus (HRV) in 3 patients (4%), and influenza B virus in 1 patient (1%). In addition, the patients who had the viruses were significantly older than those who did not. Infections with hMPV and HRV have been associated with a risk of severe illness and death among older adults. Therefore, differentiating SARS-CoV-2 from other respiratory viruses, such as hMPV and HRV, is necessary to prevent and control the spread of infection, especially in older adults.

Effects of Manufacturing Conditions on Pharmaceutical Properties of Petrolatum Ointment-Distribution of Hydrocarbon.

Petrolatum ointment, which is an oleaginous ointment, is generally produced through manufacturing processes such as melting, mixing, and cooling. In this type of semisolid formulation, the manufacturing conditions of each process are empirically known to affect the quality of the resultant preparation; however, in many cases, the details of the factors are unclear. To clearly investigate the influence of the pharmaceutical properties of petrolatum ointments, we manufactured several ointments while changing the conditions of the mixing and cooling process after melting white petrolatum. As a result, the temperature at the termination was determined to influence the pharmaceutical properties of the final product. To investigate these phenomena, each petrolatum ointment sample was examined via digital microscopy and laser Raman analysis, and the distribution of the liquid-solid parts of samples was investigated. The internal structure of the ointment sample manufactured at a mixing-stop temperature of 40 °C, the needle crystals and the spherical aggregates surrounding them appropriately coexisted, while the structure exhibited a state wherein the two were linked in a semisolid phase. Meanwhile, for the ointment sample manufactured under the lowest mixing-stop temperature of 25 °C, the liquid part and the spherical aggregates were clearly separated, indicating that the liquid part was easily separated from ointments. In addition, the distribution of the hydrocarbons among the samples was measured via GC-MS; no significant difference in chemical structure was observed. In conclusion, the internal structure of the petrolatum ointment was changed by the manufacturing conditions, and this affected the pharmaceutical properties.

Detection of Borrelia DNA in Tick Species Collected from Vegetation and Wild Animals in Fukuoka, Japan.

We screened for the presence of Borrelia spp. in ticks collected from vegetation by flagging and from wild animals between May 2017 and November 2018 in Fukuoka, located in the northern Kyushu region of Japan. A total of 1,601 ticks were collected and separated based on morphology into nine species, namely Ixodes turdus, I. ovatus, Amblyomma testudinarium, Haemaphysalis flava, H. formosensis, H. kitaokai, H. longicornis, H. hystricis, and H. megaspinosa. The ticks were segregated into 561 pools and nested PCR was used to detect borrelial DNA. Borrelia turdi and Borrelia sp. HM were identified in two of the 561 pools. This is the first report of the presence of the Lyme disease group of Borrelia and of the relapsing fever group of Borrelia in Fukuoka, Japan.

[Clinical Research on the Effects of Differences in Properties of External Preparations for Skin on Application Amount: An Exploratory Investigation of the Relationship between the Base Usability and Application Amount by Questionnaire in Adult Subjects].

Compared to oral medication, the base plays a large role in the external preparation for skin, and dermatologists select the dosage form based on understanding of the effect of the base as well as according to skin symptoms and conditions, application site, age, season, etc. Further, in treatment with external preparations, it is important for patients to understand the application method and continue to apply an adequate amount to achieve the treatment goal. However, there is little evidence regarding the relationship between base properties or usability and the application amount. In this study, we investigated the usability and application amount of three bases with different properties (ointment base, cream base, and lotion base) in 62 adult subjects and exploratively examined the effect of the different base properties on the application amount. The results of this clinical study showed that the usability and preference for the base used for external preparations varies, and poor usability and low preference may lead to a reduction in the application amount. Even with good usability and high preference, there were many cases in which an adequate amount was not applied due to lack of specific instructions on external use. When selecting or changing the base in an external therapy, it is important for not only dermatologists but also pharmacists providing instructions on external use to be aware of the importance of the base and actively instruct patients to apply an adequate amount of the preparation.

[Influence of Bases for External Medicines with Different Coatability and Water Retentivity on Wound Healing].

When selecting external medicines for the treatment of skin diseases, it is thought to be very important to consider differences in characteristics of their bases, because the bases may influence the clinical efficacy of the medicines. In this study, we investigated whether the differences in characteristics of three kinds of bases, white petrolatum, macrogol ointment, and aqueous gel affect wound healing. In vitro moisture permeability tests demonstrated that these bases have different characteristics in coatability and water retentivity, with the rank order of the intensity of coatability as white petrolatum>macrogol ointment>aqueous gel, and that of water retentivity as macrogol ointment>white petrolatum>aqueous gel. Similar rank order of these bases was observed for transepidermal water loss and stratum corneum water content in the dry skin on the abdomen of guinea pigs induced by topical application of acetone/ether mixture, followed by water. In addition, we found that treatment with macrogol ointment, but not white petrolatum or aqueous gel, significantly accelerated wound healing in rat skin, and that the contents of basic fibroblast growth factor and epidermal growth factor in the skin treated with macrogol ointment were significantly higher compared with non-treated skin. In conclusion, these results imply an important role of the bases of external medicines in the treatment of skin diseases.

Molecular epidemiology of coxsackievirus A6 derived from hand, foot, and mouth disease in Fukuoka between 2013 and 2017.

Coxsackievirus (CV)-A6 has been the primary causative agent of hand, foot, and mouth disease (HFMD) in Japan since 2011. In Fukuoka, CV-A6-associated HFMD caused epidemics in 2013, 2015, and 2017. This paper reports the genetic characteristics of the CV-A6 entire viral protein 1 (VP1) derived from patients with HFMD in Fukuoka between 2013 and 2017. CV-A6 was detected in 105 of 280 clinical specimens, and the entire VP1 sequences could be analyzed for 90 of the 105 specimens. Phylogenetic analysis revealed that the CV-A6 strains were classified into clade A and subgrouped into subclade A3 or subclade A4. Each subclade strain carried amino acid substitutions in the presumed DE and GH loops of the VP1, and no amino acid substitutions were identified as deleterious to the protein function. No significant difference was found in the clinical symptoms between the genetic subclades using statistical analyses. In conclusion, this study clarified the genetic diversity of CV-A6 in Fukuoka from 2013 to 2017. The emergence of the CV-A6 strains was classified into derived new subclades based on phylogenetic analysis of the VP1 gene that may cause CV-A6-associated HFMD epidemics approximately every 2 years.

Effects of Manufacturing Conditions on Pharmaceutical Properties of Petrolatum Ointment.

Oleaginous white petrolatum ointment (WP ointment) is one of the most commonly used dosage forms in the preparation of topical products. In general, WP ointments containing medium chain fatty acid triglycerides (MCT) are manufactured through a process of melting, mixing, agitating, and cooling. To investigate the pharmaceutical properties of WP ointments in greater detail, we examined manufacturing factors which could potentially influence the pharmaceutical properties of the finished product. WP ointment samples containing 10% MCT were stirred with a homogenizer and a paddle mixer at 65°C, then the homogenizer was stopped. Next, the paddle-mixer was stopped at several planned temperature points at which different samples were taken. Each sample was then cooled under the following planned conditions: rapid-cooling [-50°C/h] and slow-cooling [-7.5°C/h]. The pharmaceutical properties of each WP ointment sample, along with the appearance (Optical/digital microscope), hardness (Rheometer), and bleeding ability (100 Mesh wire-net cone) were measured. Then, release profiles were performed with a WP ointment using the model active ingredient Vitamin D. As a result, high hardness, low bleeding ability and low release profile were observed in the WP ointment samples that were manufactured under the condition of stopping the paddle-mixer at 40°C. However, the influence of cooling speed was observed to affect only hardness. Through optical microscopic observation, it was found that the appearance of WP ointment samples differed depending on the conditions under which they were manufactured. In this study, it was clear that the pharmaceutical properties of WP ointment samples were particularly influenced by the paddle-mixer stopping temperature.

[Blood PCB concentrations and their tendencies examined in Fukuoka 2007-2010 annual inspections for Yusho].

We performed PCB congener specific analysis using HRGC/HRMS and determined their concentrations and blood chromatogram classifications in 275 individual blood samples collected during annual Yusho examinations between 2007 and 2010. When both blood total PCB concentrations and blood chromatogram classifications were compared among eleven Yusho patients undergoing examinations between 2007 and 2010, the longitudinal concentrations and classifications in the respective individuals hardly changed over these years. In a subset of persons suspected of Yusho, it was found that the mean total blood concentrations of three index congeners, that is, 2, 3', 4, 4', 5-pentaCB (PCB118), 2, 2', 4, 4', 5, 5'-hexaCB (PCB153), 2, 3, 3', 4, 4', 5-hexaCB (PCB156) and mean blood total PCB concentrations gradually decreased between 2007 and 2010. Mean concentration of PCB118 in blood from persons suspected of Yusho was calculated as 0.035 ng/g in the 2010 examination, which was 36% lower than the mean value of 0.054 ng/g in the 2007 examination: the concentration of PCB118 showed the highest degree of decrease among the three index congeners. Among persons suspected of Yusho, occupational rates of persons with blood PCB concentrations below one ppb in the entire population increased from approx. 50% in 2007 to approx. 70% in 2010. The lowest concentration of PCB156 in blood of persons suspected of Yusho in 2010 was 0.43 pg/g, which was almost equivalent to the limit of the determination value on HRGC/HRMS, S/N = 10.

[The rapid analysis of polychlorinated quaterphenyls in blood using different diameter capillary column with the high-resolution gas chromatograph high-resolution mass spectrometer].

The polychlorinated quaterphenyl (PCQ) concentrations in blood are important discriminative parameters in yusho patient. In this study, a rapid analytical method for PCQ using different diameter capillary column (rapid-Rtx65TG) with high-resolution gas chromatograph high-resolution mass spectrometer (HRGC/HRMS) instead of the gas chromatograph electron capture detector (ECD/GC) was developed. Using different diameter capillary columns, the analysis time of the HRGC/HRMS was drastically shortened, and the detection sensitivity was improved. In the rapid-Rtx65TG column, a small-bore capillary column (length 1m, I.D. 0.1mm) was connected with the inlet side of the GC, and behind that column, a large-bore capillary column (length 15mm, I.D. 0.53mm) for octadecachloroquaterphenyl (ODCQ) analysis was connected. In the HRGC/HRMS measurement of ODCQ by the rapid-Rtx65TG column, the minimum limit of detection for the apparatus was 0.4 pg, and the minimum limit of determination for the blood was 0.008 ppb. On ECD/GC in the conventional method and HRGC/HRMS in this study, the PCQ concentration in blood including yusho patients and yusho suspected persons was almost equivalent.

Determination of brominated flame retardants and brominated dioxins in fish collected from three regions of Japan.

The concentrations of brominated dioxins which are polybrominated dibenzo-p-dioxins/polybrominated dibenzofurans (PBDD/DFs) and mono-bromo polychlorinated dibenzo-p-dioxins/dibenzofurans, polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA) were investigated in a total of 45 fish samples collected from three regions in Japan. In the brominated dioxins, 1,2,3,4,6,7,8-heptabromodibenzofuran (HpBDF) was the most abundant congener, and it was found in seven fish samples at 0.10-25.6 pg/g wet weight (ww). The highest concentration of 1,2,3,4,6,7,8-HpBDF was found in the pike eel. Regarding other congeners, 2,3,7,8-tetrabromodibenzo-p-dioxin was detected in the sea bream at 0.02 pg/g ww, and 2,3,7,8-tetrabromodibenzofuran was detected in the conger eel at 0.03 pg/g ww. 3-Bromo-2,7,8-trichlorodibenzofuran was detected in the Sardinella zunasi and the conger eel at 0.01 pg/g ww and 0.02 pg/g ww, respectively. Using toxic equivalency factors of chlorinated dioxins, we calculated the PBDD/DFs concentrations of these fish samples at 0.001-0.256 pg TEQ/g ww. PBDEs were detected in all of the fish samples. The concentrations of total PBDEs were 0.01-2.88 ng/g ww. The seerfish and the yellowtail containd PBDEs in high concentrations. The most dominant congener in most of the fish was 2,2',4,4'-tetrabromo diphenyl ether. TBBPA was detected in 29 fish samples at 0.01-0.11 ng/g ww. The mean level of TBBPA was about one-tenth or less of the total level of PBDEs. A good correlation was obtained between total PBDEs and fat content. On the other hand, no correlation was obtained between TBBPA and fat content. The daily intakes from fish were estimated to be 0.58 ng/kg body weight (bw)/day for total PBDEs, 0.03 ng/kg bw/day for TBBPA, and 0.01 pg TEQ/kg bw/day for brominated dioxins in the case assuming that the average bw of a Japanese adult person is 50 kg and that the average fish consumption is 82 g/day. For PBDEs, the provisionally calculated value was much less than the lowest observed adverse effect level value (1 mg/kg bw/day). For brominated dioxins, the daily intake was at a very low level compared with the Japanese daily intake of polychlorinated dioxins from fish. Even if the value of PBDD/DFs is added to the amount of chlorinated dioxin exposure, it was estimated that it is less than the tolerable daily intake (4 pg TEQ/kg bw/day) in Japan.

[Blood PCB concentrations and their characteristics examined in Fukuoka 2006 annual inspection for Yusho].

We analyzed sixty-eight PCB isomers and determined their concentrations and characteristics in fifty-eight individual blood samples collected during the annual Yusho inspection in 2006. The method used in this study consists of a rapid GPC clean-up and sensitive PCB isomer-specific identification with HRGC/HRMS. As a result, the highest total PCB concentration in inspected subjects was 6.6 ppb on a blood weight basis, which was 8.1 times higher than that of a control blood sample. When blood PCB patterns were compared among twenty-seven persons in undergoing examinations both 2004 and 2006, the longitudinal classifications in the respective individuals hardly changed between these years. Mean concentrations of each PCB isomer in the blood of pattern A subjects (n=4) were compared with those in the blood collected from Fukuoka prefecture residents (n=127). Consequently, the respective blood concentrations of PCB #157, #156, #189, #137, #195, #170, #99 were obviously higher than those of Fukuoka residents, while those of PCB #118, # 05 was evidently lower than those of Fukuoka residents.

[Analysis of polychlorinated quaterphenyls in Yusho diagnosis in 2005].

The polychlorinated quaterphenyl (PCQ) concentrations in the blood are important discriminative parameters in Yusho patients. The blood samples of 81 persons were analyzed for PCQ in the 2005 annual inspection for Kanemi rice oil poisoning. PCQ was detected in 28 blood samples (ranging from 0.02 to 4.05 ppb). The means of PCQ concentration in the blood were 1.80 ppb (0.33-4.05 ppb) in pattern A, 0.71 ppb (<0.02-2.26 ppb) in pattern B, 0.03 ppb (< 0. 02-0.14 ppb) in pattern BC and 0.02 ppb (<0.02-0.45 ppb) in pattern C, respectively. PCQ was still present at high concentration in the blood of patterns A and B. Since PCQ concentration is a useful diagnostic criteria, it is important to follow up on this survey of blood PCQ levels.

[Analysis of polychlorinated quaterphenyls in Yusho diagnosis].

The polychlorinated quaterphenyl (PCQ) concentrations in the blood are important discriminative parameters in Yusho patients. In this study, the analytical method of PCQ was improved, and a high speed and precision analysis could be achieved. A recovery test showed satisfactory recoveries of 99.5% (RSD4.8%, n = 5). Using this method, the authors analyzed 62 blood samples which were collected from the 2004 annual inspection for Kanemi rice oil poisoning. PCQ was detected in 20 blood samples (ranging from 0.03 to 4.15 ng/g). The average concentration was 2.07 ng/g in the pattern A (typical Yusho patients), 0.76 ng/g in the pattern B, 0.18 ng/g in the pattern BC and 0.01 ng/g in the pattern C, respectively.

[Congener specific determination of PCBs in human blood using gel permeation chromatography (GPC) and high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS)].

We studied the progressive analytical method for polychlorinated biphenyls (PCBs) in blood samples collected from Yusho patients. This study established a systematic procedure for determining both "total concentration of PCBs and their profiles" and "concentrations of mono-ortho PCBs as dioxin-like compounds", providing a significant index for diagnosis. The method we developed consists of a rapid clean-up using gel permeation chromatography (GPC) and sensitive identification with high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). This method provides identification of all of PCB congeners typically detected in human blood samples, especially it is worth noting that nine significant congeners on Yusho diagnosis (IUPAC #105, #114, #118, #123, #153, #156, #157, #167, #189) were detected from a five-gram blood sample. Using the present method, we analyzed sixty-six individual blood samples collected during the annual Yusho inspection in the 2004, and could identified sixty-eight PCB isomers in all samples. As a result, ten patients were classified into pattern A, the typical Yusho pattern. The mean total PCB concentration of pattern A subjects was 2.95 ppb, while that of control subjects was 0.77 ppb, at whole blood basis respectively.

Determination of polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins/dibenzofurans in marine products.

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in plastics and textile coatings, and these compounds have been recognized as ubiquitous environmental contaminants. Furthermore, it is considered a serious problem that polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs), having toxicities similar to those of chlorinated dioxins, are generated by the manufacture of brominated flame retardants (BFRs) such as PBDEs, and formed by the combustion of substances containing BFRs. Several congeners of PBDD/DFs and PBDEs have been detected in the adipose tissue of the Japanese. Although food is suspected as an exposure source, little information is available regarding the levels of these brominated compounds in food, as compared with information regarding dioxin or polychlorinated biphenyls. It is necessary to investigate the levels of these brominated organic compounds in various foods and to estimate their influence in the case of human exposure. We developed an efficient method of analyzing PBDEs and PBDD/DFs contents in food samples using accelerated solvent extraction and determined the concentrations in several marine products such as raw fish, processed foods, and seaweed purchased in Japan. A recovery test (n = 5) using the method and involving dried fish showed acceptable recoveries of 57.7-78.5% (RSD 5.4-15.9%) for PBDEs and 50.0-56.4% (RSD 1.5-7.9%) for PBDD/DFs. In the analysis of marine product samples, several congeners of PBDEs were detected in raw fish, processed fish, and seaweed; the highest concentration of sigmaPBDEs was detected in yellowtail (1161 pg/g whole basis), followed by mackerel (553.5 pg/g whole basis). The most dominant congener present in these marine samples was 2,2',4,4'-tetraBDE (#47).

[Equality as Yusho diagnosis indices between blood PCB constitution data from using a conventional analytical method and a PCB isomer specific analytical method].

This paper reports that we can replace a conventional PCB analytical method with an isomer specific analytical method in Yusho diagnosis. The authors analysed 109 blood samples from people who had the 2002 annual inspection for Kanemi rice oil poisoning using both methods. The data from these methods were compared and proved to be almost the same in the so-called "PCB pattern" which has been adopted as a Yusho diagnosis index since early 1970s.