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REVIEW ARTICLE
Year : 2022 | Volume
: 29
| Issue : 3 | Page : 183-191
Carbapenemase producing Enterobacteriaceae: Environmental reservoirs as primary targets for control and prevention strategies
Ifeyinwa Nkeiruka Nwafia1, Anthony Chibuogwu Ike2, Ibuchukwu Nkeonyenasoya Orabueze1, Walter Chukwuma Nwafia3
1 Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka; Department of Medical Microbiology, University of Nigeria Teaching Hospital Enugu, Enugu State, Nigeria
2 Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria
3 Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria
Correspondence Address:
Ibuchukwu Nkeonyenasoya Orabueze
Department of Medical Microbiology, University of Nigeria Teaching Hospital Enugu, Enugu State
Nigeria
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/npmj.npmj_95_22
Carbapenemase-producing Enterobacteriaceae (CPE) have become one of the greatest public health challenges globally. In the past decade, antimicrobial resistance (AMR) was viewed as a clinical problem in many parts of the world; hence, the role and magnitude of the contribution of the environment were not well appreciated. This review article was done with online published articles extracted from different databases using search terms related to the work. Evidence has shown that there exists the presence of carbapenemase genes in the environment, consequently fuelling the dissemination with alarming consequences. CPE when acquired causes life-threatening infections in humans. The health and economic impact of these infections are numerous, including treatment failure due to limited therapeutic options which hamper the containment of infectious diseases, further contaminating the environment and worsening the public health challenge. It is a well-known fact that the rate of emergence of resistant genes has outpaced the production of new antimicrobial agents, so it is pertinent to institute effective environmental measures to combat the spread of AMR organisms before it will completely gain a foothold and take us back to 'the pre-antibiotic era'. Environmental sources and reservoirs of resistant genes should therefore be amongst the primary targets for the control and prevention of the spread of resistant genes in the environment. This calls for the effective implementation of the 'one health' strategy with stakeholders committed to the design and enforcement of environmental mitigation policies and guidelines.
Keywords: Antimicrobial resistance, carbapenemase, carbapenemase-producing Enterobacteriaceae, environment, multi drug resistance
One of the greatest feats in the history of modern medicine is the discovery of antibiotics which reformed medicine and saved many lives.[1] Today, the emergence and dissemination of antimicrobial resistance (AMR) is about to wipe out this hard-fought historic gain, threatening the reversal of this century's advancement in modern medicine globally. Due to the priority and public health challenges of AMR, the World Health Organization has ranked AMR amongst the top ten public health threats[2] and has also predicted that people may die of common infections as was seen in the era before the discovery of antibiotics.[3] This is because the rate of emergence of AMR by several pathogens has outpaced the production of new antibiotic agents, thus worsening the clinical and economic impact of AMR. It has become pertinent to institute effective measures to combat the spread of antimicrobial-resistant organisms globally before they completely gain a foothold and take humanity back to the pre-antibiotic era.
The cost of AMR on both human and financial resources is on the increase. The Global Research on AMR estimated that 1.27 million people died in 2019 due to multi-drug resistant diseases.[4] This figure has been projected to rise to 10 million with the annual cost increasing from $300 billion to more than $1 trillion by 2050.[5],[6] It is anticipated that by 2030, AMR may have forced up to 24 million people into excessive poverty, starvation and malnutrition.[4] There is therefore the need to institute urgent effective measures to ensure that these projections do not turn out to be a reality.
Antibiotic resistance has been referred to as a 'tragedy of the commons' because over utilisation due to self-interest has exhausted the limited resources.[7],[8] The development of resistance in the microorganism is a natural phenomenon but widespread misuse and overuse of antimicrobial agents in both humans and animals worsen the situation. Antimicrobial-resistant genes are frequently located on mobile genetic elements that can easily be transmitted from one bacterium to another. It is well-known that mobile genetic elements carry several genes that confer resistance to multiple classes of antimicrobial agents.[9],[10]
Carbapenem is considered the last resort in the treatment of life-threatening infections caused by extended-spectrum beta-lactamase-producing Gram-negative organisms.[11] Carbapenem is classified in the Watch group of the WHO AWaRE classification, so carbapenem resistance is a significant public health concern of global importance. Resistance to carbapenems has been shown to increase 10–20 fold as Gram-negative organisms are exposed to this antibiotic.[12] The production of the enzyme, carbapenemase is a major mechanism underlying carbapenem resistance in carbapenem-resistant Enterobacteriaceae.[13] Carbapenem-resistant organisms are under the WHO priority group of pathogens that need urgent antibiotic development. Carbapenemases are beta (β) lactamase enzymes which hydrolyse the β-lactam ring of carbapenem antibiotics thereby rendering them inactive. This has made carbapenemases to be recognised as a new threat, because of their role in inactivating the last line in antibiotic defence. Carbapenemase production is majorly seen in Enterobacteriaceae. Enterobacteriaceae are a large group of Gram-negative, rod-shaped bacteria accounting for up to 80% of all clinically important Gram-negative isolates and up to 50% of all clinically significant bacteria.[14],[15]
Carbapenemase-producing Enterobacteriaceae (CPE) is of particular importance with epidemiological relevance because the genes are frequently located on mobile genetic elements, such as transposons, integrons and plasmids, and can be transferred horizontally from one bacterial species to another. The threats encountered in the spread of CPE are probably one of the greatest public health and developmental challenges, and it is gradually shifting the world to the post antibiotic era.
The problem of AMR is global, affects all people irrespective of their level of development and riches. AMR is being driven by antimicrobial use and abuse in human, animal and environmental sectors. In 2008, the 'One Health' approach was adopted by international organisations and professional bodies to reduce the spread of AMR. The one health tripartite approach highlights the importance of the environment, human and animal health interface as an effective way in mitigating the increase and spread in the rate of AMR.[16] Amongst these three sectors, the environmental sector is the most neglected in the fight against AMR,[17] because AMR has naturally been viewed as a clinical problem. Environmental reservoirs are potential sources of infection by AMR organisms[18],[19] because the mobile genetic elements (MGEs) which carry the resistant genes can be retained and harboured in the environment. The fight against the spread of AMR genes needs a good understanding of the role played by environmental reservoirs such as sinks, fomites, water, food and soil.
In developing countries in Africa, antimicrobial use and abuse are common in both humans and animals because of the unregulated drug supply chains including purchase without prescriptions.[20] Some of these consumed antimicrobials maybe excreted unchanged and end up in the environment, thus contributing to the selection pressure that may lead to the emergence of novel antimicrobial resistant genes in the environment. The increase in knowledge and awareness is important, and one of the targets of the Global action plan to tackle this menace, hence this review will provide deep insight and foster good knowledge of the role of environmental reservoirs in fuelling the scourge of CPE. This initiative is geared towards tackling the AMR problem by providing evidence-based information on environmental reservoirs of carbapenemase-producing organisms, to enhance decision-making and infection prevention and control practices in combating the spread of AMR.
MethodsSearch criteria
The information was obtained from published articles using databases such as PubMed, Web of science, EMBASE, ScienceDirect, Hinari, Scopus, Google Scholar and African journals on line (AJOL).
The search terms used include 'carbapenem', 'carbapenemase genes', 'antimicrobial resistance' and 'environmental sources of carbapenemase genes and antibiotic resistance'. Amongst the articles found by the entry criteria, only those showing data on AMR, carbapenem, carbapenemase genes and environmental reservoirs of carbapenemases were selected in this review. Published articles in the English language were selected for this study and emphasis was not laid on the study location or year of publication. Articles written in other languages and those related to carbapenemases were excluded. The flow diagram of selected articles included in this review is depicted in [Figure 1].
Structure and mechanisms of action of carbapenem
Carbapenems are a class of antimicrobial agents used mainly in the treatment of infections caused by multidrug-resistant bacteria. The first carbapenem antibiotic was thienamycin, produced in 1976, with activity against aerobic and anaerobic organisms including methicillin-resistant Staphylococcus aureus[21] Unfortunately, this great discovery was undermined by the chemical instability of thienamycin in aqueous solution: Sensitivity to mild base changes and high reactivity to nucleophiles and even thienamycin's own primary amine.[22],[23] In order to overcome the instability of thienamycin, other analogous derivatives with increased stability were then developed.
Carbapenems have a concentration-independent killing effect on the bacteria[24],[25] with a unique structure that confers protection against most beta-lactamases.[26] It is a bactericidal antibiotic, possessing a β-lactam ring. The carbapenems have a methylene group in the dihydrothiazine ring of the cephalosporin nucleus (e.g. imipenem, meropenem, ertapenem and doripenem) contain a carbon double bond instead of sulphur in the 5-membered thiazolidine ring and have a side chains ®, which differs from penicillin by having a double bond between C-2 and C-3 and a carbon atom as shown in [Figure 2]. Furthermore, both carbepenems and cephalosporins have two-side chains which differentiate the spectrum of activity seen amongst the various antibiotics while penicillins have only one side chain.[27],[28]
The cell wall of bacteria is complex structures composed of peptidoglycan polymers consisting of alternating N-acetylmuramic acid and N-acetylglucosamine units. The polymer is linked by transglycosidases and forms 'giant macromolecules' after the cross-linkages which provide shape and rigidity that protects the cell from lysis due to osmotic pressure changes.
The last step in the synthesis of peptidoglycan is transpeptidation, catalysed by transpeptidase enzymes, which are penicillin-binding proteins (PBPs). Carbapenems irreversibly bind to PBPs located on the bacterial cell wall because they have structural similarity with acylated D-alanyl-D-alanine which is the terminal amino acid residue of the peptidoglycan. In Gram-negative bacteria, it transverses through the outer membrane proteins before binding to the PBPs.[29] The binding of carbapenems to PBPs will inhibit the activity of D-alanyl-D-alanine carboxypeptidase thereby preventing the formation of peptide cross-linkages and other similar peptidase activities, hence interfering with the formation of the bacterial cell wall. Thus, the inhibition of cell wall formation together with its autolysis creates holes through which the cell membrane is pushed out eventually leading to rupture and death of the organism.[30],[31]
The evolution of carbapenemases
Carbapenem antibiotics serve as the last resort in the treatment of infections caused by MDR organisms owing to their stability to many of the beta-lactamases and their high penetration through the Gram-negative bacterial outer membrane.[32],[33] However, its widespread use has led to a rapid increase in the number of carbapenem-resistant organisms globally.[34],[35] Bacteria have a plethora of mechanisms to resist the action of carbapenems. Three major known mechanisms are: (1) porin-mediated decreased uptake of carbapenems, (2) efflux pumps: Which push the antibiotics out of the cell and (3) enzyme-mediated resistance which can either be chromosomally mediated and/or acquired through genetic mobile transfer elements.[36] Acquisition of carbapenemase genes is the main mechanism underlying carbapenem resistance in bacteria worldwide, especially in Enterobacteriaceae.[37] Carbapenemase encoding genes are usually located on plasmids, which can easily be transmitted vertically as well as horizontally between different strains and species of microorganisms.[38],[39],[40] Furthermore, carbapenemase genes are frequently associated with other smaller mobile genetic elements such as the transposons and integrons which help to promote horizontal genetic transfer, expedite the spread of carbapenemase genes and increase the resistance capacity to multiple antibiotics.[41]
The first identified carbapenemase produced in Enterobacteriaceae was the non-metallo-enzyme carbapenemase A (NMC-A) detected in 1993. NMC-A is a chromosomal-mediated carbapenemase that is acquired through clonal transfer.[12] In 1996, Klebsiella pneumoniae carbapenemase (KPC) was the first plasmid-mediated carbapenemase identified in North Carolina, the United States of America, and has disseminated at a fast rate globally.[42] Subsequently, several CPE has been described and currently, more than 350 carbapenemase variants have been identified in Enterobacteriaceae (http://www.lahey.org/Studies/).
Environmental reservoirs of carbapenemase-producing Enterobacteriaceae
Broadly speaking, the environment (hospital and community environment) plays a pivotal role in the dissemination and emergence of carbapenemase genes as shown in [Figure 3].[43],[44],[45] The so-called 'big five' clinical carbapenemase genes; KPC, Oxaciliinase-48, imipenemase, Delhi metallo-β-lactamase (NDM) and Verona integron-encoded metallo-β-lactamase[46] have been isolated from environmental samples globally. These genes can be introduced directly to the environment or emerge by selection pressure as a result of constant exposure of bacteria to antibiotic residues. Approximately 30%–90% of antibiotics consumed by humans, livestock and aquaculture are usually excreted unchanged or as active metabolites in urine or stool within some hours.[47],[48] These excreted antibiotics have half-life that vary from a few hours to 100 days in the environment.[49]
Figure 3: Environmental dissemination of carbapenemase-producing EnterobacteriaceaeWhen the antimicrobial residue is discharged into the environment, it results in several harmful effects at different concentrations including enhancing biofilm formation,[50],[51] virulence, gene transfer and quorum sensing.[52] Of interest is the low sub-inhibitory concentration of the excreted antibiotic residues, which could be 1000 times lower than the therapeutic or bactericidal level,[53] but adequate to induce AMR.[54] There has been evidence that low sub-inhibitory concentrations lead to increase in the frequency of mutations, overexpression of certain resistant genes and ultimately lead to persistent of the resistant mutants in the environment.[55],[56] Furthermore, the antibiotics may reduce the number of susceptible microbes thereby increasing the nutrient available and enhancing the growth of resistant strains.[57],[58] Long-term exposure of environmental bacterial strains to low concentrations of antibiotics significantly modulates the bacterial genomes' expression.
The antibiotic residues can enter the environment through municipal solid and liquid waste, pharmaceutical industry, agriculture and aquaculture. This is illustrated in [Figure 3]. It is noteworthy that antibiotics can also be produced and released in natural microbial systems by bacteria that reside in the soil. Apart from these routes, air-borne aerosols and dust are also important vectors promoting environmental dissemination.[59],[60]
Hospitals as well as other health-care facilities create a unique environment for the acquisition of antimicrobial-resistant genes and this has been widely reported. The hospital environment harbours sick patients, who consume carbapenems and health-care workers who are carriers of CPE. Furthermore, the hospital environment promotes proliferation of biofilm in the presence of sub-optimal antibiotic concentrations[51],[52] and enhance bacterial interaction, replication and dissemination of the resistant genes.[61] CPE has been isolated from contaminated surfaces such as mattresses, tables, infusion pumps, bed linens, sinks, stethoscopes and switches[62],[63],[64] In France, it was reported that the persistence of blaOXA-48 producing K. pneumoniae in the intensive care unit was due to contamination of mattresses and sinks by the CPE.[61] Other researchers[65],[66],[67] have also identified CPE in hospital sinks and have proven that the replacement of affected sinks and related pipes eradicated the CPE. Recent studies have not only reported hospital environment as a potential reservoir of CPE but have also linked them as a causative route of transmission of healthcare-associated infections. This was evidenced in a study of the molecular epidemiology of environmental and clinical carbapenemase-producing Gram-negative bacilli in Guelma, Algeria.[66] In this study, the strains of blaOXA-48, blaNDM-1, blaOXA-23 genes isolated from the hospital environment had a clonal relationship with the strains isolated from clinical samples.[66]
Hospital sewage is not left out; it is a reservoir and hotspot for genetic transfer of AMR genes. Several researches conducted in different parts of the world have identified high rates of carbapenemase genes in hospital sewage.[67],[68],[69] These reports highlight hospitals sewage as a potential reservoir of carbapenemase genes. The hospital sewage is a complex matrix containing faeces and urine of patients undergoing antibiotic treatment as well as wastewater containing antibiotic residues.[43] It serves as a good medium for the increase in selection pressure and horizontal transfer of carbapenemase genes between the organisms present in the sewage. The literature indicates that hospital wastewater, as compared to municipal wastewater, has a significantly higher diversity and quantity of antibiotic resistance genes (ARGs) carrying carbapenemase-encoding genes.[70] A study conducted in Ireland reported more of the carbapenemase genes in hospital wastewater than in pre-hospital water.[44] In studies conducted in China, the researchers identified blaNDM-1, blaKPC-2 and blaOXA-58 in hospital sewage.[67] Similarly, blaNDM-4 and blaNDM-1 genes were isolated from hospital sewage in India and Lebanon, respectively.[68],[69] Most hospitals do not have a wastewater-treated plant despite WHO recommendation that all hospital waste should be treated before releasing to the environment.[71] Globally, about 80% of hospital wastewater are discharged into the environment untreated and this is more in low- and middle-income countries.[72] Even treated wastewater has been shown to carry the resistant genes as reported by studies in Brazil that identified blaKPC in already treated hospital wastewater.[73],[74] Hospital wastewater is a major source of carbapenemase genes and has continued to fuel its dissemination in the environment.
Surprisingly, several studies have detected carbapenemase genes in municipal water. The treatment of municipal water targets the removal of organic and inorganic contaminants but has no effect on antibiotic residues or ARG.[75] The presence of CPE in a sample of treated drinking water connotes a serious threat to public health. A study done in the United States identified carbapenemase genes in drinking water samples taken from 6 different states.[76] Also in Sudan, several carbapenemase genes have been isolated from drinking water.[77] In addition, municipal solid waste may contain unused or expired drugs, when discharged into the environment endanger the raising trend in emergency of carbapenemase-producing organisms in the soil or aquatic environment.
We seem to neglect the huge contribution of the pharmaceutical companies to AMR. Several studies have shown that the dumping of pharmaceutical effluent which normally contains antibiotics on a large scale contaminates the surrounding soil and water; thereby worsening the danger of the dissemination and emergence of antibiotic-resistant organisms yet it has received less attention. It has been reported that pharmaceutical effluent has the highest concentrations of antibiotics and ARGs in the environment when compared to discharges from other sources.[78],[79] In India, researchers analysed water samples collected from villages surrounding Hyderabad's industrial suburb Pattancheru, where a lot of pharmaceutical industries were located. They detected antibiotics in all the water samples, with some samples containing very high antibiotics concentrations more than the concentrations found in the blood of patients' on blood on antibiotics therapy.[80]
Rivers and lakes are the natural receptacles of wastewater containing antibiotic residues released from domestic, livestock, hospital pharmaceutical companies and from other environmental sources. These water bodies are sources of water for drinking, domestic activities, recreational and irrigation purposes; there contamination with antibiotic doses and carbapenemase genes is a potential concern to human health. This is evidenced by previous studies which have been done in several regions and continents that reported presence of carbapenemase genes in lakes, streams and rivers.[81],[82],[83] with the detected carbapenemase genes being similar to those isolated from clinical isolates worldwide. Bleichenbacher et al.[81] reported an epidemiologic link between a clinical NDM-5 producing Escherichia coli ST410 strain from China and a non-clinical strain isolated from surface water in Switzerland.
Another important environmental reservoir of carbapenemase genes is infected animals and livestock faeces even companion animals too. These infected faeces can either contaminate the surface water bodies or use as manure or can potentially directly infect humans in close contact such as farm workers, animal caretakers and their family members. All these cumulatively can lead to easy transmissibility of carbapenemase genes. The presence of carbapenemase-producing Escherichia coli have been reported from faecal samples collected in pig barns in Germany.[84] Highly mobile and migratory birds can pick up carbapenemase genes from contaminated environment, travel large distances and deposit it in another environment thereby facilitating the dissemination of the resistant genes.
Humans can become exposed to carbapenemase-producing organisms by consuming contaminated vegetables. Fresh vegetables and fruits can be contaminated through manure with infected faeces or irrigation with contaminated water during cultivation. This was evidenced by WHO report that linked DNA fingerprinting from sick people and an agricultural source.[85] In Algeria[86] and Switzerland,[87]blaOXA-48 producing K. pneumoniae were isolated in fresh vegetables. Likewise in Japan, Soliman et al.[88] isolated 19 different AMR genes including blaNDM-1 type carbapenemase gene from fresh vegetables. Escherichia coli isolate co-producing blaNDM-1 and blaKPC-2 was identified in lettuce in China.[89] Other vegetables that have been implicated are tomatoes, spinach, carrot and parsley.
DiscussionThe alarming trend in the rise of AMR is worrisome. The aforementioned evidence has made it clear that carbapenemase-producing organisms are circulating and polluting the environment, emphasising the role the environment plays in the spread of carbapenemase genes. The outcome of infection with CPE are far reaching with dire consequences affecting nations and limiting the achievement of the WHO's sustainable development goals. The burden of AMR is disproportionately distributed across the world with low- and middle-income countries bearing the greatest impact.[90] It is worthy to note that sub-Saharan Africa suffers the highest-burden of AMR amongst all the continents. With proactive measures in place, the clinical, social and economic impact of AMR will be ameliorated. Urgent measures should be instituted and succinctly put; the time to act is now.
This is a clarion call to all nations to institute stringent measures to curb the amount of antimicrobial waste entering the environment. Curtailing AMR is not the sole responsibility of a group of people; all persons are involved in this fight because AMR affects everyone irrespective of wealth or status. WHO advocated the one health approach which encompasses the health, animal and environmental sectors. This leaves no stone unturned in the fight against AMR and should be one of the priorities of every government across the world.
Development and implementation of safety measures in the disposal of antimicrobial waste from human, animal and environmental systems including the establishment of standard antimicrobial safety levels should be enforced at global, national and state levels to minimise the public health impact of environmental discharges. Concordant with this is pharmacy regulations which should stipulate that antibiotics are only issued with prescriptions.
In human health, the behaviour and attitude of the prescribers/patients should be changed through the practice of effective antimicrobial stewardship (AMS) programmes which promote responsible antibiotic use and procurement of quality drugs. AMS has been proven to be a strategy that promotes responsible antibiotic use. The strategy has been well established in developed countries but grossly underutilised in developing nations.[91]
The use of antibiotics for humans consumed in animal husbandry should be discouraged or banned, though re-enforcement of this law may vary from one country to the other. Many developed countries have instituted and established these laws in animal husbandry; some have banned all antimicrobials used in humans while others have banned a few. In Africa, Namibia was the first country to ban the routine use of antibiotics in animal husbandry.[92] However, many of the developing countries in Africa are yet to key into this initiative for obvious reasons,[93] resources are limited with so little or none channelled to implement and enforce these laws including punishment of offenders.
The implementation of effective infection prevention and control measures in environmental sector, including water, sanitation and hygiene (WASH) measures should be foremost. Policies, guidelines and standard operative procedures on environmental cleaning should be put in place to guide compliance and sustainability of the programme. The WHO has estimated that two billion people do not have basic sanitation facilities with 673 million practicing open defaecation method.[94] Open defecation is worse in developing countries, so faecal contamination of water will lead to the spread of resistant genes.
The importance of public education cannot be overemphasised. The general population and other stakeholders in society need to be informed and educated on the threat of AMR, risk factors and preventive measures in lay language, simple for all to understand through multiple media channels.[95] For effective treatment of infected patients, financial resources should be invested in research and development of new antibiotics and vaccines. Continuous monitoring and surveillance systems to identify CPE in the environment will inform decision-making and rightful channelling of resources. In addition, there is also need to consider other emerging treatment alternatives such as use of bacteriophage, quorum sensing inhibitors and probiotics.[96],[97]
There are a number of limitations in this work. The principal limitation is the retrospective design. Second, the articles assessed were the ones published in the English language, papers published in other languages were not considered. Third, the authors assessed only CPE, regardless of the fact, that there are other major resistant phenotypes that contribute greatly to the rising trend of AMR. Finally, most of the articles assessed were from developed countries because of paucity of data in developing regions.
ConclusionCPE pose a serious health concern. Knowledge of environmental reservoirs of the resistant organisms and resistance genes is important to combat their spread and is also one of the national action plans. There is the presence of clinically relevant carbapenemase genes in the environment. These genes are easily acquired to cause life-threatening infectious diseases in humans. The environmental spread of carbapenemase-producing organisms will surely lead to escalation of the threat to human health. Environmental reservoirs should, therefore, be one of the primary targets for control and prevention strategies. Efforts should be made to remove or at least decrease the amount of antibiotic-resistant organisms and ARGs in the final effluent, especially in developing countries. This calls for effective implementation of 'one health' strategies with stakeholders committed to the design and enforcement of environmental mitigation policies and guidelines. In health-care settings, AMS and infection prevention and control programmes should be established with promotion of education, awareness and environmental measures while leveraging on administrative support.
Recommendations
AMS should be instituted in all hospitals in developing countries with strong management support for sustainability.
Policies and guidelines on waste disposal and WASH should be strictly implemented and monitored.
Financial resources should be channelled to antimicrobial surveillance, research and development particularly in developing countries.
Public education should be promoted and encouraged.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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