It was over sixty years ago that Sir Richard Doll’s pioneering work first demonstrated a causal link between tobacco smoking and an increased risk of lung cancer. The lessons drawn from it have undoubtedly saved millions of lives over the years, but it is disappointing that according to the recently published European cancer mortality predictions for 2013, lung cancer remains the biggest cause of cancer death in male EU residents, and  is predicted to become the biggest cause of cancer mortality in women in the near future, overtaking deaths from breast cancer.
The trend is similar in the US. A recently published paper in the New England Medical Journal, which involved data from more than two million women at three different time periods, showed that women who smoke currently are at a far greater risk of death from lung cancer than were women who smoked in the 1960s and the 1980s; the risk is now equal for both genders. While other factors that increase the risk of lung cancer, such as asbestos and radon gas exposure, have now been identified, tobacco smoke is still thought to be responsible for around 90% of lung cancer cases.
During the decades since Doll’s work it has, of course, been demonstrated that the risk of death from many other diseases, including other cancers, ischemic heart disease, stroke, chronic obstructive pulmonary disease and asthma, is augmented by smoking tobacco. More recently it has been recognized that passive smoking can also increase the risk of smoking-related diseases, and that prenatal exposure to tobacco smoke increases the risk of low birth weight and premature neonates, as well as SIDS and asthma in infancy. But in spite of the concerted efforts that have been made to educate the public about the dangers of tobacco smoke over more than half a century, a substantial minority of the population, including many physicians, still smokes.
Now for the good news. Several comparative studies indicate that public smoking bans now operating in much of the developed world are already affecting the rate of cardiovascular and respiratory disease. And a very recent robust study from Belgium, giving data from the three phases of the ban in that country, where smoking was first prohibited in the workplace (2006), then in restaurants (2007) and finally in bars serving food (2010), demonstrates a fall in the premature birth rate after each phase. So finally at least those of us who have heeded the oft-repeated health message may benefit fully from our prudence!

Clinical decisions need to be made at the earliest possible time to facilitate the administration of quick and accurate treatment plans. Point of care testing (POCT) enables tests to be convenient and fast, making them suitable for use with a broad range of patients, including diabetics. Glycated hemoglobin (HbA1c) is commonly tested in diabetics as it provides a reliable measure of glycemic control (Figure 1). However, the role of HbA1c in the diagnosis of diabetes has only more recently been documented. HbA1c levels reflect average circulating glucose levels over the lifespan of red blood cells (2-3 months). Once hemoglobin molecules have been glycated, they become highly stable, enabling a greater level of clinical information to be obtained from them than a single glucose measurement taken at a particular point in time.

by Gavin Jones, Diabetes Product Manager, EKF Diagnostics

By taking serial HbA1c measurements, an individual’s control over their glucose levels can be assessed in response to changes in management strategies. Measurements should be taken every 2-6 months with target HbA1c levels set individually and therapy adjusted accordingly to provide the most effective treatment (1). The target ranges of HbA1c for diabetic patients, depending on their risk of severe hypoglycemia, cardiovascular status and co-morbidities, should be set between 6.5 – 7.5% DCCT (48 – 58 mmol/mol), with the non-diabetic reference range being 4.0 – 6.0% DCCT (20 – 42 mmol/mol). One point for consideration is that HbA1c results may be affected by any condition that leads to a change in red blood cell survival. But even then, HbA1c can be used to detect trends in a patient’s glycemic control.

HbA1c in POCT-based diabetes monitoring
HbA1c determination was originally based on methods such as ion exchange and affinity chromatography with alternative affinity and immunological methods following later, taking HbA1c into the POC environment.

Typically, when using laboratory-based testing, patients with existing diabetes are monitored for HbA1c every 2-6 months, requiring a visit to a nurse or phlebotomist and a follow-up appointment 1 to 2 weeks later to discuss the results. Use of POCT would mean that after just one visit, patients can leave with their results, eliminating the need for a follow-up appointment. By enabling an earlier therapeutic decision, diabetes control can be improved whilst also providing economic benefits in terms of cost and time.

Diabetes diagnosis
The benefits of HbA1c in the management of diabetes can also be directly applied to the diagnosis of diabetes. Unlike glucose levels, which are affected by what has been eaten and drunk in the previous 2-3 hours, the measurement of HbA1c levels does not require fasting. As a simple and immediate test for diabetes, POC HbA1c can support the early identification of at-risk individuals. This would rapidly enable them to make small changes to their lifestyle to significantly reduce the risk of developing type 2 diabetes.

Patients diagnosed with diabetes who are able to maintain low blood HbA1c levels also have a significantly reduced chance of complications after diagnosis (2); early detection by POCT can reduce this risk even further. The ability to rapidly assess and change these risk outcomes has significant health benefits and reduces the costs associated with recurrent leg ulcers, blindness, heart disease and stroke, for example, all of which are conditions and complications commonly associated with type 2 diabetes.

The World Health Organization (WHO) has recommended the use of HbA1c for the diagnosis of diabetes (3). In the UK for example, the National Institute for Clinical Excellence (NICE) has published guidelines for diabetes prevention which aim to identify people at high risk of type 2 diabetes and offer cost-effective, appropriate interventions to prevent or delay onset (4). Used in conjunction with a lifestyle health risk assessment, these guidelines advocate the monitoring of HbA1c levels to allow healthcare providers to  advise individuals on treatment regimens, depending on their classification as low, moderate or high risk. Current guidance, therefore, supports the use of HbA1c in screening for type 2 diabetes, and in the management of patients with diabetes. The use of POCT could improve the management of patients with established diabetes in both primary and secondary care settings and enable earlier type 2 diabetes diagnosis.

What to look for in a POC HbA1c analyser
Most POC HbA1c analysers use a single drop of blood (4-10 µL), which is applied to a reagent cartridge. The cartridge is then directly inserted into a desktop device for analysis. Time to results is generally between 3 to 10 minutes, depending on the analyzer. This quick turn-around time, in combination with simple operation, is key to maintaining effective POC testing.

Simplicity
To minimize human error and the subsequent need for repeat testing, a POC analyser should be as easy as possible to use. Also the analyser should be highly intuitive, requiring little user training. Features that support ease-of-use include ready-to-use reagent cartridges which can be inserted straight into the analyser. The blood sample can then be added directly, without the need for premixing or pipetting. Minimizing the number of steps in the procedure not only reduces the potential for user error, but also helps to standardize results by eliminating variation from different users.

Audit trails
For patient safety purposes, audit trails must be readily available. Use of barcode scanning for patient and user identification, as well as confirmation of the batch of reagents and controls used, ensures an analyser can provide such information in a timely manner. Two levels of quality controls that are recorded and held within the analyser’s memory are also ideal for auditing purposes.

Certification
Certification of the analyser in order to confirm delivery of accurate, standardized results should also be a key consideration. In an effort to standardize HbA1c results, the AACC set up the ’National Glycohemoglobin Standardization Program’ (NGSP) in 1996. In parallel, the International Federation of Clinical Chemistry (IFCC) developed reference methods for glycated hemoglobin. In 2006 and 2007, an international consensus between IFCC and AACC was agreed upon (5).

The calibration and certification of laboratories and manufacturers to the same standards has improved the conformity of results. However, in practice, differences can still be observed among technologies and between individual systems. These observed differences arise because of heterogeneity of hemoglobins, underlying differences in technologies (e.g. ion exchange, boronate affinity, immunoassay), calibration drifts or lot to lot variability. Providing the manufacturer follows the recommendations of the IFCC and NGSP to ensure instruments and reagents are accurately aligned and traceable to the reference method, this should not be a problem.

Methodology
There are POC HbA1c analysers available (e.g., the Quo-Lab, EKF Diagnostics, Cardiff, UK) (Figure 2) where results are not affected by hemoglobin variants (which do not result in reduced erythrocyte life span), labile glycated hemoglobin or hematocrit levels. Such analysers use Boronate Fluorescence Quenching Technology (BFQT) (6) (Figure 3) which is associated with simple, yet powerful multiple optical measurements. This is based on well-documented boronate affinity chromatography systems used in reference laboratories. However, as BFQT does not require chromatographic separation, the methodology allows for fast, simple and accurate POC measurement of HbA1c to deliver comparable results to chromatography-based techniques.

Summary
Type 2 diabetes can be managed easily and effectively through the monitoring of HbA1c levels, as opposed to blood glucose. POC diagnosis enables early detection in higher risk patients, before any additional complications arise. POCT therefore not only improves patient access to testing, but provides accurate diagnoses there and then. Treatment strategies can be determined immediately, eliminating the need for a follow-up visit to discuss the results. The ability for diagnosis to occur near to the patient provides greater convenience, thus increasing the likelihood of compliance.

When selecting an analyser for use at the POC, users need to bear in mind that it needs to be a convenient and appropriate option. The focus should be on meeting regulatory requirements, as well as ease of use in order to ensure rapid testing, with accurate, standardized resulting data.

References:
1. Diabetes UK. HbA1c Standardization: Information for Clinical Healthcare Professionals. 2009. http://www.diabetes.org.uk/Guide-to-diabetes/Monitoring/Blood_glucose/Glycated_haemoglobin_HbA1c_and_fructosamine/HbA1c_Standardisation_Information_for_Clinical_Healthcare_Professional.
2. Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977-86.
3. World Health Organization. Use of glycated hemoglobin (HbA1c) in the diagnosis of diabetes mellitus. 2011. www.who.int/diabetes/publications/report-hba1c_2011.pdf.
4. National Institute for Health and Clinical Excellence. Preventing type 2 diabetes: risk identification and interventions for individuals at high risk. 2012. www.nice.org.uk/nicemedia/live/13791/59951/59951.pdf.
5. Geistanger A, Arends S, Berding C, Hoshino T, Jeppsson JO, Little R, Siebelder C, Weykamp C; on behalf of the IFCC Working Group on Standardization of Hemoglobin A1c. Statistical Methods for Monitoring the Relationship between the IFCC Reference Measurement Procedure for Hemoglobin A1c and the Designated Comparison Methods in the United States, Japan, and Sweden. Clin Chem. 2008 Aug;54(8):1379-85.
6. Wilson DH, Bogacz JP, Forsythe CM, Turk PJ, Lane TL, Gates RC and Brandt DR. Fully automated assay of glycohemoglobin with the Abbott IMx analyzer: novel approaches for separation and detection. Clinical Chemistry October 1993 vol. 39 no. 10 2090-2097

Some species of non-diphtheriae Corynebacterium bacteria are opportunistic pathogens responsible for lower respiratory tract infections primarily in immunocompromised patients or in patients with chronic respiratory diseases. In the last years an increasing number of reports have demonstrated their role as emerging pathogens causing pneumonia or exacerbations of chronic pulmonary diseases. Thus, these species should not always be considered as mere colonizers.

by Dr M. Díez-Aguilar, Dr R. Cantón, Dr M. A. Meseguer

Non-diphtheriae Corynebacterium species are considered to be colonizers of the skin, nasopharyngeal tract and mucous membranes. However, in the last decade there have been an increasing number of reports that recognize these microorganisms as opportunistic pathogens that can cause disease in certain circumstances [1–3]. Since the population of immunocompromised patients is constantly growing, due to AIDS, age, use of invasive devices and immunosuppressive regimens, e.g. after transplantation, the clinical relevance of these opportunistic pathogens is rising.

A broad range of infectious diseases caused by non-diphtheriae Corynebacterium species have been reported including endocarditis, bacteriemia, pneumonia, tracheobronchitis, necrotizing tracheitis, exudative pharyngitis, rhinosinusitis, osteitis, conjunctivitis, and skin and urinary tract infections.

Lower respiratory tract infection, typically occurs in the context of underlying immunosuppressive conditions (such as diabetes, malignancy, corticoid therapy) and in patients with pre-existing pulmonary diseases such as chronic obstructive pulmonary disease (COPD), bronchiectasis and cystic fibrosis. In these patients non-diphtheriae Corynebacterium species can cause pneumonia and acute exacerbations of COPD. Previous hospitalization, wide-ranged antibiotic therapy and presence of multiple medical devices are risk factors for acquiring non-diphtheriae corynebacterial infection. Nosocomial outbreak of infection or colonization has been also observed [4]. Nevertheless, community acquired bronchitis in elderly patients with COPD have been reported.

Typically, Corynebacterium pseudodiphtheriticum, Corynebacterium striatum, and Corynebacterium propinquum are the species more frequently involved in lower respiratory tract infections [1–4]. The role of other Corynebacterium species in lower respiratory tract infections could have been underestimated, as only a few cases have been reported. The various non-diphtheriae Corynebacterium species that have been involved as responsible for respiratory tract infections are shown in Table 1. After appropriate antibiotic treatment a favourable outcome was achieved in most patients.

Pathogenesis
The respiratory tract damage caused by these microorganisms is probably the result of their opportunistic overgrowth and their possible virulence factors in patients with immune impairment and/or compromised pulmonary function.

Patients with chronic respiratory infections, such as obstructive pulmonary disease and bronchiectasis are predisposed to a persistent and non-innocent colonization of the lower respiratory tract by several non-pathogenic microorganisms. The high density of microorganisms covering the surface of the bronchial mucosa results in consistent pathogenic effects throughout the respiratory epithelium. Such effects include reduction of the supply of oxygen, water and organic nutrients to cells of the bronchial epithelium, as well as the liberation of potentially bioactive molecules which induce pro-inflammatory processes leading to accumulation of immune inflammatory cells. Defective pulmonary defences (impaired mucociliary clearance, airway inflammation and permanent dilatation within the bronchial wall), periodic infectious exacerbations caused by other respiratory infecting pathogens, and local immune disorders can cause a ‘vicious cycle’ of infection and inflammation of the airway. In these conditions the replacement of the pharyngeal resident microbiota with the opportunistic overgrowth and predominance of corynebacterial organisms in the respiratory tract can take place resulting in disease.

However, these microorganisms could express virulence factors that would contribute to the infection. Still, the virulence factors of non-diphtheriae Corynebacterium infection remain poorly understood, but recent in vitro studies on  Corynebacterium pseudodiphtheriticum behaviour with epithelial cells have demonstrated the capacity for adherence, internalization, intracellular survival and persistence of the organism [5]. Therefore, in vivo C. pseudodiphtheriticum not only multiplies at and remains on the surface of the epithelial host cells, but also could reach the cytoplasm. This ability of C. pseudodiphtheriticum to survive within host cells highlights the potential capacity of other non-diphtheriae Corynebacterium to act as opportunistic pathogens.

Microbiological diagnosis
The key for the microbiological diagnosis of respiratory tract infection caused by non-diphtheriae Corynebacterium species is the microscopic observation of the predominant presence of Corynebacterium morphotype in a Gram stained purulent respiratory sample [Fig. 1], together with an abundant growth in the culture [6]. To determine the quality of the sputum it is important to follow the scoring system of Washington and Murray, which assesses a good quality of samples when there are more than 25 leukocytes and less than 10 squamous epithelial cells per field.

Identification of Coryneform bacteria
It is important to correctly identify Coryneform bacteria to the species level in order to reach the microbiological diagnosis, but also to detect unsuspected species, investigate potential pathogenicity and describe new species that could be clinically relevant.

Phenotypic characteristics such as colony size, pigmentation, catalase, and motility are useful for establishing the genus. For identification to the species level, biochemical testing performed using commercially available identification systems such as API Coryne, API CH50 plus, API 20 E and Rap IDCB Plus method, as well as automated systems such as Vitek2 (bioMèriux) and Biology systems could be employed. However, these methods are unreliable for some species (Corynebacterium accolens, C. striatum).

Nowadays an accurate and definitive identification is reached by the use of sequence-based identification techniques: 16s RNA and rpoB gene are the two approaches used for the characterization of non-diphtheriae Corynebacterium species. In fact, in recent years, many new species of the Corynebacterium genus have been described thanks to molecular biology techniques [7]. The use of mass spectrometry technology like MALDI-TOF MS is acquiring an increasingly important role in identifying and detecting these microorganisms [8]. This technology requires neither extensive training nor cost and it has been reported that it provides identification to genus and species level with an accuracy that approaches that of genetic methods.

Antimicrobial susceptibility
It is essential to test the antimicrobial susceptibility in all clinically relevant isolates due to the variable susceptibility of these microorganisms. Overall, non-diphtheriae Corynebacterium species are constitutively resistant to macrolides, lincosamides and type B streptogramins; susceptible to cefotaxime, amoxicillin/clavulanate, rifampin, and vancomycin (the recommended drug to treat severe infections) and have variable susceptibility to other antibiotics. C. striatum is the species which exhibits the highest resistance pattern.

According to CLSI (Clinical and laboratory Standard Institute) guidelines the reference method is the broth microdilution technique. This committee provides interpretive criteria for penicillin and erythromycin based on minimum inhibitory concentration (MIC) values following testing by this method, and for cephalosporin and linezolid the criteria are currently adapted from those from Streptoccocus and Enteroccocus, respectively, and remaining criteria are adapted from those from Staphyloccocus.

Although some laboratories use the disc diffusion method for susceptibility testing, the interpretative categories for zone diameters need to be established. The diffusion gradient tests (i.e. Etest) showed a good correlation of MICs with the broth microdilution method.

Conclusion and future perspectives
It is clear that due to the increasing number of immunocompromised patients and those with pre-existing pulmonary diseases, non-diphtheriae Corynebacterium species should be considered as an emerging cause of lower respiratory tract infection. A rapid and accurate laboratory detection, identification and assessment of these opportunistic microorganisms are critical for the correct diagnosis, taking into consideration that some of them are resistant to multiple antibiotics. Although more studies are need to enhance the understanding of the clinical significance of these microorganisms, clinicians should be aware of the potential pathogenic role of these species in the context of immunosuppression or chronic respiratory disease and they should not be always considered as mere colonizers.

References
1. Díez-Aguilar M, Ruiz-Garbajosa P, Fernández-Olmos A, Guisado P, Del Campo R, Quereda C, Cantón R, Meseguer MA. Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen. Eur J Clin Microbiol Infect Dis 2012; doi: 10.1007/s10096-012-1805-5.
2. Nhan TX, Parienti JJ, Badiou G, Leclercq R, Cattoir V. Microbiological investigation and clinical significance of Corynebacterium spp. in respiratory specimens. Diagn Microbiol Infect Dis 2012; 74(3): 236–241.
3. Otsuka Y, Ohkusu K, Kawamura Y, Baba S, Ezaki T, Kimura S. Emergence of multidrug-resistant Corynebacterium striatum as a nosocomial pathogen in long-term hospitalized patients with underlying diseases. Diagn Microbiol Infect Dis 2006; 54(2): 109–114.
4. Renom F, Garau M, Rubí M, Ramis F, Galmés A, Soriano JB. Nosocomial outbreak of Corynebacterium striatum infection in patients with chronic obstructive pulmonary disease. J Clin Microbiol 2007; 45(6): 2064–2067.
5. Souza MC, Santos LS, Gomes DL, Sabbadini PS, Santos CS, Camello TC, Asad LM, Rosa AC, Nagao PE, Hirata Júnior R, Guaraldi AL. Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells. Mem Inst Oswaldo Cruz 2012;107(4): 486–93.
6. Funke G, von Graevenitz A, Clarridge JE 3rd, Bernard KA. Clinical microbiology of coryneform bacteria.Clin Microbiol Rev 1997; 10(1): 125–159.
7. Bernard K. The genus corynebacterium and other medically relevant coryneform-like bacteria. J Clin Microbiol. 2012; 50(10): 3152–3158.
8. Gomila M, Renom F, Gallegos Mdel C, Garau M, Guerrero D, Soriano JB, Lalucat J. Identification and diversity of multiresistant Corynebacterium striatum clinical isolates by MALDI-TOF mass spectrometry and by a multigene sequencing approach. BMC Microbiol 2012;12: 52.

The authors
María Díez-Aguilar* MD; Rafael Cantón MD, PhD; and María Antonia Meseguer MD, PhD

Department of Clinical Microbiology, Ramón y Cajal University Hospital, Madrid, Spain

*Corresponding author
E-mail: maria_diez_aguilar@hotmail.com