Study design, period and area
This was laboratory based (in-vitro) study where bacteria S. aureus and E. coli were employed in testing the filtration efficiency of the selected cotton cloth mask made locally (Dar es Salaam, Tanzania) and surgical masks during Covid-19 pandemic. Dar es Salaam, is the biggest business city in Tanzania with approximately 6 million people. According to the current Covid-19 status in Tanzania, Dar es Salaam region is leading in terms of morbidity and fatality [11]. In this regard, Dar es Salaam Regional Commissioner has declared mass masking [7]. Tanzania, a low income country, hence the majority of its citizens cannot afford buying medical facemasks available in the Dar es Salaam market, therefore most rely on locally made cloth masks which are cheaper and re-usable [15]. Hence, a need to carry out this study.
Study subject
Facemasks were purposively selected from the Dar es Salaam market to include different cloth masks designs made up of 100% cotton and medical facemasks as described below [13]; Sample 1 (S1); Was made from thick wax cotton fabric, commonly known as “kitenge”; Swahili local name”. It consisted of two layers of the kitenge fabric; an inner and outer surface of the mask. It had no middle filter layer. S2; composition for sample 1 plus middle layer/filter made of fabric lining commonly known as canvas material. S3; composition for sample 1 plus laminate breathable middle layer or filter made of non-woven fabric lining commonly known as stiff material (Kiarasheba tailoring mart in Dar-es-Salaam, Tanzania). S4; N95 respirator (Kimberly Clark Corp, USA). S5 Surgical mask (BBL, China) S6: Locally made surgical mask (PMCL, Tanzania). S7: Was made from thick wax cotton fabric, commonly known as “kitenge”; It consisted of two layers of the kitenge fabric; an inner and outer surface of the mask. The kitenge was pleated both sides but had no middle filter layer.
Experimental design
Bacterial filtration efficiency, in vitro is widely accepted method for evaluating face masks [13,16], in this case bacteria penetrating the face masks are collected, cultured and counted to determine colony forming units. In this experiment, a 100 ml spray plastic bottle (Aroma, India) was used for spraying bacterial suspension to determine facemask filtration efficiency. A single spray by 100ml plastic spray bottle was equivalent to 250 μl with an approximated flow rate of 31.5 ft3/min (A. Singl, personal communication, April 28, 2020). The flow rate used in this study was greater than the normal range of human respiration and higher than the flow rate of 1 ft3/min as recommended American Society for Tropical Medicine [16]. A single spray of bacteria suspension was performed to every test facemask to represent an average number of times a normal person can sneeze within 4 hours [17], a time recommended to change a facemask. During spraying the facemask and spray bottle were kept in contact. This was done to simulate the a person wearing a facemask to prevent or slow the spread of aerosol.
Laboratory procedures
The mask was prepared and labelled according to mask descriptions above (S1-7). Test bacteria were selected to represent gram positive and negative, spherical and rod- shaped, and of sizes smaller than SARS-CoV-2-respiratory droplet; S. aureus ATCC 25923 and E. coli; ATCC 25922 (American Type Culture Collection Inc., Rockville, Md), respectively. Briefly, each bacteria suspension which consisted of normal saline (NaCl; 0.9%w/v [Aculife Health Care, India]) and the test bacteria were prepared by keeping the turbidity comparable to 0.5 MacFarland [13] using both visual comparison to turbidity standards and a Densichek photometer (bioMérieux). A 0.5 MacFarland standard inoculum was prepared from 48 hours cultures using cystine–lactose– electrolyte-deficient agar (CLED) (Oxoid LTD, England) incubated at 37°C in ambient air. Since cloth masks were locally manufactured sterilization using an hot air oven at 170 °C for 1 hour was performed to avoid possible contamination from the manufacturer. While in contact, each test facemask (S1-7) was sprayed once using 100 ml plastic spray bottle. Then using regular cotton swabs (Medico, China), dipped in normal saline, swabbing was performed to unsprayed side of the mask immediately (at 0 hours) and after 4hrs. The swab was streaked by spreading it over the surface of an agar plate containing CLED followed by 48 hours incubation at 37oC in ambient air [13].
Quality control
A duplicate bacteria culture were performed to represent each design of the facemask (S1-7) for every type of bacteria strain (S. aureus/ E. coli). In vitro tests uses positive and negative controls to determine the initial number of bacteria. The control was prepared by inoculating 250 μl of 0.5 MacFarland S. aureus ATCC 25923 and E. coli; ATCC 25922 (volume of single spray by 100 ml plastic spray bottle); this was done in assumption that no innervation was applied. To enable enumeration of colonies a serial dilution to 106 by diluting was used as an inoculum by inoculating 1 μl of each bacteria suspension to CLED media followed by 48 hours incubation at 37°C in ambient air.
Interpretations
The piles of bacteria cells observed after an incubation period were termed as a colony forming units (CFUs). For each plate, colonies were counted using the Protocol Bacteria Colony Version 2.05 to obtain the CFUs counts from 0.5 MacFarland E. coli and S. aureus suspensions for the test (T) and control (C) per single 100 ml plastic spray bottle. The results were expressed as CFUs per 250 μl of 0.5 MacFarland E. coli or S. aureus. Growth on culture were determined by comparing the colony characteristics, i.e., colony color and texture, any growth out of the streaked line was regarded as contamination. Absence of growth were determined by comparing with a negative control (un-inoculated plate). Two independent readers observed the culture plates to determine the colony growth and number of colonies. Discrepancies were resolved by consulting an experienced microbiologist. Then, bacteria filtration efficiency was mathematically calculated by BFE = 100% (C-T)/C [14].
Data analysis
Data from laboratory sheet data collection was entered in Microsoft excel sheets (Microsoft Corporation, Redmond, WA). BFE was expressed as a percentage of differences in CFUs between the control and the test face mask divided by CFUs of the control.
Ethical consideration
Ethical approval was sought from the Institutional Review Board of the Muhimbili University of Health and Allied Sciences (MUHAS). This was an in-vitro study where no human subject was investigated, the study used standard organisms, S. aureus; ATCC 25923 and E. coli; ATCC 25922). Additionally, all methods were carried out in accordance with relevant guidelines, regulations and good laboratory practice of MUHAS.