Limitations of chlorine disinfection of human excreta: implications for Ebola disease control

Various NGO guidelines suggest that human excreta may be disinfected by the application of concentrated (e.g., 0.5%) chlorine solutions. However, chlorine-based disinfectants are thought to rapidly lose their bactericidal and virucidal properties in contact with high levels of organic matter and chlorine application results in the production of toxic chlororganic compounds. To evaluate the disinfection efficacy of chlorine solutions (HTH, NaDCC and household bleach) against viruses and bacteria within excreta matrices, laboratory-scale disinfection experiments were undertaken. Human excreta matrices containing raw wastewater, with 0%, 10% and 20% (w/v) added faecal sludge, were disinfected with chlorine solutions at a ratio of 1:10 (chlorine solution: excreta matrix). Contact time was set at 30 minutes and bacterial (FC and IE) and viral (SOMPH) indicators were used to measure disinfection efficacy. Results demonstrated that at high levels of solids content, disinfection efficacy was significantly reduced. These results support the need to find a more effective means of disinfecting human excreta in future Ebola outbreaks

Box-plots displaying overall log reduction levels for viral indicators (C, D & E) following the addition of chlorine and hydrated lime to excreta matrices (pooled data n = 36; 2 contact times (Ct) x 2 mixing methods x 3 excreta matrices x 3 repetitions).

<p>* = Indicates a significant statistical difference in log reduction.</p

Flow chart of the human excreta containment experiments.

<p>Flow chart of the human excreta containment experiments.</p

Assessment of recommended approaches for containment and safe handling of human excreta in emergency settings

<div><p>Ebola and cholera treatment centres (ETC and CTC) generate considerable quantities of excreta that can further the transmission of disease amongst patients and health workers. Therefore, approaches for the safe handling, containment and removal of excreta within such settings are needed to minimise the likelihood of onward disease transmission. This study compared the performance and suitability of three chlorine-based approaches (0.5% HTH, NaDCC and NaOCl (domestic bleach)) and three lime-based approaches (10%, 20% and 30% Ca(OH)₂). The experiments followed recent recommendations for Ebola Treatment Centres. Three excreta matrices containing either raw municipal wastewater, or raw municipal wastewater plus 10% or 20% (w/v) added faecal sludge, were treated in 14 litre buckets at a ratio of 1:10 (chlorine solutions or lime suspensions: excreta matrix). The effects of mixing versus non-mixing and increasing contact time (10 and 30 mins) were also investigated. Bacterial (faecal coliforms (FC) and intestinal enterococci (IE)) and viral (somatic coliphages (SOMPH), F⁺specific phages (F+PH) and <i>Bacteroides fragilis</i> phages (GB-124PH)) indicators were used to determine the efficacy of each approach. Lime-based approaches provided greater treatment efficacy than chlorine-based approaches, with lime (30% w/v) demonstrating the greatest efficacy (log reductions values, FC = 4.75, IE = 4.16, SOMPH = 2.85, F+PH = 5.13 and GB124PH = 5.41). There was no statistical difference in efficacy between any of the chlorine-based approaches, and the highest log reduction values were: FC = 2.90, IE = 2.36, SOMPH = 3.01, F+PH = 2.36 and GB124PH = 0.74. No statistical difference was observed with respect to contact time for any of the approaches, and no statistical differences were observed with respect to mixing for the chlorine-based approaches. However, statistically significant increases in the efficacy of some lime-based approaches were observed following mixing. These findings provide evidence and practical advice to inform safe handling and containment of excreta and ensure more effective health protection in future emergency settings.</p></div

Residual free chlorine (mg L^-1) following the addition of chlorine solutions to excreta matrices.

<p>Residual free chlorine (mg L^-1) following the addition of chlorine solutions to excreta matrices.</p

Comparison of raw and treated wastewater quality from full-scale treatment (batch volumes ranged from 10 to 15 m³).

<p>*^<b>1</b> Performed when adequate monitoring equipment had become available in the field.</p><p>*^<b>2</b> Calculated with reference to an average value for untreated wastewater in the absence of quantitatively sufficient data.</p><p>Comparison of raw and treated wastewater quality from full-scale treatment (batch volumes ranged from 10 to 15 m³).</p

Schematic overview of the high pH (Protocol A) treatment process.

<p>Schematic overview of the high pH (Protocol A) treatment process.</p

Comparison of consumption rates of chemical reagents, residual aluminum and volume of sludge produced from full-scale treatment.

<p>Comparison of consumption rates of chemical reagents, residual aluminum and volume of sludge produced from full-scale treatment.</p

Incidence of sequences homologous to ΦB124-14 and ΦB40-8 human gut metagenomes.

<p>Percentage of individual metagenomes in which sequences homologous to φB124-14 or φB40-8 were identified (≥80% identity over ≥100 nucleotides, 1e⁻⁵ or lower). The microbial metagenomes examined were derived from individuals of European (MetaHit) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Qin1" target="_blank">[28]</a>, Japanese <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Kurokawa1" target="_blank">[8]</a> and American <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Gill1" target="_blank">[60]</a> origin, alongside the combined viromes from 12 individuals of American descent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053-Reyes1" target="_blank">[6]</a>. <b>MH MetaHit</b>– All individuals represented in the MetaHit dataset; <b>Jap</b> – All individuals of Japanese origin; <b>AM</b> – All individuals of American descent; <b>Virome</b> – All viromes from individuals of American origin. <b>B.</b> Scatter plots illustrating the relationship between size of individual metagenomes searched and detection of sequences homologous to φB124-14. r² =  Pearson correlation co-efficient. **<i>P</i><0.0001.</p

Comparative genomic analysis of ΦB124-14 and ΦB40-8 (ATCC 51477-B1).

<p><b>A.</b> Nucleotide sequences of φB124-14 and φB40-8 were compared using the Artemis Comparison Tool (ACT). Shaded areas between linear phage genome maps represent areas of high nucleotide identity (90% or greater). Colour scale represents level of nucleotide identity at each region of homology. The ORF map for φB40-8 corresponds to the annotations available in the GenBank submission (FJ008913.1). For the purposes of this analysis, the φB124-14 genome was linearised between ORFs 29 and 30 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone-0035053-g002" target="_blank">Figure 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone.0035053.s004" target="_blank">Table S2</a>), in order to compare the circular φB124-14 genome with that of φB40-8. Colours of ORFs correspond to functional assignments as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035053#pone-0035053-g002" target="_blank">Figure 2</a>. <b>B.</b> Comparison of amino acid sequences from φB124-14 ORFs with those annotated in the φB40-8 genome. Shading between arrows indicates those sharing high amino acid sequence identity. Colour scale indicates level of amino acid identity between each homologous ORF.</p