RESUMEN
Purpose: To analyze the presence of severe acute respiratory syndrome coronavirus 2 (SARS?CoV?2) in tears/conjunctival epithelium and assess the cytomorphological changes in the conjunctival epithelium of coronavirus disease 2019 (COVID?19) patients. Methods: In this pilot study, patients with moderate to severe COVID?19 were recruited from the COVID ward/intensive care unit of the institute. Tears and conjunctival swabs were collected from COVID?19 patients and sent to the virology laboratory for reverse transcription polymerase chain reaction (RT?PCR) testing. Conjunctival swabs were used to prepare smears, which underwent cytological evaluation and immunocytochemistry for SARS?CoV?2 nucleocapsid protein. Results: Forty?two patients were included. The mean age of participants was 48.61 (range: 5–75) years. Seven (16.6%) patients tested positive for SARS?CoV?2 ribonucleic acid in tears samples, four (9.5%) of which were positive on conjunctival swab by RT?PCR in the first test. Cytomorphological changes were observed significantly more in smears from patients with positive RT?PCR on tear samples, including bi?/multi?nucleation (p = 0.01), chromatin clearing (p = 0.02), and intra?nuclear inclusions (p < 0.001). One case (3.2%) showed immunopositivity for SARS?CoV?2; this patient had severe disease and the lowest Ct values for tear and conjunctival samples among all positive cases. Conclusion: Conjunctival smears from patients with COVID?19 revealed cytomorphological alterations, even in the absence of clinically significant ocular infection. However, viral proteins were demonstrated within epithelial cells only rarely, suggesting that although the conjunctival epithelium may serve as a portal for entry, viral replication is possibly rare or short?lived.
RESUMEN
Three insect cell lines, namely, DZNU-Bm-1, DZNU-Bm-12, and NIAS-MaBr-92, were tested for susceptibilityto Bombyx mori nucleopolyhedrovirus (BmNPV). The cytopathic effects of infection on cultured cells wereobserved. BmNPV was then serially passaged in these cell lines to assess their long-term ability to supportBmNPV replication. The cell lines were cultured in MGM-448 medium supplemented with 10% fetal bovineserum. Virus inoculum was prepared from B. mori larvae infected with BmNPV. The early signs of infectionstarted appearing within 16–36 hours post inoculation (hr pi). DZNU-Bm-1 and DZNU-Bm-12 cells showedheavy clumping. NIAS-MaBr-92 cells lost their motility and tended to form loose aggregations. Cytopathiceffects such as hypertrophy of nuclei and increase in cell size were prominent in all three cell lines. Beginningof occlusion body (OB) formation in DZNU-Bm-1 was first observed at about 36–42 hr pi, whereas, inDZNU-Bm-12, they were observed only after 42 hr pi. NIAS-MaBr-92 cells exhibited OB formation onlyafter 84 hr pi. The large mature OBs were visible in DZNU-Bm-1 cells by 66 hr pi, whereas, in DZNU-Bm-12cells by 72 hr pi. Mature OBs were observed in NIAS-MaBr-92 cells only after 120 hr pi. A number of OBsper cell varied from 12 to 82 in DZNU-Bm-1, 3 to 23 in DZNU-Bm-12, and 3 to 14 in NIAS-MaBr-92 cells.In this study, DZNU-Bm-1 and DZNU-Bm-12 cell lines were found to be better than NIAS-MaBr-92 interms of percentage infection. The yield of OBs per infected cell is also the highest in DZNU-Bm-1, whereasDZNU-Bm-12 and NIAS-MaBr-92 have comparable yields of OBs per cell.
RESUMEN
The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.