This contribution describes the possibility of applying liquid chromatography coupled to tandem mass spectrometry for analysing sewage in order to track down the use of new psychoactive substances.
by J. Kinyua, Prof. A. Covaci, Prof. A. L. N. van Nuijs
Introduction
Sewage-based epidemiology (SBE) is an alternative method of monitoring population drug use by the analysis of excretion products of drugs in sewage (Fig. 1). SBE has been applied since 2005 as a complementary approach to classical investigation methods, such as interviews with users, medical records, population surveys, and crime statistics for estimating illicit drug use in communities [1–3]. Data obtained from SBE provide information on drug use in a direct, quick and objective way.
New psychoactive substances (NPS) are substances that are not controlled by the 1961 United Nations Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances and that may pose a threat to public health [4, 5]. These compounds mimic effects of illicit drugs like cocaine, cannabis and amphetamines and are produced to evade law enforcement by introducing slight modifications to chemical structures of controlled substances [6]. Currently, more than 450 NPS are being monitored by the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) with 101 new substances reported for the first time in 2014 to EU Early Warning System (EWS). Synthetic cannabinoids and synthetic cathinones are the largest groups in the NPS scene [7]. NPS are easily acquired through online vendors and in smart shops where they are sold with misleading information about their effects and safety [8]. They are considered a growing problem in many communities and are responsible for numerous fatal intoxications [9]. SBE has the potential to be usefully applied for the detection and quantification of NPS to document their occurrence to appropriate authorities. Being an emerging issue, only a few studies have applied SBE for the analysis of NPS [10–13]. In this contribution, the optimization, validation and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC–ESI-MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine, 4-methoxymethamphetamine (PMMA), and 4-methoxyamphetamine (PMA) is described together with a critical evaluation of the methodology.
LC-MS/MS methodology
An LC-MS/MS method was developed and validated using a Phenomenex Luna HILIC (hydrophilic interaction liquid chromatography) 200A (150 x 3 mm, 5 µm) column, with a mobile phase composed of A) 5 mM ammonium acetate in ultrapure water and B) acetonitrile. The mass spectrometer compound dependent parameters, fragmentor voltage and collision energy, were optimized to acquire two multiple reaction monitoring (MRM) transitions (qualifier and quantifier) for each compound, and one MRM for the internal standards (IS). The method was validated, assessing accuracy and precision, using blank sewage (samples collected prior to 2009 in which NPS have not been detected). A linear range with lower limits of quantification (LLOQ) of 0.5 ng/L (MXE and methylone) and 2 ng/L (all other compounds) and upper limits of quantification (ULOQ) of 200 ng/L was achieved for investigated compounds. The limit of detection (LOD) was between 0.02 and 0.2 ng/L for all compounds.
Sample collection and preparation
24-h composite influent sewage samples were collected from different wastewater treatment plants (WWTPs) in Belgium and one WWTP in Zurich. Before sample extraction, 50 mL sewage was filtered through a 0.7 µm glass filter to remove solid particles. After filtration, the samples were brought to pH 2 using a 6 M HCl solution and spiked with deuterated IS at a concentration of 100 ng/L. Thereafter the solid-phase extraction (SPE) procedure was performed using a mixed-mode strong cation exchange sorbent-Oasis MCX (Fig. 2).
Application of the procedure
The method could reliably differentiate the analytes and IS from endogenous components. MXE, methylone and ethylone could be detected. The method revealed the presence of MXE in sewage from five urban centres within two counties in Belgium. Methylone was detected and quantified in only two samples from Switzerland at levels slightly higher than LLOQ (Fig. 3). The compounds that were not detected could be absent in the sewage or present in the form of metabolites which were not targeted in the present study.
Advantages/limitations of the SBE methodology
Phenylethylamine-based compounds (synthetic cathinones and amphetamine-like substances) form a large group of NPS and they are very polar. Hydrophilic interaction was found to be a good and robust LC stationary phase to obtain retention for these high-polarity compounds. Furthermore, we showed for the first time in SBE that the use of a more realistic matrix for method development, such as real sewage, can help in overcoming challenges associated with matrix effects in MS detection. The results from these samples demonstrate the importance of developing highly sensitive analytical methods that can detect and quantify NPS at very low concentrations (<10 ng/L).
Limitations of the present methods
It is difficult to determine if the low drug concentrations in sewage are related to low popularity of the NPS or due to the presence of an unknown form of the parent drug in sewage, urinary metabolites or transformation product from other in-sewer processes. SBE requires a specific, reliable and stable biomarker for the NPS of interest. Further studies on the metabolism and in-sewer transformation processes (which may affect stability of drug residues) of NPS needs thus to be carried out to provide SBE with information regarding additional biomarkers of NPS parent drugs.
Future of SBE in NPS analysis
Concentrations of NPS in sewage may be low depending on the area served by the WWTP and on the prevalence of its use [14]. Therefore, pooled urine analysis would be useful in detecting the occurrence of NPS before dilution into sewage [15]. It would be a valuable approach to combine pooled urine analysis and SBE to track down the actual use of NPS in communities.
Conclusion
In conclusion, SBE can help in revealing the occurrence of NPS within catchment areas of urban centres and showed the need to develop very sensitive analytical methods to detect NPS in sewage.
References
1. Bijlsma L, Sancho JV, Pitarch E, et al. Simultaneous ultra-high-pressure liquid chromatography-tandem mass spectrometry determination of amphetamine and amphetamine-like stimulants, cocaine and its metabolites, and a cannabis metabolite in surface water and urban wastewater. J Chromatogr A 2009; 1216: 3078–3089.
2. Boleda MR, Galceran MT, Ventura F. Trace determination of cannabinoids and opiates in wastewater and surface waters by ultra-performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 2007; 1175: 38–48.
3. Huerta-Fontela M, Galceran MT, Ventura F. Ultraperformance liquid chromatography-tandem mass spectrometry analysis of stimulatory drugs of abuse in wastewater and surface waters. Anal Chem 2007; 79: 3821–3829.
4. United Nations Office on Drugs and Crime (UNODC). Global synthetic drugs assessment. (United Nations publication, Sales No. E.14.XI.6), 2014. http://www.unodc.org/documents/scientific/2014_Global_Synthetic_Drugs_Assessment_web.pdf
5. King LA, Kicman AT. A brief history of ‘new psychoactive substances’. Drug Test Anal. 2011; 3: 401–403.
6. Dargan PI, Wood DM. Novel psychoactive substances classification, pharmacology and toxicology. Elsevier/Academic Press, 2013. ASIN: B00FK8HYY2.
7. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). New psychoactive substances in Europe. An update from the EU Early Warning System, 2015. http://www.emcdda.europa.eu/publications/2015/new-psychoactive-substances
8. EMCDDA. EMCDDA–Europol 2013 Annual Report on the implementation of Council Decision 2005/387/JHA, 2014. http://www.emcdda.europa.eu/publications/implementation-reports/2013
9. Vevelstad M, Øiestad E.L, Middelkoop G, et al. The PMMA epidemic in Norway: Comparison of fatal and non-fatal intoxications. Forensic Science International 2012; 219: 151–157.
10. Kinyua J, Covaci A, Maho W, et al. Sewage-based epidemiology in monitoring the use of new psychoactive substances: validation and application of an analytical method using LC-MS/MS. Drug testing and analysis 2015 ( In press).
11. Reid M.J, Derry L, Thomas K.V. Analysis of new classes of recreational drugs in sewage: Synthetic cannabinoids and amphetamine-like substances. Drug Test Anal. 2014; 6: 72–79.
12. Van Nuijs ALN, Gheorghe A, Jorens PG, et al. Optimization, validation, and the application of liquid chromatography-tandem mass spectrometry for the analysis of new drugs of abuse in wastewater. Drug Test Anal. 2014; 6: 861–867.
13. Kankaanpää A, Ariniemi K, Heinonen M, et al. Use of illicit stimulant drugs in Finland: a wastewater study in ten major cities. Sci Total Environ. 2014; 487: 696–702.
14. Archer JRH, Dargan PI, Lee HMD, et al. Trend analysis of anonymised pooled urine from portable street urinals in central London identifies variation in the use of novel psychoactive substances. Clinical Toxicol (Phila). 2014; 52: 160–165.
15. Archer JRH, Dargan PI, Hudson S, et al. Analysis of anonymous pooled urine from portable urinals in central London confirms the significant use of novel psychoactive substances. QJM 2013; 106: 147–152.
The authors
Juliet Kinyua MSc, Adrian Covaci PhD, Alexander L.N. van Nuijs* PhD
Toxicological Center, University of Antwerp, Belgium
*Corresponding author
E-mail: alexander.vannuijs@uantwerpen.be
In spite of increased publicity in the Western world about malaria and drives to provide mosquito nets, the disease is still endemic in a large part of the world. This article discusses different methods of malaria diagnosis and the role that point-of-care tests can play in the ultimate goal of malaria elimination.
by Dr Jackie Cook
Finding the balance: over- and under-diagnosing malaria
Malaria remains a huge burden in many parts of the world, particularly in sub-Saharan Africa. Despite increased availability of effective treatments and interventions, malaria elimination is still out of reach for many countries. Whilst availability of effective interventions that reduce contact with infected mosquitoes, such as insecticide treated bed-nets or indoor spraying with insecticide are key to reducing malaria prevalence, case management also plays a key role. Many who need treatment are unable to get it, either through lack of access to healthcare, or because infections remain undiagnosed. Conversely, some studies suggest that many patients are receiving anti-malarials unnecessarily due to a tendency to diagnose based solely on clinical symptoms, many of which are similar to other infections, rather than using a diagnostic. In under-resourced settings, this can result in any child presenting with a fever being prescribed malaria drugs. This simultaneously means non-malaria fevers remain undiagnosed and untreated, as well as a large proportion of unnecessary prescriptions for malaria drugs, which increases healthcare costs and the risk of drug resistance, a very potent threat. In order to counteract this, the last few decades have brought a push from health officials, researchers, donors and governments alike to confirm every suspected case of malaria before prescribing treatment.
Microscopy
For many, malaria diagnosis is performed using microscopy, a procedure that is relatively cheap but requires a skilful operator. Malaria is caused by the plasmodium parasite and it undergoes several developmental and replication stages in the human. These stages can be seen through a microscope when blood is prepared on either a thin or thick film and stained, normally, with Giemsa or Wright’s stains. Experienced microscopists can detect down to 1 parasite per microlitre of blood, although the typically quoted sensitivity for microscopy is approximately 100 parasites per microlitre. In reality, the sensitivity of the test depends greatly on the microscopist. In areas where malaria transmission is declining, microscopists can go months without seeing a positive slide, and as such, skills may begin to decline. In addition, the need for well-maintained microscopes and access to slides and stain can mean microscopy is not always available.
Rapid diagnostic tests
The first malaria rapid diagnostic test (RDT) was developed in 1993 and in the decades since many variations have proliferated on the market. RDTs are typically immunochromatograhic tests that use monoclonal antibodies to detect the presence of plasmodium antigens (proteins produced by the parasite) which are present in the blood of infected, or recently infected, individuals. They are generally stable at a range of temperatures and do not require special storage conditions. RDTs require significantly less training for use than microscopy and a positive infection is easy to identify by visualization of a ‘positive’ line, meaning the results are much less subjective. Most RDTs require 15–20 minutes for development, meaning treatment can be given while patients wait at health facilities.
However, there are a few downsides to the use of RDTs. The presence of parasite antigen doesn’t always equate with a current infection, but can signify a recently cleared infection from within the previous two weeks. In addition, several studies have reported the deletion of certain antigens detected by RDTs in plasmodium parasites, meaning false-negative results may be obtained in areas using these types of RDTs [1]. The World Health Organization (WHO), in collaboration with the Foundation for Innovative New Diagnostics (FIND), has set up an RDT product testing programme, an essential quality assurance component considering the huge influx of RDT brands that have popped up in the past 20 years [2]. The reports from the programme make worrying reading with very low sensitivity for some brands, differences between batches of RDT and a general lower sensitivity for non-falciparum infections for nearly all brands.
The hidden reservoir: asymptomatic, low-density infections
In general, the limited sensitivity of both microscopy and RDT (unreliable detection in infections with a parasite density less than 100 parasites per microlitre) is not an issue for symptomatic malaria infections, the majority of which will consist of high parasite densities. However, asymptomatic infections are numerous, in high and low transmission settings. These asymptomatic infections pose a problem for control programmes. The carriers do not feel unwell so have no reason to present to a health facility for testing and yet, they may be infectious to mosquitoes, meaning they pose a risk for onward transmission. In order to detect and treat these asymptomatic infections, malaria programmes are now taking their diagnostics into the community in a strategy termed Mass Screening and Treatment (MSAT). This involves testing everyone within a community regardless of whether they have symptoms. Many of these infections are asymptomatic and therefore also likely to be low-density; hence which test you use can mean the difference between detecting 10 infections or 100 infections. Whilst RDT is ideal for field conditions, studies have shown that they can miss a large proportion of infections that are present [3].
Molecular tests
Polymerase chain reaction
More sensitive diagnostics are available in the terms of molecular tests. The most commonly used is polymerase chain reaction (PCR). Numerous PCR assays have been developed, many based on amplifying the 18S ribosomal RNA (18SrRNA), first published by Snounou and colleagues in 1993 [4]. PCR detects parasite nucleic acids and can detect much lower parasite densities than RDT or microscopy, with tests reportedly able to detect down to 1 parasite per microlitre of blood, as well as being able to accurately distinguish between plasmodium species. However, the number of assays available has resulted in calls for a standardized test so results can be compared across the world. PCR tests are generally performed on blood collected on filter paper but the equipment required for PCR and the expense of maintaining a sterile lab environment precludes PCR from being available in many health facilities. This means that samples need to be sent away, with an often long wait for results. Although more field-friendly PCR methods are in the pipeline, currently, PCR is not generally considered suitable for a point-of-care test, although it’s use in epidemiological studies is undisputed.
Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) was first developed in 2000, with the aim to amplify DNA in a sensitive, specific and speedy manner (Figs 1, 2). One of the main advantages is the fact it can be performed under isothermal conditions, and thus averting the need for a thermocycler. LAMP can be thought of as a ‘rough-and-ready’ PCR, as it is also less sensitive to inhibitors present in biological samples, and therefore allows the use of simple and cheap DNA extraction methods. The fast time-to-results and the minimal equipment required make LAMP an attractive option for field diagnosis. In order to make this a viable option, FIND and partners Eiken Chemical Ltd, Japan, and the Hospital for Tropical Diseases (HTD), London, UK have developed a field-stable kit with all reagents freeze-dried into the lid of the reaction tube, which means minimal processing is required. Although still in the development and testing stage, current results of the use of the kit are promising, with strong agreement with PCR results and a considerably higher sensitivity than RDT [5-8]. Whilst seemingly the most sensitive of the point-of-care tests available, there are some downsides to LAMP. Results still take considerably longer than RDT, requiring patients to wait at clinics for 2 hours for results, or leaving the health facility staff with the complicated task of contacting and following up any positive patients. In addition, electricity is required for the processing of samples, making it not practical for many places.
Future for point-of-care diagnostics for malaria
These advances in molecular diagnostics mean infections that would previously have remained undetected can now be confirmed, treated and cleared. Identifying and treating all infections becomes a greater priority as transmission reduces and the possibility of elimination comes into focus. This is occurring in areas around the world such as Swaziland and Zanzibar in Africa and in South East Asia, where the need to eliminate has become ever more important with the emergence of drug-resistant parasites. In these areas, identification of every last parasite is the aim and development of a quick, sensitive and reliable diagnostic is key to that.
As more studies reveal the extent of the low-density parasite reservoir, there is a sense of ‘the more we look the more malaria we will find’. But do we need to find all these infections in order to eliminate malaria? It should be noted that these ‘super-sensitive’ tests are a relatively recent phenomena and that countries have succeeded in malaria elimination without them. The role these low-density parasitemias play in transmission is not fully understood but for now the aim remains to clear the last parasite standing.
References
1. Houze S, Hubert V, Le Pessec G, Le Bras J, Clain J. Combined deletions of pfhrp2 and pfhrp3 genes result in Plasmodium falciparum malaria false-negative rapid diagnostic test. J Clin Microbiol. 2011; 49(7): 2694–2696.
2. WHO, FIND, CDC. Malaria rapid diagnostic test performance: Results of WHO product testing of malaria RDTs: Round 5. 2013; http://www.who.int/malaria/publications/atoz/9789241507554/en/.
3. Cook J, Xu W, Msellem M, Vonk M, Bergström B, Gosling R, Al-Mafazy AW, McElroy P, Molteni F, Abass AK, Garimo I, Ramsan M, Ali A, Mårtensson A, Björkman A. Mass screening and treatment on the basis of results of a plasmodium falciparum-specific rapid diagnostic test did not reduce malaria incidence in Zanzibar. J Infect Dis. 2015; 211(9): 1476–1483.
4. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993; 61(2): 315–320.
5. Hopkins H, González IJ, Polley SD, Angutoko P, Ategeka J, Asiimwe C, Agaba B, Kyabayinze DJ, Sutherland CJ, Perkins MD, Bell D. Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda. J Infect Dis. 2013; 208(4): 645–652.
6. Polley SD, González IJ, Mohamed D, Daly R, Bowers K, Watson J, Mewse E, Armstrong M, Gray C, Perkins MD, Bell D, Kanda H, Tomita N, Kubota Y, Mori Y, Chiodini PL, Sutherland CJ. Clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria. J Infect Dis. 2013; 208(4): 637–644.
7. Aydin-Schmidt B, Xu W, González IJ, Polley SD, Bell D, Shakely D, Msellem MI, Björkman A, Mårtensson A. Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias. PLoS One 2014; 9(8): e103905.
8. Cook J, Aydin-Schmidt B, González IJ, Bell D, Edlund E, Nassor MH, Msellem M, Ali A, Abass AK, Mårtensson A, Björkman A. Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar. Malar J. 2015; 14(1): 43.
The author
Jackie Cook PhD
London School of Hygiene and Tropical Medicine, London, UK
E-mail: Jackie.cook@lshtm.ac.uk
In spite of some exceptions, the clinical microbiology lab has been a late starter as far as automation is concerned. It has also traditionally been viewed as ‘low tech’, especially when compared to its cousins in clinical chemistry or pathology. A variety of factors, however, have been converging to reverse such a situation.
Automation hampered by process complexity
One of the most important barriers to the automation of a clinical microbiology lab is process complexity. Unlike hematology or chemistry labs, which have little diversity in specimens and generally use standard collection tubes, microbiology laboratories need to work with a vast range of specimen types in a multitude of transport containers. The complex nature of specimen processing and culturing and the ensuing lack of standardization have been major deterrents to automation.
Nevertheless, growth in the presence of automated technologies in clinical microbiology labs is now expected to accelerate as a result of several factors, above all rising demand. This requires agility and high responsiveness, making automation indispensable.
Ageing populations drive demand
Ageing populations with more-complex diseases and conditions require a growing number of tests – for example, to monitor implants and prosthetic devices for infections. The elderly also need greater care in medicating, since they are more prone to adverse drug events.
In the year 2000, an article by Dr. Thomas T. Yoshikawa of the King-Drew Medical Center in Los Angeles noted that though “the major focus in infectious diseases for the past decade has been on young adults”, in the future “the vast majority of serious infectious diseases will be seen in the elderly population.”
Infectious disease, resistant bacteria
The rise in infectious disease outbreaks in recent decades is another factor driving demand for early detection by clinical microbiology labs, to contain their spread.
On their part, multidrug-resistant pathogens pose their own specific challenges. Delays in obtaining lab results leads to over-treatment of many patients – and increased antibiotic resistance. In 2008, a team from Erasmus University Medical Centre in Rotterdam found that quicker microbiological lab turnaround led to “a significant reduction in antibiotic use” in a study of almost 1,500 patients. This finding assumes considerable significance when one takes account of the fact that 5 years later, another study found that just 3% of community-acquired respiratory infections in the UK were guided by laboratory results.
The role of budgets and cutbacks
In an era of budgetary cutbacks, financial considerations too have reinforced demands for the automation of clinical microbiology labs. There is some irony here. Given the nature of a hospital business, it has been easier for administrators to assess the productivity of their clinical laboratories, determine return on investment (RoI) and justify new outlays – via quantifying and benchmark tests and staff numbers. Such an exercise has, in general, already been conducted for other hospital labs. It is now the clinical microbiology laboratory’s turn.
The above considerations are summed up in an article in the December 2013 issue of the journal ‘Clinical Chemistry’ which quotes Gilbert Greub of the Institute of Microbiology at the University Hospital in Lausanne, Switzerland. He says that the key reasons for the Hospital’s decision to move toward a fully automated laboratory consisted of a shortage of financial resources and the concomitant increase in activity of the Hospital’s clinical diagnostic microbiology laboratory “of about 4% to 12% per year.”
Workflow improvement, staff shortages
Indeed, improvements in workflow and quicker test results are also directly related to growing automation. One of the most important collateral effects of this is the freeing up of staff for other work.
In May 2009, the ‘Wall Street Journal’ warned about “the shrinking ranks of skilled lab workers” in the US, which pose “a potential threat to the safety and quality of health care”. Hospitals, it continued, said that “it can take as much as a year to fill some job openings,” while an American Society for Clinical Pathology (ASCP) survey found average job-vacancy rates topping 50% in some states.
The ASCP survey also illustrated another interesting fact. Laboratories which were affected by new technologies found a decreased need for as large a staff. However, 75% of respondents said they were not affected by new technologies. In other words, not only does automation seem to be an answer to staff shortages. There is also a lot of untapped room for growth.
Europe faces staff shortages too. A report by Belgium’s University Hospital at Leuven highlights the challenges of an ageing workforce, alongside major waves of retirement which have started recently and are expected to continue for several years. The problem is exacerbated by a decline in interest in labs as a career and the presence in the workforce of fewer young recruits. As a result, the paper warns, there is a “trend towards employing less-trained technicians.”
Transferring skills to points of need
The benefit of automation in the face of labour shortages is to utilize the skills of medical laboratory professionals where they are most needed and to automate tasks that are repetitive and do not require the comprehensive skill set of a trained professional.
For example, a laboratory could use an automated system for mundane and repetitive tasks such as “planting and streaking of urine samples and other liquid specimens,” while assigning a lab technician “to perform Gram stain review and processing of more-complex specimens, such as tissue.”
At the other end, boredom can also be a problem. In a non-automated environment, lab staff frequently complain of poor turnaround (TAT), referring to the duration or idling time between inoculation of media and microbial growth. By shifting monotonous tasks to automation, while assigning higher-skill tasks to a technologist, the laboratory reduces boredom and increases productivity.
The May 2009 article by the ‘Wall Street Journal’ quotes Dr. Carol Wells, director of the clinical laboratory sciences programme at the University of Minnesota in Minneapolis: “Many tests are automated, but that doesn’t mean a lab monkey can do them.” The machines, she continues, need careful monitoring. Should they “spit out a result” which does not make sense, only a skilled lab technician can catch a possible discrepancy and determine what is wrong.
Liquid-based microbiology, MALDI-TOF mass spectrometry drive demand
Automation of the clinical microbiology lab is also being driven by supply-side factors.
Among the first is the advent of new technologies, such as liquid-based microbiology and mass spectrometry. Liquid-based microbiology allows specimens of varying viscosities (e.g. stool or sputum) to be homogenized into a liquid phase, in order to enable greater consistency in the inoculation of medium. Specimen elution from recent flocked-style swabs into liquid phase has also resulted in a significant increase in the release of viable organisms from the swab, in other words resulting in greater sensitivity for detection of microorganisms.
The second technology is matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. This permits the accurate and rapid identification of microorganisms isolated from clinical specimens. MALDI-TOF procedures “are highly amenable to automation because they are relatively simple, do not change based on organism, and are reproducible.” In addition, target plate spotting and extraction of proteins “can be standardized for most organisms, and when combined with automation, automated crude extraction using the on-plate formic acid extraction method can be performed with minimal staffing.”
Specimen processing as entry point
One of the first points of entry by new technology in a clinical microbiology lab consists of front-end instrumentation to automate and standardize initial specimen processing. Automation of the front-end makes it possible for tests to be conducted as soon as specimens arrive, obviating the need for a separate ‘stat’ lab.
Nevertheless, the impact of automation in specimen processing is not necessarily uniform, and depends on the needs of a particular laboratory. One study by researchers at Penn State College of Medicine and Medical College of Wisconsin estimated break-even point of more than 4 years for a barcode-driven, conveyor-connected automated specimen processing system which included plating and streaking, incubation, image acquisition, and digital microbiology.
Full- versus partial automation
As with other types of clinical labs, automation of the clinical microbiology laboratory can be classified into total (or full) laboratory automation or modular automation. Most automation systems have traditionally been designed for the larger, high-volume laboratory with substantial specimen throughput requirements. More recently, automated processing units have been designed for the pre-analytical section of smaller/medium-sized laboratories.
In effect, smaller labs can choose to automate only some of the processing steps. The investment required for automation makes such a choice imperative. The report by the University Hospital at Leuven cited previously notes that its own implementation of ‘full lab automation’ cost €3.2 million with €1.7 million in capital investment and another €1.5 million for refurbishment. Time needs to be also factored in. The Leuven study notes that their automation entailed 1 year in preparatory work and 1 year for adaptation. Against this, the savings achieved were equivalent to 3.8 full time employees (FTE).
Some hospitals begin small and scale up. For example, the University Hospital in Lausanne, Switzerland, started with two stand-alone automated systems for microbial identification. It moved after a few years “to a fully automated laboratory, by adding the missing pieces to the puzzle, i.e., smart incubators, high-quality digital imaging, an automated colony-picking system, and all required transport belts in between.”
A glimpse of the future
Today, state-of-the-art clinical microbiology labs have the potential to automate nearly all areas of testing, including inoculation of primary culture plates, detection of growth on culture media, identification of microorganisms, susceptibility testing, and extraction and detection of nucleic acids in clinical samples. In the future, process standardization is expected to be reinforced by high-resolution digital imaging and robotics, and take automation in the clinical microbiology lab to wholly new frontiers.
The aim of the first annual World Antibiotic Awareness Week, held in November, was to raise recognition of the growing problem of bacterial resistance to antimicrobials and to disseminate information on how these drugs can be used more prudently. Is it still possible, though, to prevent an antibiotic apocalypse?
The development of drug-resistant bacteria is the inevitable result of natural selection, but formerly the discovery of novel compounds kept pace with microbial evolution; this is no longer the case. During the past decade numerous academic articles have reported alarming examples of antibiotic resistance in microorganisms, including multidrug-resistant Staphylococcus aureus and Mycobacterium tuberculosis, as well as extensively drug-resistant tuberculosis, and the mass media has duly disseminated this information to the general public. Around 5 years ago the NDM-1 gene, which confers resistance to the potent carbapenem antibiotics used against multi-resistant strains of Gram-negative bacilli, was found in Enterobacteriaceae including the ubiquitous Escherichia coli. The latest catastrophe is the emergence of the MCR-1 mechanism that allows polymixin-resistance plasmids to be transferred between strains of Enterobacteriaceae. And polymixins are (or should be) the drugs of last resort to treat infections with bacteria that are multidrug resistant, including carbapenem-resistant strains.
As well as over-liberal medical prescription of unnecessary antibiotics without prior diagnostic testing, premature cessation of treatment and unregulated sources of drugs enabling “self-prescription”, the routine use of antimicrobials in industrialized agriculture has greatly exacerbated the resistance problem. The recently reported polymixin resistance was first observed in China during routine testing of commensal E. coli in food animals, prompting a robust study that discovered the MCR-1 mechanism in 15% of E. coli isolates from raw meat, 21% of isolates from livestock and 1% of isolates from infected patients. Although the problem is currently confined to China, this type of mechanism spreads resistance so easily between bacteria that it will soon become a global problem. Should polymixin-resistance plasmids be transferred to Enterobacteriaceae that are already multidrug-resistant, truly untreatable Gram-negative bacterial infections would result.
Initiatives to prevent the further squandering of antibiotics coupled with rigorous infection control procedures are highly unlikely to prevent an antibiotic apocalypse now. But a worldwide ban on the veterinary use of medical antimicrobials might just stem the tide until new drugs (such as teixobactin for multidrug-resistant Gram-positive pathogens) have been approved.
November 2025
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