Cleaning Procedures at INMI for EBOV Patient Rooms

According to internal protocols, daily cleaning of occupied patient rooms and management of spills of body fluids were performed using wipes and mops saturated with 0.5% sodium hypochlorite; the adequacy of daily cleaning was not assessed. After patient discharge, careful terminal cleaning of all room surfaces, reusable equipment, and everything downward to the floor was performed using manual scrubbing with wipes and mops saturated with 0.5% sodium hypochlorite, as suggested by international guidelines. ⁷

Summary of Clinical Courses

On November 25, 2014, the first EBOV patient was hospitalized in the high isolation unit (HIU) at INMI.Reference Petrosillo, Nicastri, Lanini, Capobianchi, Di Caro and Antonini ⁸ On December 4, his condition worsened and he was transferred to the intensive care high isolation unit (IC-HIU), where he remained for 7 days. On December 11, he returned to the HIU, and he was discharged on January 2, 2015. Profuse vomiting and diarrhea dominated the clinical course; EBOV was detectable in this patient’s blood until day 25 and in his urine until day 35 from admission.

The second patient was admitted on May 13, 2015, in the HIU. In the meanwhile, no patients were admitted to this room. The patient was discharged on June 10; intensive care was not required. EBOV was detectable in this patient’s blood until day 9 and urine until day 21.

Sample Collection and Timing

Sampling activities were performed wearing the same personal protective equipment used for medical care. Environmental samples were collected by slowly swiping the nylon-flocked swabs (Copan Italia, Italy) on the surfaces horizontally, vertically, and diagonally ≥10 times, then the swabs were re-suspended in polypropylene tubes containing 0.5 mL phosphate-buffered saline. The samples were immediately transported to the BSL-4 laboratory, which is located in the same compound. The sites and timing of the sampling are summarized in Table 1.

TABLE 1

Results of Environmental Samples Collected from Patient Rooms at the Lazzaro Spallanzani National Institute for Infectious Diseases

NOTE. IC-HIU, intensive-care, high-isolation unit; HIU, high-isolation unit; PCR, polymerase chain reaction; i.v., intravenous; ND, not done.

^a Different sites of floor and frequently touched surfaces, 1 sample for each site.

^b Patient with negative viremia for EBOV but PCR-positive in other body fluids (ie, urine, sputum and material from oropharyngeal and rectal swabs).

^c All floors were made of polyvinyl chloride.

^d In both cases, the culture of viable virus on VERO E6 cells resulted negative. According to the clinical records, areas where PCR results were positive were intensively contaminated by vomit during the patient stay.

^e Sampled on the day of discharge, without any previous cleaning.

Laboratory Testing

After inactivation in BSL 4, the samples were extracted and tested for the presence of EBOV genomes using a commercial quantitative real-time RT-PCR specific for filoviruses, targeting the L gene (RealStar Filovirus Screen RT-PCR Kit 1.0, Altona Diagnostics, San Francisco, CA). This test was approved by the World Health Organization for United Nations procurement. ⁹ PCR-positive samples were tested for the presence of infectious viral particles. Confluent monolayers of Vero E6 cells in T-25 flasks were inoculated with 0.1 mL of undiluted sample in 1 mL of minimum essential medium (MEM). After absorption at 37°C for 1 h, excess inoculum was removed, and complete medium with 2% fetal calf serum (FCS) was added to the flasks. Cells were observed each day for signs of cytopathic effect (CPE). If no CPE appeared, sequential passages were performed every 5 days by detaching and co-culturing with fresh cells, which were observed for another 5 days. At 20 days after inoculation, the cells were removed from the flask by scraping and both supernatants and cells were tested for the presence of EBOV-RNA.