Comparison of Immunochemical Reactions of Infliximab Innovator and Biosimilars on an Infliximab Detection Kit Used for Therapeutic Drug Monitoring.

Monitoring serum infliximab (INF) concentrations is crucial for designing appropriate doses for patients with rheumatoid arthritis. It is recommended to maintain the serum trough INF level at least 1.0 µg/mL. In Japan, an in vitro diagnostic kit using immunochromatography has been approved to determine whether the serum INF concentration is over 1.0 µg/mL or not, and to support the determination of the necessity of increasing the dose or switching to another drug. Biosimilars (BS) of INF may have immunochemical properties different from those of its innovator product, which may show different reactivities on the diagnostic kit. In this study, the responses of the innovator and five BS products on the kit were compared. Based on visually comparing the intensity of color development between the test and control samples, differences were found in the judgment results depending on the analyst. In particular, 1.0 µg/mL was not determined as positive in some cases, whereas 2.0 µg/mL was reliably determined as positive. Overall, no significant difference in reactivity was found between the innovator and five BS products. To further compare the differences in immunochemical properties, the reactivity of these products with three enzyme-linked immunosorbent assay (ELISA) kits was compared. The results confirmed that there were no significant differences among the innovator and BS products in reactivity with the examined kits. When using that diagnostic kit, the users need to be aware that the judgement around 1.0 µg/mL INF may differ depending on the test conditions, including the analyst.

Evaluation of the analytical performance of anti-SARS-CoV-2 antibody test kits distributed or developed in Japan.

Background: With the spread of COVID-19, anti-SARS-CoV-2 antibody tests have been utilized. Herein we evaluated the analytical performance of anti-SARS-CoV-2 antibody test kits using a new reference standard prepared from COVID-19 patient sera. Methods: Fifty-seven kits in total (16 immunochromatography types, 11 ELISA types and 30 types for automated analyzers) were examined. By measuring serially diluted reference standards, the maximum dilution factor showing a positive result and its precision were investigated. Results: The measured cut-off titers varied largely depending on the antibody kit; however, the variability was small, with the titers obtained by each kit being within twofold in most cases. Conclusion: The current results suggest that a suitable kit should be selected depending on the intended purpose.

Characterization of Prototheca CYP51/ERG11 as a possible target for therapeutic drugs.

Prototheca spp. are achlorophyllous algae, ubiquitous in nature. An increasing number of human and animal cases of Prototheca infection (protothecosis) are reported, and antifungal azoles, which inhibit sterol 14α-demethylase (CYP51/ERG11) involved in ergosterol biosynthesis, have empirically been used for the treatment of protothecosis. Although Prototheca, like fungi, has ergosterol in the cell membrane, efficacy of the antifungal azoles in the treatment of protothecosis is controversial. For investigating the interaction of azole drugs with Prototheca CYP51/ERG11, the CYP51/ERG11 genomic genes of four strains of P. wickerhamii and one strain each of P. cutis and P. miyajii were isolated and characterized in this study. Compared with the CYP51/ERG11 gene of chlorophyllous Auxenochlorella Protothecoides, it is possible that ProtothecaCYP51/ERG11 gene, whose exon-intron structure appeared to be species-specific, lost introns associated with the loss of photosynthetic activity. Analysis of the deduced amino acid sequences revealed that Prototheca CYP51/ERG11 and fungal CYP51/ERG11 are phylogenetically distant from each other although their overall structures are similar. Our basic in silico studies predicted that antifungal azoles could bind to the catalytic pocket of Prototheca CYP51/ERG11. It was also suggested that amino acid residues away from the catalytic pocket might affect the drug susceptibility. The results of this study may provide useful insights into the phylogenetic taxonomy of Prototheca spp. in relationship to the CYP51/ERG11 structure and development of novel therapeutic drugs for the treatment of protothecosis. LAY SUMMARY: Cases of infection by microalgae of Prototheca species are increasing. However, effective treatment has not been established yet. In this study, gene and structure of Prototheca's CYP51/ERG11, an enzyme which might serve as a target for therapeutic drugs, were characterized for the first time.

Elucidation of the statistical factors that influence anti-drug antibody cut point setting through a multi-laboratory study.

Aim: Appropriateness of anti-drug antibody (ADA) assay is critical for immunogenicity assessment of biopharmaceuticals. Although cut point setting in ADA assay has a large impact on the results, a standard statistical approach for its setting has not been well established. Methodology: In this multi-laboratory study, to elucidate factors influencing the cut point setting, we compared the statistical approaches and calculated cut points for multiple datasets of ADA assays using the individual procedure employed at each laboratory. Conclusion: We showed that outlier exclusion, false-positive rate and investigating data distribution have the greatest impact on both screening and confirmatory cut points. Our results would be useful for industry researchers and regulators engaged in immunogenicity assessment of biopharmaceuticals.

Case report of cutaneous protothecosis caused by Prototheca wickerhamii designated as genotype 2 and current status of human protothecosis in Japan.

An 85-year-old Japanese woman presented with infiltrative erythema and ulceration on the extensor surface of her right forearm. Direct microscopic examination demonstrated spherical and morula-like sporangia, while histopathology revealed numerous microorganisms with a mulberry-like appearance in the dermis. Staining of the microorganisms also showed mulberry-like sporangia that resembled the spokes of a wheel. The isolated yeast-like microorganism had been identified as Prototheca wickerhamii genotype 2 in another independent study on the basis of its morphological, biochemical and genetic analysis. This case of protothecosis was recorded in Kyushu, Japan, and oral treatment with itraconazole 200 mg/day for 2 months was effective. Herein, we also summarize and analyze 39 cases of human protothecosis reported in Japan since the first record in 1983.

Comparison of different immunoassay methods to detect human anti-drug antibody using the WHO erythropoietin antibody reference panel for analytes.

Development of an appropriate assay to detect anti-drug antibody (ADA) is important for assessing immunogenicity to therapeutic protein products. However, characterizing ADA assay methods is difficult because human ADA as a reference standard is not available in most cases. We compared the analytical performance of three ligand-binding assay methods for ADA, namely, surface plasmon resonance (SPR), electrochemiluminescence (ECL), and biolayer interferometry (BLI) methods, by using the anti-erythropoietin (EPO) monoclonal antibody reference panel developed by the World Health Organization (WHO) in 2015. Dose-dependent binding responses were observed for all nine anti-EPO antibodies in the anti-EPO panel by the SPR and BLI methods. In contrast, the ECL method did not clearly detect binding of low-affinity anti-EPO antibodies. Regarding IgG2 and IgM antibodies derived from the same clone, IgG2 exhibited a higher binding response in the SPR assay, whereas the IgM binding response was higher than that of IgG2 in the ECL assay. In the case of the BLI method, there was no consistent pattern observed in the binding responses of IgG2 or IgM. Results of the anti-EPO antibody reference panel, which contains a variety of monoclonal antibodies, indicated that the ability to detect ADAs differed among these assay methods. Therefore, with ligand-binding assays, differences in assay platforms can affect the sensitivity and other characteristics of assays to detect ADAs. These results show that understanding the analytical performance of ADA assays is important for an appropriate assessment of immunogenicity. Our study also indicated the benefits of using the established human ADA reference panel to assess the assay methods for ADA detection.

Immunogenicity of therapeutic protein products: current considerations for anti-drug antibody assay in Japan.

Immunogenicity assessment is an important issue for ensuring the safety and efficacy of therapeutic protein products. Although the reliability of the anti-drug antibody (ADA) assay is one of the key points, there are some difficulties in assessing its validity because the analytes are polyclonal antibodies with variable and unknown characteristics. To elucidate the points to consider for the ADA assay, a Japanese research group was established that discusses the issues raised on the immunogenicity assessment. In this review, we first introduce the current situation regarding the development and immunogenicity assessment of therapeutic protein products in Japan. We then present our current view and recommendations on the ADA assay by considering its unique features.

Molecular Characterization of Prototheca strains isolated in China revealed the first cases of protothecosis associated with Prototheca zopfii genotype 1.

In this study, six strains of Prototheca isolated in China from human patients diagnosed as protothecosis and cows with mastitis were characterized by polymerase chain reaction (PCR) and nucleotide sequencing of the ribosomal RNA gene (rDNA) and by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The results indicated that three strains isolated from the human patients were P. zopfii genotype 1, revealing the first cases of human protothecosis associated with P. zopfii genotype 1. The remaining three strains were shown to be P. zopfii genotype 2. Interestingly, one strain isolated from the cerebrospinal fluid of the human patient appeared to have both of the genotype 1- and 2-specific alleles in the small subunit (SSU) rDNA although it was classified by MALDI-TOF MS as genotype 2. For genotyping of certain strains of P. zopfii, it may be necessary to comprehensively evaluate the diversity in the SSU rDNA sequences and the MALDI-TOF MS results.

Eosinophilic plasmacytic conjunctivitis concurrent with gingival fistula caused by Schizophyllum commune in a captive cheetah (Acinonyx jubatus).

We describe for the first time the diagnosis of Schizophyllum commune infection in a captive cheetah. Eosinophilic plasmacytic conjunctivitis was detected histopathologically in a biopsy specimen. Both a second surgical specimen and drainage fluid from a gingival mass and fistula contained fungal hyphae in giant cells with granulomatous inflammation. Allergic S. commune mycosis was suspected at this point. A monokaryotic isolate was characterized morphologically, and then identified genetically. Treatment with itraconazole and pimaricin was effective.

Population Pharmacokinetics of Diazoxide in Children with Hyperinsulinemic Hypoglycemia.

BACKGROUND: Diazoxide is the first-line treatment for pediatric hyperinsulinemic hypoglycemia (HI). This study aimed to elucidate the pharmacokinetics of diazoxide in children with HI. METHODS: We obtained 81 blood samples from 22 children with HI. Measured serum diazoxide concentrations were used for population pharmacokinetic analysis. Patient factors influencing pharmacokinetics were estimated using nonlinear mixed-effects model analysis. Relationships between drug exposure and adverse drug reactions were also investigated. RESULTS: Diazoxide disposition in the body was described by a 1-compartment model. Oral clearance (CL/F) and the volume of distribution were proportional to body weight (WT), as expressed by CL/F in males (liters/h) = 0.0358 + 0.00374 × WT (kg). CL/F in females was 39% greater than that in males. Steady-state concentrations of diazoxide were similar following twice- and 3 times-daily dosing when the total daily doses were comparable. A patient whose serum diazoxide concentration exceeded 100 µg/mL over a 4-month period developed hyperglycemia. No significant correlation was observed between severity of hirsutism and diazoxide concentration. CONCLUSION: We have proposed for the first time a population pharmacokinetic model for diazoxide in children with HI. The potential risk of diabetes mellitus and/or hyperglycemia increases when serum concentrations of diazoxide exceed 100 µg/mL.

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis.

Species of the genus Prototheca are achlorophyllous algae and ubiquitous in nature, and so far, six species have been listed in this genus: Prototheca wickerhamii, Prototheca zopfii, Prototheca blaschkeae, Prototheca cutis, Prototheca stagnora and Prototheca ulmea. A strain of the genus Prototheca, IFM 53848T, was isolated in Japan from a patient with systemic protothecosis and had been designated P. wickerhamii. Our previous study, by using PCR analysis, revealed that its SSU rRNA gene (rDNA) was distinctively larger than that of P. wickerhamii and other species of the genus Prototheca. In this study, molecular analysis showed that the exceptionally large SSU rDNA of IFM 53848T contains four group I introns. The morphology of IFM 53848T was indistinguishable from those of P. wickerhamii or P. cutis, and phylogenetic analyses, based on the sequences of the SSU rDNA exons and the D1/D2 region of the large subunit rDNA, indicated that IFM 53848T was closely related to P. cutis. On the other hand, unlike P. cutis, IFM 53848T failed to assimilate fructose or lysine and grew well at higher temperatures of up to 42 °C. In addition, the nucleotide sequence of the ribosomal internal transcribed spacer and the matrix assisted laser desorption ionization time-of-flight mass spectrometry profile of IFM 53848T were clearly distinct from those of P. cutis. The results strongly suggest that IFM 53848T represents a novel species, and so the seventh member of the genus Prototheca, which we have named Prototheca miyajii sp. nov. The unique characteristics of the strain may provide useful insights into the systematic taxonomy of the genus Prototheca.

Wickerhamomyces anomalus fungal keratitis responds to topical treatment with antifungal micafungin.

We describe a 91-year-old woman who suffered from fungal keratitis after corneal transplantation. The causative organism was identified as Wickerhamomyces anomalus (formerly Pichia anomala or Hansenula anomala) on the basis of morphological characteristics and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. The patient was successfully treated with topical micafungin (MCFG) only. We present the first report of a case of W. anomalus fungal keratitis that responded to topical treatment with the antifungal MCFG.

Ribosomal internal transcribed spacer of Prototheca wickerhamii has characteristic structure useful for identification and genotyping.

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms.

Intraocular penetration of intravenous micafungin in inflamed human eyes.

Eight eyes of 7 patients with fungal disease received intravenous injections of 150 to 300 mg micafungin, and samples of blood, cornea, retina-choroid, aqueous humor, and vitreous humor were collected. The micafungin levels in all collected samples exceeded the MICs; however, the levels in the vitreous and aqueous humors were lower. Our findings suggest that intravenous micafungin should be given in combination with intravitreal antifungal agents after vitrectomy in severe cases of intraocular fungal diseases.

Rare case of fungal keratitis caused by Corynespora cassiicola.

We describe a 76-year-old male farmer with no diabetes mellitus and no history of ocular trauma from soil or plants who developed a corneal infection from a plant pathogen. The organism was identified as Corynespora cassiicola based on both the morphological characteristics and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. The patient was treated successfully with a combination of topical and systemic voriconazole. This is the first reported case of keratomycosis caused by C. cassiicola.

Rare case of fungal keratitis caused by Plectosporium tabacinum.

A rare case of fungal keratitis caused by Plectosporium tabacinum is reported. A 78-year-old female gardener presented with conjunctivitis and an oval infiltrate with irregular margins in the nasal half of the cornea in the right eye. Light microscopy of corneal scrapings revealed a filamentous fungus, and a diagnosis of fungal keratitis was made. The patient was admitted into our hospital on February 19, 2008. Treatment with topical miconazole, topical fluconazole, pimaricin ointment, intravenous miconazole, and corneal debridement was commenced. One week later, the infiltrate improved, but the central part of the infiltrate was still deep. Topical fluconazole was switched to topical voriconazole, and intravenous miconazole was switched to intravenous voriconazole. One month after admission, the causative organism was identified by morphology and molecular biological analysis as Plectosporium tabacinum. The corneal infiltrate resolved 3 months after admission. A stromal scar persisted for 3 months after the patient was discharged. This is the first detailed report of fungal keratitis caused by P. tabacinum. Voriconazole was effective in treating this refractory keratitis.

[Clinical manifestations, treatment and outcome of ocular infections caused by Paecilomyces species].

PURPOSE: To report the predisposing factors, patient background, treatment and outcomes of ocular infections caused by Paecilomyces spp. in Japan. METHODS: We reviewed the medical records and the published literature in Japan on ocular infections caused by microbiologically-proven Paecilomyces spp. Nineteen eyes of 18 patients; 4 eyes of 4 of our patients and 15 eyes of 14 published Japanese patients were studied. RESULTS: Ten men and 8 women (9 OD, 8 OS, 1 OU) were diagnosed with ocular infections caused by Paecilomyces spp. The mean age was 69 years (range 33 to 90 years). The initial diagnosis of ocular infection caused by Paecilomyces spp. was keratitis in 14 eyes and endophthalmitis in 5 eyes. The final diagnosis was keratitis in 2 eyes, keratitis with involvement of the anterior segment of the eye, i.e., hypopyon or corneal rupture, in 12 eyes, and endophthalmitis in 5 eyes. The predisposing factors had a history of ocular surgery, corneal trauma and soft contact lens use. Other factors were diabetes and old age. The infections developed more often in the fall and winter. The identified Paecilomyces spp. had a high sensitivity to micafungin and voriconazole, but low sensitivity to amphotericin B, flucytosine and fluconazole. The prognosis of Paecilomyces spp. ocular infections was very poor, viz., final vision was counting fingers or worse in 60%, ocular perforation in 42%, and eye enucleation in 11%. The percentage of eyes with a final visual acuity worse than counting fingers was 0% in those with keratitis, 50% in those with involvement of the anterior segment, and 100% in those with endophtalmitis (p = 0.0446). Among the antifungal agents, the percentage of cases with final vision of counting fingers or worse was 90% with fluconazole, 80% with itraconazole, 100% with miconazole, and 71% with voriconazole. CONCLUSION: The results show that if the Paecilomyces spp. infection spreads intraocularly, it is difficult to mitigate the clinical damage even with antifungal agents with high sensitivity.