Jinja Regional Referral Hospital (RRH) is located in southeastern Uganda, approximately 87 kilometers east of Kampala, Uganda. It is the largest hospital in eastern Uganda, with a bed capacity of 600 and serves as referral for Bugiri, Iganga, Jinja, Kaliro, Kamuli, Mayuge, Kayunga and parts of Mukono district. It has an infection prevention committee and locally made hand sanitizer in addition to commercial hand sanitizer for use by its staff. The hospital had a star 4 Laboratory that also served as a referral for 23 sites. The Laboratory was preparing for accreditation.

This was an in in vitro laboratory-based experimental study. A suspension test for the demonstration of bactericidal activity of disinfectants in vitro (prEN12054) is used in Europe and EN 1500 for in vivo protocols [9] .

These were; Locally Made ABHS and Saraya Hand sanitizer, Purposive sampling method was applied. We tested two brands of hand sanitizers that were available at the hospital during the time of the study which were compared to a reference standard (60% IPA).

The standard organisms that represent the commonly isolated pathogens causing nosocomial infections in Uganda were used in the experiment. These included; Klebsiella pneumoniae (ATCC 13883), Escherichia coli (ATCC 25922), and S. aureus (ATCC 25923). The culture media used in the study were blood agar and Muller Hinton Agar; they will be prepared using SOPS and the Microbiology laboratory at JRRH. Verified timers, autoclaves, water baths, biosafety cabinets, weighing scales and pH meters of the study site laboratory were used.

Data collection forms; Percentage bacterial growth reduction and Time-kill log.

Two brands of alcohol-based hand sanitizers were found at Jinja Hospital, Saraya and locally made Alcohol based sanitizer.

60% Isopropyl alcohol and neutralizer that contained Polysorbate80 were purchased from Joint medical stores, Kampala.

All instruments used in the study were validated by competent qualified service providers.

There was double entry of data into Microsoft excel software.

American Type Culture Collection (ATCC) Standard organisms were used and no expired products were used in the study.

Positive and negative control samples were included in the assays.

The obtained raw data were double entered into Microsoft Excel, Version 5.0 (Microsoft Redmond, WA, USA) and then graphs plotted. Data were exported to graph pad prism software to plot a time to kill curve.

Log and Percent reduction for the standard microorganisms were calculated and presented using tables.

The study protocol was approved by Mbarara University of Science and Technology, Department of Medical Laboratory Science (Reference: MUST/MLS/031) and Jinja Regional Referral Hospital Research Ethics Committee (REC) granted Permission/ethical waiver where the study was conducted.

The study was limited to bacteria; no viruses and yeasts were handled because of capacity to isolate viruses and a method EN1275 is used for testing efficacy against yeasts and viruses.

The study was in vitro and did not closely stimulate real world conditions of Hand Sanitizer use.

Results from this study were shared with Jinja Regional Referral Hospital (JRRH) Administration and Infection Control and Prevention Committee (IPC) and also compiled into a dissertation, a copy of which was submitted to the Library in the Department of Medical Laboratory Science of Mbarara University of Science and Technology Abstracts of this work have been sent and accepted at Colorado WASH symposium and ICREID conference.

The JRRH locally made ABHS was effective against all the test organisms. The antibacterial effectiveness was assessed by counting Colony Forming Units (CFU) after treatment with the hand sanitizer for 15 seconds contact time. Logarithmic reduction factors and percentage growth reductions calculated as shown

in Table 2. The highest reduction factors were recorded against E. coli and K. pneumoniae at 7.18 and S. aureus at 7.16 whose baseline counts were 1.5 × 10⁷, 1.5 × 10⁷ and 1.45 × 10⁷ CFU respectively (Table 2).

Saraya hand sanitizer was effective against all the test organisms. The antibacterial effectiveness was assessed by counting colony forming units (CFU) after treatment with the hand sanitizer for 15 seconds contact time. Logarithmic reduction factors and percentage growth reductions calculated as shown in Table 3. The highest reduction factors were recorded against E. coli and K. pneumoniae at 7.18 and S. aureus at 7.16 whose baseline counts were 1.5 × 10⁷, 1.5 × 10⁷ and 1.45 × 10⁷ CFU respectively and post counts after treatment with sanitizer were 1 CFU across all organisms with percentage growth reduction of 99.99999%.

The controls hand sanitizer (60% IPA) was satisfactory for suspension test for all the test organisms used in the studyin agreement with prEN 12054 method (Table 4).

Both Locally made Alcohol Hand sanitizer and Saraya had a bacterial percentage reduction of 99.99999% at 15 and 30 seconds contact time (Table 5 and Table 6) against E. coli, K. pneumonia and S. aureus whose baseline counts were 1.5 × 10⁷, 1.5 × 10⁷ and 1.45 × 10⁷ CFU respectively whose post counts after treatment with sanitizers were 1 CFU across all organisms.

The highest reduction factors were recorded against E. coli and K. pneumoniae at 7.18 each and S. aureus at 7.16 whose baseline counts were 1.5 × 10⁷, 1.5 × 10⁷ and 1.45 × 10⁷ colony forming units respectively (Figure 1).

Both sanitizers reduced the baseline counts from 1.5 × 10⁷ CFU to 1 CFU within 15 seconds of contact time, post exposure counts remained constant (1 CFU) at 30 and 45 seconds contact time (Figure 2).

The neutralizer used in the study was found not to be toxic to the microorganisms used; more than 80% of the organisms were recovered after being treated with the neutralizer for 10 seconds contact time (Table 7).

Both Saraya and JRRH Locally made ABHS mentioned ethanol as their active ingredient. Efficacy was independent of the ethanol concentration in each brand (Saraya 70% and locally made 80% alcohol). In this study alcohol concentrations were not adjusted, sanitizers were used in their original form from the manufacturer/producers. Results of this study are similar to Sax et al., (2009) where Alcohol based products typically contained between 60% and 95% alcohol, in form of ethanol or Isopropanol. At those concentrations, alcohol immediately denatured proteins effectively neutralizing certain types of organisms [14] .

In agreement with prEN 12054, locally made ABHS and Saraya were found to exceed a 10⁵-fold reduction of all the three ATCC strains (Klebsiella pneumoniae (ATCC 13883), Escherichia coli (ATCC 25922), and S. aureus (ATCC 25923)

within 15 s (Table 1 and Table 2). This study is in compliance with WHO (2009) that describes an efficacious locally produced hand sanitizer that reduced microorganism by a 10⁵-fold reduction and above [9] .

According to the label on Saraya hand sanitizer that shows the hand sanitizer can kill 99.999% of bacteria, bacterial percentage reductions were calculated and this study is in agreement with the label however there was no data on efficacy of the locally produced hand sanitizer nevertheless this study found out that the sanitizer can reduce bacteria by 99.99999% (Table 4 and Table 5).

The two hand sanitizers have a broad spectrum antimicrobial activity. A study by Vardhaman showed that alcohol based hand sanitizers were effective against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis [15] .

The Positive control (60% Isopropyl alcohol) had a percentage growth reduction of 99.999%, while the negative controls (Neutralizer) had no effect on the growth reduction hence these were satisfactory for suspension test for all the test organisms used in the study.

This study showed that the use of a neutralizer that contained Polysorbate 80 (Tween 80) was non-toxic on both gram negative and gram positive bacteria since more than 80% of the bacteria were recovered after being treated with the neutralizer for 10 seconds contact time. Acceptable neutralizer efficacy and neutralizer toxicity ratios are defined as ≥0.75 (75%) recovery [13] .

Adams et al. (2005) reported that mixture of 0.2% Tween 80, 1.17% Lecithin, 0.1% triton in distilled water is an effective neutralizer for Alcohol hand gel with whom we are in agreement according to our results. Another study by Norhan Sheraba et al. (2012) shows that Tween 80 in 1% physiological peptone was the most effective neutralizer contrary, Mohamed (2004) found Letheen broth to be an effective neutralizer [16] .

The hand sanitizers used had 100% efficacy that meets World Health Organization (WHO) standards that require 5 log₁₀ CFU/mL Reduction of bacteria within 30 seconds of contact time. From this study we recommend the use of the JRRH locally made hand sanitizer and Saraya. Hand rubbing time can be reduced from 30 s to 15 s.

Local production of ABHS should be adopted in tertiary facilities as the preferred means of providing for ABHS for their staff at the “point of care” (POC). This is recommended by WHO and satisfies the urgent need to make available hand sanitizers in healthcare facilities at the POC to help in reducing transmittable infections [1] .