Absence of transmission of NDM and OXA-48 carbapenemase genes in a chronic care unit of a long-term care facility

Infection prevention and control measures are used to contain outbreaks of carbapenemase-producing Enterobacteriaceae. We report the absence of transmission of Klebsiella pneumoniae carrying New Delhi metallo-β-lactamase and oxacillinase-48 genes among 19 screened contacts of an index case after 14 months of routine practices in a long-term care facility.


Background
The emergence of carbapenem resistance among Gram negative bacteria heralds a future of potentially untreatable infections due to extreme antimicrobial resistance (Paterson and Doi, 2007). The hydrolysis of carbapenems by carbapenemase-producing Enterobacteriaceae (CPE) is an emerging mechanism of resistance in hospital-acquired infections. There are limited antimicrobial treatments existing for patients infected with these pathogens (Tacconelli et al, 2018). Infections due to CPE are associated with greater mortality than those caused by carbapenem-susceptible bacteria (Falagas et al, 2014).
Canadian rates of CPE colonisation increased nearly five-fold from 2014 to 2017; however, an increase in the rate of CPE infection has not yet been documented in Canada (Public Health Agency of Canada, 2019). The implementation of institutional control measures is deemed essential to limit the spread of CPE locally, nationally and internationally (French et al, 2017;Public Health Agency of Canada, 2019).
Combined control measures are typically required to prevent and control outbreaks with CPE. These measures include routine practices and additional precautions such as contact precautions, hand hygiene, staff education, patient isolation, enhanced environmental cleaning and disinfection, and active surveillance (French et al, 2017). Considering that silent CPE transmission has occurred in the context of contact isolation, identifying asymptomatic Structured observation has been found to be the best indicator to assess handwashing practices in Indian households (Biran et al., 2008).

Outcome variable
The outcome variable considered for the analysis was 'the use of soap/detergent and water for handwashing'. It is defined as the presence of soap/detergent along with water in the usual place of handwashing among the households, where the place of handwashing was observed.

Predictor variables
The predictor variables used in the analysis were chosen based on the extensive literature review and available information in the NFHS-4. Specifically, the predictor variables used were the schooling of the household head (< 5 years including the illiterates, 5-9 years, 10-11 years, ⩾ 12 years), sex of the household head (male, female), religion of the household head (Hindu, Muslim, Christian and Others), caste/tribe of the household head (scheduled caste [SC], scheduled tribe [ST], other backward classes [OBC] or non-SC/ST/OBC), household size (< 5 members, ⩾ 5 members), house type (kuccha, semi-pucca, pucca), location of water source (in own dwelling, elsewhere), ownership of the house (not own house, own house), wealth index (poorest, poorer, middle, richer, richest), place of residence (urban, rural) and region (north, central, east, northeast, west, south).

Statistical analysis
In the present study, cross-tabulations between the outcome and predictor variables were done using the appropriate sample weights. The binary logistic regression was carried out to understand the predictors of handwashing practices. For this regression analysis, the dependent variable 'Soap/ detergent and water used for handwashing' was categorised into two, i.e. 1 = yes, 0 = no. The variables 'house type' and 'ownership of house' were dropped from the regression analysis to avoid multicollinearity. The Statistical Package for Social Sciences (SPSS-25, IBM Corp., Armonk, NY, USA) was used for analysis. The choropleth map was prepared at the district level using the ArcMap (version 10.4) to assess the regional scenario. The local indicators of spatial association (LISA) cluster map and Moran's I scatter plot were calculated through GeoDa (version 1.14) to understand the spatial clustering in the use of soap/detergent and water for handwashing.

Type of handwashing elements observed at the usual place of handwashing
Soap/detergent and water were observed in the usual place of handwashing in three-fifths (60%) of the households ( Figure 1). In 16% of the households, only water was observed in the usual place of handwashing. Seven out of every ten households were observed to have water and any cleansing element in their regular handwashing place. Nine percent of the households were found to have no water, no soap or any other cleansing agent at their usual place for handwashing. Table 1 presents the bivariate analyses to understand the individual association between the predictors and outcome variable. Of the male-headed households, 61% use soap and water for handwashing compared with 55% of the female-headed households. Use of soap and water for handwashing was found to increase with increasing education of  Journal of Infection Prevention 00 (0) patients colonised with CPE through active surveillance is essential to curtail outbreaks (French et al, 2017). Diligent use of additional precautions and appropriate equipment cleaning and disinfection for patients acting as asymptomatic reservoirs has led to successful cluster extinction (Calfee and Jenkins, 2008). Considering the presumed prolonged colonisation with CPE, most public health guidelines suggest that contact precautions be kept indefinitely. The longevity of colonisation is now being questioned (Tucker et al, 2019).

Handwashing through soap and water by background characteristics of the households
Here, we report the findings of a programme of active contact surveillance for a CPE positive index case who had been residing in the chronic care unit of a long-term care facility (LTCF) for 14 months following the isolation of CPE during a brief hospital admission. The patient's status as an individual infected or colonised with a CPE was not known to the chronic care unit. As a result, only routine practices were implemented during this time.

Methods
This study was performed at the chronic care unit of a LTCF within Winnipeg, Manitoba, Canada. Subjects were patients who were in contact with a CPE colonised individual prior to the initiation of specific infection prevention and control (IPC) measures. Subjects were screened for CPE colonisation via rectal swabs collected on days 0, 7 and 21 after exposure was identified. Swabs were cultured on CHROMagar mSuperCARBA agar (CHROMagar, Paris, France), and Gram negative, carbapenem-resistant organisms were identified to species level using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Billerica, MA, USA). Phenotypic confirmation of carbapenemase production by Enterobacteriaceae isolates was performed using the Neo-Rapid CARB kit (Rosco Diagnostica; Taastrup, Denmark), a chromogenic assay based on the hydrolysis of imipenem. The carbapenemase type was initially determined by a polymerase chain reaction (PCR) screen capable of detecting KPC, NDM, VIM, OXA-48, IMP and GES genes. Phenotypic susceptibility testing was performed on all CPE isolates using the Vitek 2 N208 or N391 cards (bioMérieux, St-Laurent, Québec, Canada) and disk diffusion and interpreted according to current Clinical and Laboratory Standards Institute (CLSI) and US Food and Drug Administration (FDA) breakpoints (for tigecycline). Whole genome sequencing of carbapenemase-producing Enterobacteriaceae was performed with the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sequencing data have been deposited in NCBI as BioProject PRJNA511988 and BioSamples SAMN13659125 (Klebsiella pneumoniae) and SAMN13659126 (Escherichia coli). Contigs were generated using the assembly and annotation pipeline of the rapid infectious disease analysis platform (IRIDA v19.09), which combines SPAdes-based de novo assembly with Prokka-based annotation. Carbapenemase-producing genes were characterised using ResFinder 3.2 (Center for Genomic Epidemiology, https://cge.cbs.dtu.dk/services/) and CARD (the comprehensive antibiotic resistance database, https:// card.mcmaster.ca/home).
Research ethics approval for retrospective chart review was approved as well as institutional approval at the LTCF.

Results
Our index patient was first found to have a CPE-producing isolate of K. pneumoniae from a urine sample obtained in May 2017 during an acute care hospitalisation. As per regional practice, this patient's CPE status was identified in the electronic patient record (EPR) to ensure that staff are aware of the need for additional infection control measures. This organism demonstrated phenotypic antimicrobial susceptibility only to tigecycline (Table 1). Carbapenemase activity was confirmed phenotypically and by PCR detection of both NDM and OXA-48 genes. During repeat acute hospital admissions in February 2018 and May 2019, two additional urine isolates from this same index case demonstrated the presence of K. pneumoniae containing both NDM and OXA-48 carbapenemases. Our patient was admitted in April 2018 from the community to a chronic care unit at a LTCF in Winnipeg where this individual lived in a single room. This facility did not have access to the EPR system, so the patient's CPE status was unknown to the site. During temporary admission to hospital in August 2019, a blood culture from the index case yielded a CPE isolate of Klebsiella aerogenes. All urine and blood samples were obtained during repeated acute care hospitalisations. Between admissions, our patient returned to the chronic care unit of the same LTCF.
Rectal screening for CPE carriage of 19 contacts of the index case was completed three times between 10 June and 1 July 2019. Over 2 years elapsed between the first laboratory documentation of a CPE and notification of the CPE status to the chronic care unit -14 months following admission to the chronic care unit. During this 14-month period, the patient was on a unit following routine practices (standard precautions) without additional precautions or enhanced environmental disinfection.
Of the 19 screened contacts, one was colonised with a carbapenemase-producing strain of E. coli which was of intermediate susceptibility to meropenem (Table 1). OXA-48 was detected by PCR analysis. This led to another 11 contacts identified and screened for rectal carriage of CPE between 26 June and 17 July 2019. CPE was not recovered. A fourth screening for rectal carriage of CPE was completed on the initial 18 negative index unit contacts in October 2019 and CPE was not identified.
Whole genome sequencing (WGS) was undertaken of the index case's K. pneumoniae urinary isolates (May 2017 and May 2019) and K. aerogenes blood isolate (August 2019) as 2015). Globally, only 19% of people wash their hands after contact with excreta (Freeman et al., 2014).
Handwashing is practised by washing hands using the several combinations of water, solid or liquid soap, sanitiser, alcohol-based components, sand, ash and mud. Although mostly water is used for handwashing, water alone is an inefficient skin cleanser because fats and proteins are not readily dissolved in water. People in lowincome countries such as India, Bangladesh and sub-Saharan Africa use ash, mud or sand for handwashing as zero-cost alternatives to soap (Bloomfield and Nath, 2009). Although there is potential for infection transmission by using contaminated soil/mud/ash for handwashing, ash or mud is perceived to clean hands as effectively as soap (Nizame et al., 2015). Handwashing with soap can dramatically reduce the rates of common diseases, including pneumonia and diarrhoea, two of the leading causes of deaths in children. Handwashing with soap and water is a simple and efficient method for reducing the risk of infectious diseases (Burton et al., 2011). Handwashing with soap can reduce childhood mortality rates related to respiratory and diarrheal diseases by almost 50% in developing countries (Curtis and Cairncross, 2003). Handwashing with soap prevents the two clinical syndromes that cause the most significant number of childhood deaths globally; namely, diarrhoea and acute lower respiratory infections (Luby et al., 2005).
Effective national programs for changes in handwashing behaviour can be expected to reduce diarrhoea and pneumonia caused by lack of handwashing by 25% (Townsend et al., 2017). A large number of people do not wash their hands regularly or do not know how to wash their hands properly (Ali et al., 2014). Education, socioeconomic status, availability of a water source in the house, ownership of the house and rural residence are associated with handwashing (Al-Khatib et al., 2015;Halder et al., 2010;Kumar et al., 2017;Ray et al., 2010;Schmidt et al., 2009;Ssemugabo et al., 2020). Handwashing is also related to knowledge of hand hygiene and non-availability of handwashing spaces or soap among school children (Mane et al., 2016).
India, with a cumulative number of 2,905,823 cases of COVID-19, is the third-worst affected country after the USA and Brazil as of 21 August 2020 (WHO, 2020b). Experts differ on the future trend of the COVID-19 in the country, amid rapidly growing cases across the states (Application Programming Interface, 2020), and the disease transmission stage being classified as 'cluster of cases' (WHO, 2020b). Appropriate handwashing (handwashing with alcohol-based agent or soap and water for a minimum of 20 s) is recommended as one of the most important ways to prevent person-to-person transmission of COVID 19. Nevertheless, evidence suggests poor hand hygiene in hospitals /healthcare providers (Mani et al., 2010;Sureshkumar et al., 2011;Tyagi et al., 2018) and the role of hands in spreading infections in the country (Taneja et al., 2003). Handwashing through alcohol-based agent/soap and water at the household level again seems not universal, as millions of Indians do not have access to basic amenities (Kumar, 2015). With several parts of India being water-stressed, and as much as 70% of the surface water resources being contaminated (Niti Aayog, 2019), is further perceived to worsen the recommended handwashing practices. Empirical evidence on existing handwashing practices is crucial to combat infectious diseases like COVID-19. There is, however, no scientific study exploring handwashing practices, spatial clustering and its determinants at the household level using the nationally representative sample in India. The aims of the present study were to: (1) understand the pattern and predictors of handwashing using soap/detergent and water; and (2) assess the spatial clustering of handwashing through soap/detergent and water at the district level in India.

Data
The study used data from the fourth round of the National Family Health Survey (NFHS), 2015-2016. The NFHS-4 is a nationally representative survey of 601,509 households that provides information for a wide range of monitoring and impact evaluation indicators of health, nutrition and women's empowerment. The sampling design of the NFHS-4 is a stratified two-stage sample with an overall response rate of 98%. The Primary Sampling Unit (PSUs), i.e. the survey villages in rural areas and Census Enumeration Blocks (CEBs) in urban areas, were selected using probability proportional to size (PPS) sampling. Data collection was conducted in two phases from January 2015 to December 2016. The data were gathered using computer-assisted personal interviewing (CAPI) by trained research investigators. Only those respondents who gave oral/written consent were interviewed in the survey. A more detailed description of survey design, questionnaire and quality control measures can be obtained elsewhere (Paswan et al., 2017).
The NFHS-4 asked a specific question: 'Please show me where members of your household most often wash their hands'. In the households where the place of handwashing was observed, research investigators were instructed to observe the presence of water, soap/detergent (bar, liquid, powder, paste) or other cleansing agents (ash, mud, sand) or absence of any cleansing agent. The present analysis is restricted to 582,064 households where the usual place for handwashing was observed. The availability of specific handwashing materials at the usual place of handwashing is assumed to be used by the household for handwashing. There is no consensus on a gold standard for identifying handwashing behaviour (Manun'Ebo et al., 1997), though handwashing behaviour can be assessed using questionnaires, by handwashing demonstration and by direct/indirect observation. well as the positive CPE contact's E. coli rectal isolate (June 2019). Analyses revealed that the contact's E. coli isolate harbored OXA-181, not the OXA-48 or NDM of the index case's isolates. This established a lack of CPE transmission between the index case and contact. The OXA-181 enzyme shares 94% nucleotide identify with OXA-48 and crossreacts with primers for PCR-based detection of OXA-48.
Once the patient's CPE status was known to the LTFC, specific IPC precautions were immediately initiated. Staff were required to glove and gown prior to entry into the patient environment and to use dedicated equipment. If this was not possible, shared equipment was disinfected after use. Enhanced cleaning and disinfection was implemented on the unit. Education about CPE pathogens was provided to staff, residents and families. Admission screening policies were enhanced so that similar situations do not occur in the future.

Discussion
Our findings call into question the efficiency of CPE transmission. In our case, routine practices appear to have Structured observation has been found to be the best indicator to assess handwashing practices in Indian households (Biran et al., 2008).

Outcome variable
The outcome variable considered for the analysis was 'the use of soap/detergent and water for handwashing'. It is defined as the presence of soap/detergent along with water in the usual place of handwashing among the households, where the place of handwashing was observed.

Predictor variables
The predictor variables used in the analysis were chosen based on the extensive literature review and available information in the NFHS-4. Specifically, the predictor variables used were the schooling of the household head (< 5 years including the illiterates, 5-9 years, 10-11 years, ⩾ 12 years), sex of the household head (male, female), religion of the household head (Hindu, Muslim, Christian and Others), caste/tribe of the household head (scheduled caste [SC], scheduled tribe [ST], other backward classes [OBC] or non-SC/ST/OBC), household size (< 5 members, ⩾ 5 members), house type (kuccha, semi-pucca, pucca), location of water source (in own dwelling, elsewhere), ownership of the house (not own house, own house), wealth index (poorest, poorer, middle, richer, richest), place of residence (urban, rural) and region (north, central, east, northeast, west, south).

Statistical analysis
In the present study, cross-tabulations between the outcome and predictor variables were done using the appropriate sample weights. The binary logistic regression was carried out to understand the predictors of handwashing practices. For this regression analysis, the dependent variable 'Soap/ detergent and water used for handwashing' was categorised into two, i.e. 1 = yes, 0 = no. The variables 'house type' and 'ownership of house' were dropped from the regression analysis to avoid multicollinearity. The Statistical Package for Social Sciences (SPSS-25, IBM Corp., Armonk, NY, USA) was used for analysis. The choropleth map was prepared at the district level using the ArcMap (version 10.4) to assess the regional scenario. The local indicators of spatial association (LISA) cluster map and Moran's I scatter plot were calculated through GeoDa (version 1.14) to understand the spatial clustering in the use of soap/detergent and water for handwashing.

Type of handwashing elements observed at the usual place of handwashing
Soap/detergent and water were observed in the usual place of handwashing in three-fifths (60%) of the households (Figure 1). In 16% of the households, only water was observed in the usual place of handwashing. Seven out of every ten households were observed to have water and any cleansing element in their regular handwashing place. Nine percent of the households were found to have no water, no soap or any other cleansing agent at their usual place for handwashing. Table 1 presents the bivariate analyses to understand the individual association between the predictors and outcome variable. Of the male-headed households, 61% use soap and water for handwashing compared with 55% of the female-headed households. Use of soap and water for handwashing was found to increase with increasing education of prevented CPE plasmid and/or clonal spread, a surprising result for a chronic care unit in a LTCF facility where residents share common spaces with other occupants. Institutional control of CPE typically involves bundled interventions. Thus, it is difficult to tease apart the relative effect of each intervention. It has been noted that routine practices are insufficient in controlling CPE transmission considering previous documented failures of CPE control with the implementation of contact precautions and isolation (Calfee and Jenkins, 2008;French et al, 2017).

Handwashing through soap and water by background characteristics of the households
The spontaneous decolonisation of CPE colonised patients may provide an explanation for our results (Vink et al, 2019;Zimmerman et al, 2013). Considering that our screening protocol was implemented up to 14 months after initial contact with the CPE positive index case, it remains possible that contacts who were initially CPE positive spontaneously eliminated their CPE by the time of testing (Vink et al, 2019). While a mean time to CPE negativity of approximately one year has been described among CPE colonised patients after hospital discharge, further studies are necessary to understand the process of spontaneous decolonisation during continuous CPE exposure (Bar-Yoseph et al, 2016;Zimmerman et al, 2013).
Repeat hospitalisation is known to prolong CPE colonization (Zimmerman et al, 2013). Considering that hospitalisation often involves antibiotic administration, CPE's transmissibility and duration of detection may be affected by the antimicrobial selection pressure and dysbiosis caused by broad-spectrum antibiotics (Zimmerman et al, 2013). Transfer from a chronic care unit to an acute care facility often involves antibiotic initiation or broadening. Thus, CPE transmissibility may be contextual, dependent on institutional patterns of antibiotic administration.

Conclusion
While bundled interventions are typically required to control the spread of CPE, we report the absence of transmission of NDM and OXA-48 genes from a CPE index case on a chronic care unit of a LTCF after 14 months of routine practices with no additional precautions. Our findings suggest that CPE transmissibility may differ between institutional settings. Further studies involving genotypic CPE analysis, institutional antimicrobial prevalence and prolonged surveillance sampling of contacts are required to inform whether infection control measures should be tailored to the specific CPE type and institutional context. 2015). Globally, only 19% of people wash their hands after contact with excreta (Freeman et al., 2014).
Handwashing is practised by washing hands using the several combinations of water, solid or liquid soap, sanitiser, alcohol-based components, sand, ash and mud. Although mostly water is used for handwashing, water alone is an inefficient skin cleanser because fats and proteins are not readily dissolved in water. People in lowincome countries such as India, Bangladesh and sub-Saharan Africa use ash, mud or sand for handwashing as zero-cost alternatives to soap (Bloomfield and Nath, 2009). Although there is potential for infection transmission by using contaminated soil/mud/ash for handwashing, ash or mud is perceived to clean hands as effectively as soap (Nizame et al., 2015). Handwashing with soap can dramatically reduce the rates of common diseases, including pneumonia and diarrhoea, two of the leading causes of deaths in children. Handwashing with soap and water is a simple and efficient method for reducing the risk of infectious diseases (Burton et al., 2011). Handwashing with soap can reduce childhood mortality rates related to respiratory and diarrheal diseases by almost 50% in developing countries (Curtis and Cairncross, 2003). Handwashing with soap prevents the two clinical syndromes that cause the most significant number of childhood deaths globally; namely, diarrhoea and acute lower respiratory infections (Luby et al., 2005).
Effective national programs for changes in handwashing behaviour can be expected to reduce diarrhoea and pneumonia caused by lack of handwashing by 25% (Townsend et al., 2017). A large number of people do not wash their hands regularly or do not know how to wash their hands properly (Ali et al., 2014). Education, socioeconomic status, availability of a water source in the house, ownership of the house and rural residence are associated with handwashing (Al-Khatib et al., 2015;Halder et al., 2010;Kumar et al., 2017;Ray et al., 2010;Schmidt et al., 2009;Ssemugabo et al., 2020). Handwashing is also related to knowledge of hand hygiene and non-availability of handwashing spaces or soap among school children (Mane et al., 2016).
India, with a cumulative number of 2,905,823 cases of COVID-19, is the third-worst affected country after the USA and Brazil as of 21 August 2020 (WHO, 2020b). Experts differ on the future trend of the COVID-19 in the country, amid rapidly growing cases across the states (Application Programming Interface, 2020), and the disease transmission stage being classified as 'cluster of cases' (WHO, 2020b). Appropriate handwashing (handwashing with alcohol-based agent or soap and water for a minimum of 20 s) is recommended as one of the most important ways to prevent person-to-person transmission of COVID 19. Nevertheless, evidence suggests poor hand hygiene in hospitals /healthcare providers (Mani et al., 2010;Sureshkumar et al., 2011;Tyagi et al., 2018) and the role of hands in spreading infections in the country (Taneja et al., 2003). Handwashing through alcohol-based agent/soap and water at the household level again seems not universal, as millions of Indians do not have access to basic amenities (Kumar, 2015). With several parts of India being water-stressed, and as much as 70% of the surface water resources being contaminated (Niti Aayog, 2019), is further perceived to worsen the recommended handwashing practices. Empirical evidence on existing handwashing practices is crucial to combat infectious diseases like COVID-19. There is, however, no scientific study exploring handwashing practices, spatial clustering and its determinants at the household level using the nationally representative sample in India. The aims of the present study were to: (1) understand the pattern and predictors of handwashing using soap/detergent and water; and (2) assess the spatial clustering of handwashing through soap/detergent and water at the district level in India.

Data
The study used data from the fourth round of the National Family Health Survey (NFHS), 2015-2016. The NFHS-4 is a nationally representative survey of 601,509 households that provides information for a wide range of monitoring and impact evaluation indicators of health, nutrition and women's empowerment. The sampling design of the NFHS-4 is a stratified two-stage sample with an overall response rate of 98%. The Primary Sampling Unit (PSUs), i.e. the survey villages in rural areas and Census Enumeration Blocks (CEBs) in urban areas, were selected using probability proportional to size (PPS) sampling. Data collection was conducted in two phases from January 2015 to December 2016. The data were gathered using computer-assisted personal interviewing (CAPI) by trained research investigators. Only those respondents who gave oral/written consent were interviewed in the survey. A more detailed description of survey design, questionnaire and quality control measures can be obtained elsewhere (Paswan et al., 2017).
The NFHS-4 asked a specific question: 'Please show me where members of your household most often wash their hands'. In the households where the place of handwashing was observed, research investigators were instructed to observe the presence of water, soap/detergent (bar, liquid, powder, paste) or other cleansing agents (ash, mud, sand) or absence of any cleansing agent. The present analysis is restricted to 582,064 households where the usual place for handwashing was observed. The availability of specific handwashing materials at the usual place of handwashing is assumed to be used by the household for handwashing. There is no consensus on a gold standard for identifying handwashing behaviour (Manun'Ebo et al., 1997), though handwashing behaviour can be assessed using questionnaires, by handwashing demonstration and by direct/indirect observation.