Biofilm-formation capability depends on environmental oxygen concentrations in Candida species.

BACKGROUND: Although oxygen concentrations inside of the human body vary depending on organs or tissues, few reports describe the relationships between biofilm formation of Candida species and oxygen concentrations. In this study, we investigated the biofilm-forming capabilities of Candida species under various oxygen conditions. METHODS: We evaluated the adhesion and biofilm formation of Candida albicans and C. tropicalis under aerobic, microaerobic (oxygen concentration 5%), or anaerobic conditions. We also examined how oxygen concentration affects adhesion/maturation by changing adhesion/maturation phase conditions. We used crystal violet assay to estimate the approximate biofilm size, performed microscopic observation of biofilm morphology, and evaluated adhesion-associated gene expression. RESULTS: The adhered amount was relatively small except for a clinical strain of C. tropicalis. Our biofilm-formation analysis showed that C. albicans formed a higher-size biofilm under aerobic conditions, while C. tropicalis favored microaerobic conditions to form mature biofilms. Our microscopic observations were consistent with these biofilm-formation analysis results. In particular, C. tropicalis exhibited more hyphal formation under microaerobic conditions. By changing the adhesion/maturation phase conditions, we represented that C. albicans had favorable biofilm-formation capability under aerobic conditions, while C. tropicalis showed enhanced biofilm formation under microaerobic adhesion conditions. In good agreement with these results, the C. tropicalis adhesion-associated gene expression tended to be higher under microaerobic or anaerobic conditions. CONCLUSIONS: C. albicans favored aerobic conditions to form biofilms, whereas C. tropicalis showed higher biofilm-formation ability and promoted hyphal growth under microaerobic conditions. These results indicate that favorable oxygen conditions significantly differ for each Candida species.

Evaluation of a new point-of-care quantitative reverse transcription polymerase chain test for detecting severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID-19), has become a global pandemic. Although there are multiple methods for detecting SARS-CoV-2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for detecting SARS-CoV-2. METHODS: Samples from 96 patients with suspected SARS-CoV-2 infection were assessed using the real-time qRT-PCR-based POCT and the conventional real-time qRT-PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). RESULTS: The real-time qRT-PCR-based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR. All patients could be triaged within 1 day using the real-time qRT-PCR-based POCT without invalid reports. CONCLUSIONS: The real-time qRT-PCR-based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.

The effect of gut microbiota and probiotic organisms on the properties of extended spectrum beta-lactamase producing and carbapenem resistant Enterobacteriaceae including growth, beta-lactamase activity and gene transmissibility.

The gut microbiota may play a pivotal role in controlling the antimicrobial resistant (AMR) organisms although the evidences are limited. We investigated the effects of gut microbiota on the growth of AMR organisms, ß-lactamases activity and transmissibility of antimicrobial resistant properties of the extended spectrum ß-lactamase (ESBL)-producing Escherichia coli and carbapenem-resistant Enterobacteriaceae. CTX-M-15-positive, ESBL-producing E. coli and carbapenem resistant Enterobacteriaceae, Bacteroides fragilis, Bifidobacterium longum, Clostridium butyricum, Clostridioides difficile, Clostridium perfringens, Enterococcus faecium, Lactobacillus plantarum and probiotic strain of C. butyricum MIYAIRI 588 were used in this study. The growth of AMR organisms was suppressed by the supernatant of C. butyricum, C. difficile, C. perfringens, E. faecium and L. plantarum in a dose dependent manner but not by that of B. fragilis and B. longum. The ß-lactamase activity produced by E. coli was reduced by the presence of culture supernatant of certain gut microbiota during stationary phase of E. coli. Importantly, C. butyricum MIYAIRI 588 culture supernatant suppressed the transcription of blaCTX-M gene during growth phase of E. coli. The conjugation assay showed the reduction of transmissibility of antibiotic resistant gene by gut microbiota. These findings suggest that certain gut microbiota affect the antibiotic resistant activities of AMR organisms. Further studies are needed to identify the specific mechanism(s) of these actions between AMR organisms and gut microbiota.

Severe epidemic myalgia with an elevated level of serum interleukin-6 caused by human parechovirus type 3: a case report and brief review of the literature.

BACKGROUND: Human parechovirus type 3 (HPeV-3) is known to cause cold-like symptoms, diarrhea, or severe infections such as sepsis in infants and children. In adults, HPeV-3 infection is rarely diagnosed because the symptoms are generally mild and self-limiting; however, this infection has been linked to epidemic myalgia, regardless of the presence of underlying diseases, immunosuppression, or sex. CASE PRESENTATION: We describe an adult case of severe systemic myalgia and orchiodynia after infection with HPeV-3, which was transmitted from the child of the patient. Interleukin-6 (IL-6) level was found to be elevated in the patient's serum. CONCLUSION: Severe myalgia associated with HPeV-3 infection is potentially caused by an elevated serum level of IL-6.

Comparison between local and national epidemiology of antimicrobial resistance using the JANIS data.

The Japanese Ministry of Health, Labor and Welfare established the Japanese Nosocomial Infection Surveillance (JANIS) system in July 2000 to provide nationwide epidemiological information. The data of all clinically isolated bacteria at participating hospitals were collected, treated according to a protocol, and analyzed by the JANIS office. Nationwide and individual hospital data were reported to participating hospitals monthly and yearly. In this study, we surveyed local antimicrobial resistance of clinically isolated bacteria in Kawasaki City between 2014 and 2016 using JANIS data. There were 8, 14, and 16 major hospitals in Kawasaki City that participated in the surveillance in 2014, 2015, and 2016, respectively. The data were returned to each hospital monthly from JANIS, totaled for Kawasaki City, and compared with the nationwide data. The Kawasaki City data were approximately the same as the nationwide data, and most resistant bacteria decreased gradually over the three years examined. The incidence of methicillin-resistant Staphylococcus aureus (MRSA) in S. aureus (Kawasaki City, Japan) was (56.6%, 48.8%), (50.5%, 47.9%), and (51.6%, 46.9%), the incidence of penicillin-resistant Streptococcus pneumoniae (PRSP) in S. pneumoniae, was (43.4%, 37.5%), (34.3%, 38.0%), and (31.4%, 36.9%) in 2014, 2015, and 2016, respectively. Therefore, in Kawasaki City the incidence of MRSA was relatively higher and that of PRSP was relatively lower than the nationwide incidences. Both continuous local and national surveillance are important for monitoring antimicrobial resistance in clinical isolates. The JANIS database is a powerful tool for the epidemiology of nosocomial infections in Japanese hospitals.

The reduction of l-cystine to l-cysteine in the supernatant of A549 cell culture causes imipenem inactivation.

In the course of measuring the intracellular antibacterial activity of antibiotics using a human alveolar epithelial cell line A549, we discovered that the antimicrobial activity of several carbapenems (CPs) decreased in the supernatant of the cells cultured with fetal calf serum (FCS)-free RPMI1640 medium (RPMI). Further investigation revealed A549 culture supernatant inhibited the antibacterial activity of CPs but did not inactivate other types of antibiotics. CE-TOFMS and LC-TOFMS metabolomics analysis of the supernatant revealed the presence of l-cysteine (Cys), which is not an original component in RPMI. Cys is known to hydrolyze and inactivate CPs in a time- and concentration-dependent manner. In this study, the inactivating effects of A549 culture supernatant on the imipenem (IPM) were examined. Antimicrobial activity of 100 µg/mL IPM decreased to 25% with two-fold dilution of A549 supernatant incubated for 3 h. l-Cystine (CS), the Cys oxide, and an original component in RPMI did not inactivate IPM. However, the inactivating effects of A549 supernatant on IPM corresponds with the Cys concentration and depends on the CS content of the culture medium. Addition of FCS to the culture medium decreased the Cys concentration and reduced inactivation of IPM in a dose-dependent manner. Our data suggest that IPM were inactivated by Cys reduced from CS, and this CS-to-Cys conversion must be considered when evaluating the antimicrobial activity of CPs in cell culture. Further studies are needed to understand if the same inactivation occurs around the cells in the human body.

The first reported case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens.

We describe a case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens, a non-neoformans and non-gattii Cryptococcus, in a non-HIV patient. A 71-year-old man with diffuse large B-cell lymphoma receiving antineoplastic chemotherapy was febrile approximately 30 weeks after central venous port insertion, and C. liquefaciens was isolated from all three performed blood cultures as well as a central venous catheter tip culture. In vitro antifungal susceptibility tests showed that this yeast isolate was susceptible to low concentrations of amphotericin B, fluconazole, itraconazole and voriconazole yet was resistant to 5-fluorocytosine (MIC: >64 µg/ml), unlike Cryptococcus neoformans. Treatment of the patient with oral and intravenous voriconazole was effective and consistent with the susceptibility tests. Although non-neoformans and non-gattii Cryptococcus spp. are considered non-pathogenic environmental yeast, they may rarely be the causative agents of serious infections in humans, as in the present case.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2012].

The nationwide surveillance of antibacterial susceptibility to meropenem (MEPM) and other parenteral antibiotics against clinical isolates during 2012 in Japan was conducted. A total of 2985 strains including 955 strains of Gram-positive bacteria, 1782 strains of Gram-negative bacteria, and 248 strains of anaerobic bacteria obtained from 31 medical institutions were examined. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). 2. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous studies in 2009 or 2006. Therefore, the tendency to increase in antimicrobial resistance rates was not observed. 3. MEPM resistance against Pseudomonas aeruginosa was 17.8% (56/315 strains). Compared to our previous results, it was the lowest than that in 2006 and 2009. 4. Carbapenem-resistant Klebsiella pneumoniae, and multi-drug-resistant Acinetobacter species, which emerged in worldwide, were not observed. 5. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 6.2% (59/951 strains) in enterobacteriaceae, which increased compared with that of our previous studies in 2009 or before. Whereas, the proportion of metallo-beta-lactamase strains was 1.6% (5/315 strains) in P. aeruginosa, which was stable. In conclusion, the results from this surveillance suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 17 years passed after available for commercial use in Japan.

Electrostatic interaction between sulfated polysaccharides and oligopeptides from viral envelope glycoproteins using surface plasmon resonance.

Biotinylated oligopeptides from the envelope glycoproteins of dengue fever virus, influenza A and B viruses, and human immunodeficiency virus (HIV) were synthesized and their interaction with curdlan and dextran sulfates was investigated using surface plasmon resonance to evaluate the antiviral mechanisms of sulfated polysaccharides. More than two clusters consisting of basic amino acids in the oligopeptides from dengue fever virus, strongly interacted with the sulfated polysaccharides elucidated by the association- and dissociation-rate constants. Interactions decreased with the decreasing molecular weights of the sulfated polysaccharides. Although oligopeptides from influenza A virus potently interacted with the sulfated polysaccharides, no interaction was detected on a B/Hong Kong virus oligopeptide bearing few basic amino acids. For the C terminus and V3 region short and long oligopeptides from HIV gp120, the interaction was enhanced by the number of clustered basic amino acids and was inhibited by acidic and bulky amino acids.

Inhibitory effects of vaginal Lactobacilli on Candida albicans growth, hyphal formation, biofilm development, and epithelial cell adhesion.

Introduction: Antifungal agents are not always efficient in resolving vulvovaginal candidiasis (VVC), a common genital infection caused by the overgrowth of Candida spp., including Candida albicans, or in preventing recurrent infections. Although lactobacilli (which are dominant microorganisms constituting healthy human vaginal microbiota) are important barriers against VVC, the Lactobacillus metabolite concentration needed to suppress VVC is unknown. Methods: We quantitatively evaluated Lactobacillus metabolite concentrations to determine their effect on Candida spp., including 27 vaginal strains of Lactobacillus crispatus, L. jensenii, L. gasseri, Lacticaseibacillus rhamnosus, and Limosilactobacillus vaginalis, with inhibitory abilities against biofilms of C. albicans clinical isolates. Results: Lactobacillus culture supernatants suppressed viable fungi by approximately 24%-92% relative to preformed C. albicans biofilms; however, their suppression differed among strains and not species. A moderate negative correlation was found between Lactobacillus lactate production and biofilm formation, but no correlation was observed between hydrogen peroxide production and biofilm formation. Both lactate and hydrogen peroxide were required to suppress C. albicans planktonic cell growth. Lactobacillus strains that significantly inhibited biofilm formation in culture supernatant also inhibited C. albicans adhesion to epithelial cells in an actual live bacterial adhesion competition test. Discussion: Healthy human microflora and their metabolites may play important roles in the development of new antifungal agent against C. albicans-induced VVC.

Ciliates expel environmental Legionella-laden pellets to stockpile food.

When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.

Neonatal meningitis caused by Streptococcus gallolyticus subsp. pasteurianus.

We encountered a case of neonatal meningitis caused by Streptococcus gallolyticus subsp. pasteurianus. The patient was an 8-day-old boy. Gram staining of the cerebrospinal fluid (CSF) revealed gram-positive cocci in pairs or in short chains. In culture, γ-streptococcus-like colonies grew. The result of 16S rRNA sequence analysis identified S. gallolyticus subsp. pasteurianus. From these results, bacterial meningitis was diagnosed and, as a result of antimicrobial susceptibility testing, single-dose ampicillin therapy was given. Because inflammatory deterioration and spread was suspected from the CSF test results, this therapy was added by panipenem/betamipron. In response to his recovery, antibiotic treatment was stopped and the boy was discharged. This bacterium was classified as S. gallolyticus subsp. pasteurianus in the latest report in 2003. Since this change, there have only been a few cases of neonatal meningitis caused by this bacterium. Here we report this rare case.

Predicting the aggravation of coronavirus disease-19 pneumonia using chest computed tomography scans.

Presently, coronavirus disease-19 (COVID-19) is spreading worldwide without an effective treatment method. For COVID-19, which is often asymptomatic, it is essential to adopt a method that does not cause aggravation, as well as a method to prevent infection. Whether aggravation can be predicted by analyzing the extent of lung damage on chest computed tomography (CT) scans was examined. The extent of lung damage on pre-intubation chest CT scans of 277 patients with COVID-19 was assessed. It was observed that aggravation occurred when the CT scan showed extensive damage associated with ground-glass opacification and/or consolidation (p < 0.0001). The extent of lung damage was similar across the upper, middle, and lower fields. Furthermore, upon comparing the extent of lung damage based on the number of days after onset, a significant difference was found between the severe pneumonia group (SPG) with intubation or those who died and non-severe pneumonia group (NSPG) ≥3 days after onset, with aggravation observed when ≥14.5% of the lungs exhibited damage at 3-5 days (sensitivity: 88.2%, specificity: 72.4%) and when ≥20.1% of the lungs exhibited damage at 6-8 days (sensitivity: 88.2%, specificity: 69.4%). Patients with aggravation suddenly developed hypoxemia after 7 days from the onset; however, chest CT scans obtained in the paucisymptomatic phase without hypoxemia indicated that subsequent aggravation could be predicted based on the degree of lung damage. Furthermore, in subjects aged ≥65 years, a significant difference between the SPG and NSPG was observed in the extent of lung damage early beginning from 3 days after onset, and it was found that the degree of lung damage could serve as a predictor of aggravation. Therefore, to predict and improve prognosis through rapid and appropriate management, evaluating patients with factors indicating poor prognosis using chest CT is essential.

Serum gasdermin D levels are associated with the chest computed tomography findings and severity of COVID-19.

BACKGROUND: The role of programmed cell death, especially pyroptosis and apoptosis, in unfavorable immune responses in COVID-19 remains to be elucidated. METHODS: We conducted a cross-sectional analysis to investigate the association between the serum gasdermin D (GSDMD) levels, a pyroptotic marker, and caspase-cleaved cytokeratin 18 fragment (M30), an apoptotic marker, and the clinical status and abnormal chest computed tomography (CT) findings in patients with COVID-19. RESULTS: In this study, 46 patients diagnosed with COVID-19 were divided into the following three groups according to the disease severity: mild to moderate group (n = 10), severe group (n = 14), and critical group (n = 22). The serum GSDMD levels were higher in the critical group than in the mild to moderate group (P = 0.016). In contrast, serum M30 levels were lower in the critical group than in the severe group (P = 0.048). Patients who required mechanical ventilation or died had higher serum GSDMD levels than those who did not (P = 0.007). Area of consolidation only and of ground glass opacity plus consolidation positively correlated with serum GSDMD levels (r = 0.56, P < 0.001 and r = 0.53, P < 0.001, respectively). CONCLUSION: Higher serum GSDMD levels are associated with critical respiratory status and the consolidation area on chest CT in patients with COVID-19, suggesting that excessive activation of pyroptosis may affect the clinical manifestations in patients with COVID-19.

[A case of acute septic osteomyelitis onset due to Streptococcus dysgalactiae subsp. equisimilis in an elderly diabetic patient].

Group C streptococci are increasingly causing invasive infections such as that we report here. A 70-year-old man being treated for diabetes and seen at the emergency room for neck pain and fever was hospitalized for possible sepsis. His temperature was 39.8 degrees C, regular pulse 101 bpm, and pain reinforced in flexing and cervical rotation. Streptococcus dysgalactiae subsp. equisimilis (SDSE) was cultured from blood. Neck pain gradually decreased with of 2 million units PCG 6 times/day. Magnetic resonance imaging (MRI) of the cervical spine showed high-intensity areas in fat-suppression imaging at C7, Thl and intervertebral disks plus enhancement around the vertebral body, yielding a diagnosis of cervicothoracic vertebral osteomyelitis. Antimicrobial intravenous therapy continuede 6 weeks. The man was discharged after 45 days without relapse.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2009].

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.

High-Speed Quenching Probe-Polymerase Chain Reaction Assay for the Rapid Detection of Carbapenemase-Producing Gene Using GENECUBE: A Fully Automatic Gene Analyzer.

BACKGROUND: The prevalence of carbapenemase-producing organisms (CPOs) globally poses a public health threat; however, detecting carbapenemases is a challenge because of their variety. METHODS: GENECUBE, a fully automated gene analyzer, detects a target gene in a short time and simultaneously detects its single nucleotide polymorphism. We used this property to develop for the first time a rapid assay for detecting CPOs from cultured bacteria using GENECUBE. The original primer-probe sets were used to detect blaKPC, blaIMP, blaVIM, blaNDM, and blaOXA-48-like from 149 CPOs (nine types) and 61 non-CPOs. RESULTS: The sensitivity, specificity, and positive and negative predictions of the GENECUBE assay were 100%. This assay detected carbapenemase single-producers and carbapenemase co-producers with 100% accuracy. The time required for detects of four types of carbapenemase at one run was about 30 min, but it took about 1 h to detect all five types. In addition, this assay performed the rapid detection and classification of blaOXA-48, blaOXA-181, blaOXA-232, and blaOXA-244 simultaneously. CONCLUSIONS: The GENECUBE assay is a promising tool for controlling the spread of CPOs and helping to select accurate and rapid antibiotic therapies.

Rapid detection assay of toxigenic Clostridioides difficile through PathOC RightGene, a novel high-speed polymerase chain reaction device.

Nucleic acid amplification tests for diagnosing Clostridioides difficile infections (CDI) are improving to become faster and more accurate. This study aimed to evaluate the accuracy of rapid detection of toxigenic C. difficile using the novel high-speed polymerase chain reaction (PCR) device, PathOC RightGene. These results were compared and evaluated with real-time PCR (qPCR) and enzyme immunoassays (EIA) kit. For this study, 102 C. difficile and 3 Clostridium species isolated from CDI patients were used. These C. difficile isolates were 85 toxigenic and 17 non-toxigenic strains. The results of qPCR served as a standard, and sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the PathOC Right Gene were 99.2%, 99.4%, 100%, 98.8%, and 99.3%, respectively. Turnaround time of qPCR and EIA was 85 and 30 minutes, whereas PathOC RightGene was only 25 minutes including DNA extraction. This novel high-speed PCR device detected toxigenic C. difficile rapidly and accurately.

Lignosulfonate Rapidly Inactivates Human Immunodeficiency and Herpes Simplex Viruses.

Background: Very few studies of the antiviral potential of lignosulfonates have been published. With the aim of oral application, among various groups of natural products, the relative antiviral potency of lignosulfonate and its ability to rapidly inactivate viruses were investigated. Methods: As target cells, MT-4 cells in suspension and attached Vero cells were used for infections with human immunodeficiency virus (HIV) and human herpes simplex type-1 virus (HSV). Mock- or virus-infected cells were incubated for 3-5 days with various concentrations of test samples, and the viable cell number was determined with the MTT method. For the shorter exposure experiments, higher titers of HIV or HSV were exposed to test samples for 10 or 3 min, diluted to a normal multiplicity of infection (MOI), and applied to the cells. Antiviral activity was quantified by using the chemotherapy index. Results: In the long-exposure system, lignosulfonates showed comparable anti-HIV activity with those of AZT, ddC, and sulfated polysaccharides, and it exceeded those of hundreds of tannins and flavonoids. When the exposure time was shortened, the chemotherapeutic index of the lignosulfonates for HIV was increased 27-fold. At a physiological pH, lignosulfonate showed higher anti-HIV activity than commercial alkali-lignin, dealkali-lignin, and humic acid, possibly due to the higher solubility and purity. Conclusions: With their rapid virus-inactivation capabilities, lignosulfonates may be useful for the prevention or treatment of virally induced oral diseases.

Re-Evaluation of Chemotherapeutic Potential of Pyoktanin Blue.

Background: Pyoktanin blue (PB) is used for staining tissues and cells, and it is applied in photodynamic therapy due to its potent bactericidal activity. However, clinical application of PB as an antiviral and antitumor agent has been limited due to its potent toxicity. For clinical application, the antitumor and antiviral activity as well as the neurotoxicity of PB were re-evaluated with a chemotherapeutic index. Methods: Tumor-specificity (TS) was determined by the ratio of CC50 against normal oral cells/oral squamous cell carcinoma (OSCC); neurotoxicity by that of normal oral/neuronal cells; antiviral activity by that of mock-infected/virus-infected cells; and potency-selectivity expression (PSE) by dividing TS by CC50 (OSCC). Results: Antitumor activity of PB (assessed by TS and PSE) was comparable with that of DXR and much higher than that of 5-FU and melphalan. PB induced caspase-3 activation and subG1 cell accumulation in an OSCC cell line (Ca9-22). PB and anticancer drugs showed comparable cytotoxicity against both neuronal cells and OSCC cell lines. PB showed no detectable anti-HIV/HSV activity, in contrast to reverse transferase inhibitors, sulfated glucans, and alkaline extract of leaves of S.P. Conclusions: PB showed first-class anticancer activity and neurotoxicity, suggesting the importance of establishing the safe treatment schedule.