In vitro assessment and comparison of quality of alcohol-based hand rubs, pre- and peri-COVID-19 pandemic outbreak in Kenya

Sample collection

A total of 90 ABHRs samples were collected for the study period through convenience sampling method, as the total number of ABHRs brands in the Kenyan market was not known. The Kenya Medical Research Institute Innovation & Technology Division (KEMRI - ITTD) had collected ABHRs from shelves and archived them as part of its research and development activities before March 2020. A total of 55 archived sanitizers within the expiration period at the time of laboratory testing were selected for analysis. In addition, 35 different sanitizer brands from the local retail shelves manufactured during the pandemic period, i.e. after March 2020 to June 2021, were collected as peri-COVID-19 samples (Figure 1). During the peri-COVID-19 pandemic sanitizer sample collection highest priority was given to the brands found to have been already collected and archived during the pre-COVID-19 pandemic period. They were cross-checked for regulatory compliance using the Kenya Bureau of Standards (KEBS) USSD verification database.¹² All hand sanitizers collected from the market were tested. The products were visually inspected for labelling compliance on declaration of active ingredient and against the parameters specified in the Kenya Standard East Africa Standard (KS EAS) 789:2013 standard. In addition, control samples were prepared in the ITTD Laboratory using protocol as described by WHO for quality assurance so as to ensure validity and reliability of results obtained during efficacy testing.³

Figure 1. Samples of brands of alcohol-based hand rubs used in this study.

Alcohol content assay and pH of alcohol-based hand rubs

The assay profiled and quantified three analytes: ethanol, isopropyl alcohol and methanol using Shimadzu GC-2010 plus - Gas Chromatography with Flame Ionization Detector (GC-FID) following the protocol as described by Zhang.¹³ This was done as follows: the working solution (WS) was prepared by diluting glycerin in distilled water to a concentration of 4.6% (v/v). The alcohol calibration standards were prepared by adding aliquots of pure alcohols - ethanol, Isopropyl Alcoho (IPA), methanol - to distilled water. The internal standard (IS) acetonitrile (ACN) was added to the calibration solutions at a concentration of 5% (v/v). The quality control sample was prepared by diluting 25 μL of ethanol, 25 μL of IPA, and 50 μL of acetonitrile to 1 mL in distilled water. The gel ABHR samples were diluted before injection due to viscosity. These samples were analyzed in the GC-FID with the following analytical parameters:

Determination of pH of ABHRs samples was measured using Thermo Scientific Orion Star A214 pH and ISE Benchtop Meter with limits as specified by KS EAS 789:2013 standard.

Alcohol-based hand rubs efficacy testing

Efficacy testing was carried out using quality control strains of E. coli American Type Culture Collection (ATCC) 25922 and S. aureus ATCC 25923 as described in the European Standard (EN) 1500:2013¹⁴ and KS EAS 789:2013. Briefly, 0.5 Mac Farland suspensions of the microorganisms were separately prepared as per the method described by the Clinical and Laboratory Standards Institute.¹⁵ As a quality control measure, so as to ensure validity and reliability of results obtained during efficacy testing, a control sample of 80% ethyl alcohol was prepared from absolute ethanol by mixing 800 mL of pure ethyl alcohol and 200 mL of distilled water as guided by WHO³; this was further tested using an alcoholmeter to measure the alcohol percentage in the control sample. This was tested together with the ABHR samples. Means of the colony forming units were used in determining log reduction values.

Determination of logarithmic reduction

Logarithmic reduction factors (RF) were assessed based on pretreatment and post-treatment with the ABHRs and the results of each ABHRs manufactured during the pandemic period compared to those collected before the pandemic. The logarithmic reduction factors were expressed as a percent reduction. Log reduction was calculated as log10 (pretreatment A) - log10 (post treatment B) and the percent reduction was calculated as (A-B)/A% where; where A = number of viable microorganism at before treatment and B = number of viable microorganism after treatment.¹⁴

Declaration of active ingredients

In the case of active ingredients, 41.08% of pre-COVID-19 sanitizers listed the active ingredient used; of these, 16.08% specifying the percentage composition of active ingredient which included ethanol, isopropyl alcohol (IPA) and 25% did not indicate the exact percentage composition. A total of 58.9 % did not list the active ingredient. For sanitizer samples collected in the peri-COVID-19 pandemic period, the majority of the brands (94.4%) listed the active ingredients used; of these, 66.7% listed >60%, alcohol as an active ingredient and 27.7% did not declare their exact percent alcohol content; sanitizers that did not declare the active ingredient composition accounted for 5.56% (Figure 2).

Figure 2. Comparison of standards in labelling ingredients for pre-COVID-19 and peri-COVID-19 periods.

Shelf lives of the ABHRs

The shelf lives allocated to the sanitizers were highly varied and ranged from one to five years. For pre-COVID-19 alcohol-based hand rubs, 16% did not state the manufacture and/or expiry dates, as compared to only 5.56% (n = 2) for the peri-COVID-19 pandemic period. The majority of the hand sanitizers manufactured during either of the periods under review had shelf lives of between two-three years, i.e. 44.6 % for pre-COVID and 41.7% for peri-COVID-19 pandemic ABHRs. Shelf life was one-two years (10.7%), three-four years (3.6%), four-five years (1.79%), more than five years (5.36%) for pre-pandemic ABHRS and one-two years (36.1%), three-four years (11.1%), four-five years (2.8%), and more than five years (2.8%) for peri-pandemic ABHRs (Figure 3). The manufacture period for the pre-COVID samples ranged from September 2017 to March 2020 and the study sorting period for pre-COVID-19 sanitizer was capped to March 2020. The manufacturer period for peri-COVID-19 ABHRs samples ranged from March 2020 to April 2021.

Figure 3. Percentage distribution of shelf life for sanitizers before and during the COVID-19 pandemic.

Efficacy performance of ABHRs

Of the total pre-COVID-19 sanitizers tested, 12.5% (n = 7) had a performance of less than 1 log reduction, as compared to 27.8% (n = 10) peri-COVID-19 ABHRs which had a performance of less than 1 log reduction. Overall, 78.6% had between 1-6 log reduction with two samples including quality control sample displaying total microbial reduction, while most of the ABHRs manufactured during the pandemic (69.4%) showed between 1-3 log reduction with no sample manufactured during the pandemic period showing an efficacy performance above 3 log reduction (Table 1). These results display a reduction in quality performance of the ABHRs manufactured during the pandemic period in terms of efficacy.

Based on the distribution analysis for the general performance of the ABHRs, pre- and peri-COVID-19 pandemic, there was an observed clustering of the performance between <1 to 3 with an unexpected gap between 3-5 logarithmic units for peri-COVID-19 pandemic ABHRs (Figure 4). This is a strong positive correlation of log reduction between pre-COVID and peri-COVID ABHRs samples (Pearson correlation co-efficient value of R was 0.9328. The p-value was < 0.00001 implying that the result was significant at p < 0.05. This is attributed to the increased pre-COVID log reduction of the values above 3.

Figure 4. Distribution of sample comparison according to their log reduction.

Alcohol content assay and pH of ABHRs

A total of 20 peri-COVID samples were analyzed using gas chromatography with flame ionization detector (FID). The assay profiled and quantified three analytes: ethanol, isopropyl alcohol and methanol. pH was also measured as specified by KS EAS 789:2013 standard. Five samples (25%) complied and had > 60% alcohol content (Table 2). Sample PBHR 2, PBHR 9, PBHR 16 and PBHR 25 had been labelled by the manufacturer as containing IPA. However, upon analysis the first three contained ethanol as the active component while the fourth product contained a mixture of ethanol (30.41%) and methanol (31.12%).

Only five samples (25%) had an alcohol content of above 60% v/v which is the KEBS minimum limit for alcohol content in hand sanitizers. No sample met the required content of the World Health Organization (WHO) of 80% v/v of ethanol (Figure 5).

Figure 5. Alcohol content in ABHRs.

The Food and Drug Administration (FDA)-accepted limit of methanol is < 0.063,¹⁶ however, only one ABHR (PBHR 09) met the FDA requirement with a methanol content of 0.06%; meanwhile, methanol substitution was found in four samples (20%), indicative of impure ethanol being used in the production process (Figure 6). The variation of the content of methanol was high (7.29 ± 6.003) at a 95% confidence limit.

Figure 6. Methanol content in ABHRs.

One ABHR sample had a major deficiency that resulted in outlier pH results: PBHR 36 showed an acidic pH of 0.4. Most (55%, n = 12) samples had pH above the minimum limit of 6, with the range being pH 0.4–8.6 (Figure 7A). The study found no correlation between the pH of a product and the total content of alcohol in tested the samples (Coefficient correlation R was -0.1078 and p-value was 0.65) (Figure 7B).

Figure 7. (A) Percentage count of pH distribution among the products; (B) Correlation between alcohol composition and pH value.

Unexpectedly, results of the Pearson correlation analysis indicated that there was a non-significant relationship between total alcohol content and percentage log reduction, (r = - 0.167, p = 0.480) (Figure 8).

Figure 8. Correlation between ethanol content and percentage reduction.

Impact of the Kenya Bureau of Standard mark of product quality

The Kenya Bureau of Standards had a positive impact on the quality of the ABHRs. ABHRs with a KEBS standardization mark had a higher average ethanol content (57.2%) compared to average ethanol content for the ABHRs without certification (48.8%). They also had a better percentage reduction score, averaging 89% against 78.9% for uncertified ABHRs (Figure 9).

Figure 9. Ethanol content and % reduction comparison between KEBS certified and uncertified ABHRs.

Limitations of the study