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.

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.

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.

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.

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.

Kerion celsi due to Arthroderma incurvatum infection in a Sri Lankan child: species identification and analysis of area-dependent genetic polymorphism.

A three-year-old Sri Lankan boy residing in Japan developed a nodule on his scalp after visiting Sri Lanka. Two months later, the lesion increased in size to 22 × 19 mm(2), and was identified as an erythematous nodule with alopecia. Direct examination of the infected hair shafts indicated fungal hyphae outside the shafts. The fungus was identified as Microsporum gypseum following mycological examination. The sequence of the internal transcribed spacer 1 region of ribosomal RNA gene (ITS1 rDNA) exhibited 95.7-100.0% homology with that of Arthroderma incurvatum. The patient was successfully treated with a 6-week itraconazole course. We also examined DNA samples from eight clinical isolates of A. incurvatum. Alignments of ITS1 sequences of these strains and our isolate, showed gaps in the 64-bp positions 140-142 and 141-143 of the 205-207-bp ITS1 alignment. We performed phylogenetic analysis using the neighbor-jointing (NJ) method based on the ITS1 sequences of the present isolate and twenty related strains. Fifteen A. incurvatum strains were divided into East Asia and non-East Asia clusters. The present isolate belonged to the non-East Asia cluster, suggesting that the patient was infected outside Japan. Moreover, the trees suggested area-dependent genetic polymorphism of A. incurvatum.

Identification and sensitivity of two rare fungal species isolated from two patients with Fusarium keratomycosis.

We report two cases of Fusarium keratomycosis in which molecular analysis was used to identify two rare causative Fusarium species. A 65-year-old woman was diagnosed with Fusarium keratomycosis caused by F. equiseti, confirmed by sequencing the internal transcribed spacer (ITS) region and morphological characteristics. She was initially treated with topical corticosteroid but the keratitis did not improve. Even after aggressive antifungal treatment, there was a severe reduction of vision. In the end, the eye perforated. Our second case was a 75-year-old man who had diabetes mellitus. He developed keratitis after his cornea was injured by corn stalks. The ITS region identified the causative organism as F. verticillioides. Unlike the first case, he had good visual recovery after early antifungal therapy. Our findings demonstrate that both ITS and morphological characteristics can be used to identify the exact Fusarium species causing Fusarium keratomycosis. This allowed us to determine the sensitivity of these species to antifungal drugs that can be used to treat patients with these species of Fusarium.

Chlamydospores of Rhizopus microsporus var. rhizopodiformis in tissue of pulmonary mucormycosis.

Hyphae are usually the only fungal elements found in tissue of mucormycosis, and other fungal elements are quite rarely encountered. We found chlamydospores in bronchial lumina in autopsied tissue of pulmonary mucormycosis of a diabetic patient. Chlamydospores are thick-walled, asexually produced spores arising from the modification of a hyphal segment. This is the first histologic demonstration of chlamydospores in mucormycosis in which the causative fungus is culturally identified to species level. Rhizopus microsporus var. rhizopodiformis was isolated from the present autopsied pulmonary tissue. A literature review of human infection by this fungus found 27 cases with histopathologic evidence.

[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.

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.

Phaeohyphomycosis of the face caused by Exophiala oligosperma.

A 57-year-old previously healthy woman who works in the fish-processing industry presented with a 1-year history of a slightly pruritic, hyperkeratotic, brownish, erythematous lesion of the left cheek measuring 5 × 5 mm in diameter. Histopathology revealed granuloma formation in the superficial dermal layer by multinucleated giant cells that contained pale-brown septate hyphae. Periodic acid-Schiff stain showed many hyphae and catenate spores within the multinucleated giant cells. Tissue specimens and skin scrapings were obtained and incubated on mycosel agar, yielding black, velvety colonies that were morphologically identified as belonging to Exophiala species. Sequence analysis of the internal transcribed spacer region of the ribosomal RNA gene showed 99-100% homology to Exophiala oligosperma sequences. This report describes a rare case of phaeohyphomycosis of the face caused by E. oligosperma.

Ochroconis calidifluminalis, a sibling of the neurotropic pathogen O. gallopava, isolated from hot spring.

Two strains resembling the neurotropic fungus Ochroconis gallopava were isolated from hot spring river water (IFM 54738 and IFM 54739). The isolates showed optimal growth at 42 degrees C, while the maximum growth temperature was 49 degrees C, thus having temperature relationships similar to those of O. gallopava. Colonies were light olive green, with a color change to dark reddish brown after several passages, which was also observed in O. gallopava. Conidia were indistinguishable from those of O. gallopava. The antifungal susceptibility profile of the isolates was also similar to that of O. gallopava, except for a lower susceptibility to micafungin. The two isolates had 100% homologous rRNA genes including the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit. The gene fragments, as O. gallopava, could be amplified with species-specific rDNA primers, and loop-mediated isothermal amplification designed for O. gallopava yielded positive results in the two isolates. However, homologies with O. gallopava in ITS and D1/D2 regions were 79.2 and 95.9%, respectively, widely exceeding generally accepted species boundaries. These differences were corroborated in virulence tested in experimental infection. The two isolates did not kill a mouse even until 28 days. However, mortalities of four O. gallopava strains ranged from 40 to 100%. The new isolates mainly affected the kidneys; whereas O. gallopava had a strong preference for the brain. We therefore propose a new species, Ochroconis calidifluminalis, for the two isolates.

Septic shock induced by Lecythophora mutabilis in a patient with mitochondrial encephalomyopathy.

Invasive fungal infection (IFI) caused by Lecythophora mutabilis occasionally occurs in patients with impaired host immunity; such patients had eosinophilia at onset, and surviving patients were treated with fungal cell-membrane-targeted drugs. An 18-year-old man with mitochondrial encephalomyopathy accompanied with refractory anaemia and chronic renal failure developed septic shock caused by L. mutabilis, which was detected from a blood culture, and was identified morphologically and genetically. During the course of the infection, he had eosinophilia, although beta-d-glucan levels were within the normal range. He was treated with micafungin, but deteriorated and died, despite his treatment being changed to liposomal amphotericin B. On the basis of this we suggest that IFI caused by L. mutabilis should be suspected when a compromised host develops infection and eosinophilia, and that antifungal drugs that target beta-d-glucan are not advisable.

Identification of Trichosporon spp. strains by sequencing D1/D2 region and sub-typing by sequencing ribosomal intergenic spacer region of ribosomal DNA.

To re-identify and further group 25 isolates of Trichosporon spp. identified morphologically previously, sequences of D1/D2 region of large subunit (LSU) of ribosomal DNA (rDNA) of 25 tested strains for identification and those of ribosomal intergenic space 1 (IGS1) region of 11 strains for subgrouping were detected. The identifications of tested strains were changed except 6 strains. According to the alignment of the IGS1 region, 6 T. asahii isolates tested fell into 4 groups and 5 T. faecale isolates into 3 groups. Polymorphism of 2 T. japonicum isolates was found in 10 positions. With the alignments obtained in this research compared with the relative GenBank entries, it was found that T. asahii, T. faecale and T. japonicum species were divided into 7, 3 and 2 subtypes respectively. Morphological and biophysical methods are not sufficient for Trichosporon spp. identification. Sequencing becomes necessary for Trichosporon diagnosis. There is obvious diversity within a species.

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.

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.

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.