Page 255 – Clinical Laboratory int.

Diagnostic testing for Clostridium difficile: where to go from here?

, 26 August 2020/in Featured Articles /by 3wmedia

Clostridium difficile causes serious life-threatening infections but this organism has a complex pathogenesis that makes differentiating true infection from asymptomatic carriage difficult. There are a number of diagnostic testing approaches that can be used alone or in multi-step algorithms. This review discusses the impact that the type of diagnostic test has on interpretation of clinically significant infection, initiation of treatment of C. difficile infection, and how future diagnostic testing may need to differentiate asymptomatic carriage from clinically significant disease.

by Dr Michelle J. Alfa

Clostridium difficile pathogenesis
Since the initial report by Bartlett et al. in 1978 [1] that C. difficile could cause infectious diarrhoea in patients who were treated with antibiotics (in particular clindamycin), there have been significant changes in the understanding of the pathogenesis of this organism as well as the approach to the diagnosis of the illness it causes. Initially, C. difficile was thought to be solely a hospital-acquired infection associated with a history of antibiotic consumption. Subsequently it became clear that humans can have toxigenic C. difficile present asymptomatically in their gastrointestinal tract and, unlike other enteric pathogens, the concept of ‘infectious dose’ does not really apply to this gastrointestinal pathogen. C. difficile infection (CDI) is a ‘two-hit’ process (Fig. 1). The first hit is ingestion of the metabolically inactive spore form which does not produce toxin, and the second hit is an imbalance of the gut microbiome (most often due to antibiotic therapy that eradicates gut normal flora without killing the spore or vegetative form of C. difficile). These two hits allow the ingested spore to germinate in the gut to the vegetative form which then replicates and produces Toxin A (enterotoxin) and Toxin B (cytotoxin). These toxins work synergistically to cause mucosal inflammation in the colon and diarrhoea (the small intestine is not damaged). Although the toxins do not appear to spread systemically, there is evidence that humoral antibodies against C. difficile Toxin A and B are protective.

In addition to exposure to spores in the healthcare environment or on the hands of caregivers, recent evidence implicates food products (beef, pork, fowl) as a source of C. difficile spores. This food reservoir may be the basis of community-acquired CDI (CA-CDI) as it is now recognized that up to 30% of all CDIs are acquired outside of healthcare facilities and, as discussed by Humphries et al. [2], patients with CA-CDI are more likely to have mild disease, shorter hospital stay and lower rates of mortality. Unlike other enteric vegetative bacterial pathogens in food products that are killed by adequate cooking, the spore form of C. difficile is not killed by cooking [3]. Consumption of C. difficile spores via food or iatrogenic exposure does not automatically lead to disease. Indeed up to 10–20% of healthy people and up to 70% of healthy neonates may harbour this toxigenic C. difficile in their gut but be asymptomatic [3, 4]. It is unknown if this represents transient passage of the ingested spores in the gut where the microbiome keeps C. difficile spores from germinating and replicating thereby preventing toxin production, or whether there can be asymptomatic colonization by toxigenic C. difficile at such low levels that there is no mucosal damage or diarrhoea. Guerrero et al. [5] reported that 12% of asymptomatic patients screened carried toxigenic C. difficile. Although the skin levels and environmental shedding from asymptomatic carriers was lower than from patients with CDI, it has been suggested [5] that asymptomatic carriers may still represent a significant reservoir for transmission within healthcare facilities.

The unique characteristics of C. difficile that include spore formation, asymptomatic carriage and ‘two-hit’ pathogenesis present challenges in terms of optimizing and interpreting diagnostic tests.

Diagnostic testing for toxigenic C. difficile
Over the past 20 years there has been a dramatic revolution in the approach to diagnostic testing for toxigenic C. difficile. Initially in the 1970s the diagnosis of C. difficile-associated diarrhoea was made by culture and subsequent testing of C. difficile isolates to determine if they were toxigenic or not [4, 6]. This was replaced by the cytopathic effect (CPE) assay in the late 1970s and early 1980s that detected biologically active Toxin B directly from the stool sample. Some still consider the CPE assay to be the most clinically relevant diagnostic test as it demonstrates there is sufficient biologically active toxin in stool to cause mucosal damage and diarrhoea. Because culture and CPE assays were labour intensive, costly, time consuming and required specialized expertise, antigen detection assays became the diagnostic test of choice early in the 1990s [4]. However, recent studies have documented that enzyme immunoassay (EIA) for Toxin A and B alone is insensitive and should not be used as a sole diagnostic test for CDI [2, 4, 6–10]. Some researchers advocate that toxigenic culture is the most sensitive diagnostic test [9]. Isolates must subsequently be tested to confirm they are toxigenic. Because toxigenic culture is too slow for clinical testing, multi-step algorithms using glutamate dehydrogenase (GD) antigen as a screen followed by CPE or nucleic acid amplification tests (NAATs) (Table 1) have been recommended [4, 6]. Within the past 5 years there has been a push towards using NAAT alone as the most rapid and sensitive diagnostic test for toxigenic C. difficile [4, 6, 8].

Longtin et al. [7] have recommended that diagnostic testing for C. difficile should be standardized because reportable rates of CDI are dramatically affected by the diagnostic test method or test algorithm utilized. They undertook a one year prospective study and reported that using NAAT alone instead of a multi-step algorithm based on GD antigen, Toxin A/B antigen and CPE assay resulted in a greater than 50% increase in CDI rate in their facility (8.9 cases by NAAT versus 5.8 cases by multi-step algorithm per 10,000 patient days). Their study was the first to report that for patients who were test positive by NAAT alone there was a 3% complication rate compared to the 39% complication rate for patients who were positive by both NAAT and their multi-step algorithm. The lack of standardization in diagnostic testing means the incidence rates reported will vary depending on the test method(s) used. The resultant increase in CDI incidence using NAAT tests compared to other testing algorithms has implications including; Medicare reimbursement penalties in the USA, financial penalties for increased CDI rates in England, target rates in Quebec, Canada.

Additional research is needed to clarify the clinical significance of NAAT positive tests when CPE and antigen tests are negative. As suggested by a variety of published reports [6–8, 10, 11], it may be wrong to assume that higher sensitivity makes for a better C. difficile diagnostic test. Leslie et al. [12] reported that quantitation of C. difficile copy number is reliable and they suggested this added information may help determine when therapy is warranted for NAAT positive tests. They reported that 30.6% of stools that were only positive by NAAT, and had no toxin detected by CPE or antigen testing had low C. difficile copy number/ml. Their data suggests that a large portion of NAAT positive samples fall into this category of ‘questionable’ clinical significance. Vancomycin treatment of asymptomatic C. difficile carriers has been shown to itself stimulate CDI and indeed the authors warned against antibiotic treatment for asymptomatic C. difficile carriers. It may be that detection by NAAT of low organism load represents spores (i.e. no toxin present) or may represent vegetative levels that do not require antibiotic therapy. Dionne et al. [10] reported good correlation between low levels of viable C. difficile and test positivity by NAAT only (i.e. negative by CPE and antigen detection). Furthermore, they were able to demonstrate a correlation between PCR cycle time (CT) and the level of viable C. difficile in the stool sample (Table 2).

Although many published manuscripts and reviews list sensitivity and specificity values, these are all dependent upon what is used as the ‘gold standard’. For C. difficile this is not a simplistic issue. It is clear that detection of toxigenic C. difficile by culture does not always indicate clinically significant disease.

Conclusions
As summarized in Table 3, there are a number of unresolved issues relating to diagnostic testing for C. difficile. For asymptomatic carriers of C. difficile who do not have diarrhoea, the concept of NAAT admission screening and contact precautions for those who test positive has yet to be determined to be beneficial in preventing the spread of CDI. For those patients with diarrhoea it is apparent that CDI rates will vary dramatically depending on the testing algorithm used. It is clear that NAAT used alone as a sole diagnostic test will overestimate CDI rates and could lead to unnecessary antibiotic therapy. The impact is substantive as about one-third of all NAAT positive results fall into this ‘grey area’ of doubtful clinical relevance (i.e. have no detectable toxin in the stool sample and/or are toxigenic culture negative).

In conclusion, a combination NAAT test that provides a quantitative assessment of the load of C. difficile in stool along with detection of C. difficile toxin genes appears to be the ideal combination of data in order to reliably determine which patients have clinically significant CDI and require treatment. However, prospective studies that assess clinical outcome based on this quantitative NAAT testing are needed to confirm that this diagnostic approach is optimal.

References
1. Bartlett JG, Chang TW, Gurwith M, Gorbach SL, Onderdonk AB. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med. 1978; 298(10): 531–534.
2. Humphries RM, Uslan DZ, Rubin Z. Performance of Clostridium difficile toxin enzyme immunoassay and nucleic acid amplification tests stratified by patient disease severity. J Clin Microbiol. 2013; 51(3): 869–873.
3. Gupta A, Khanna S. Community-acquired infection: an increasing public health threat. Infect Drug Resist. 2014; 7: 63–72.
4. Tenover FC, Baron EJ, Peterson LR, Persing DH. Laboratory diagnosis of Clostridium difficile infection can molecular amplification methods move us out of uncertainty? J Mol Diagn. 2011; 13(6): 573–582.
5. Guerrero DM, Becker JC, Eckstein EC, Kundrapu S, Deshpande A, Sethi AK, et al. Asymptomatic carriage of toxigenic Clostridium difficile by hospitalized patients. J Hosp Infect. 2013; 85(2): 155–158.
6. Dubberke ER, Han Z, Bobo L, Hink T, Lawrence B, Copper S, et al. Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol. 2011; 49(8): 2887–2893.
7. Longtin Y, Trottier S, Brochu G, Paquet-Bolduc B, Garenc C, Loungnarath V, et al. Impact of the type of diagnostic assay on Clostridium difficile infection and complication rates in a mandatory reporting program. Clin Infect Dis. 2013; 56(1): 67–73.
8. Brecher SM, Novak-Weekley SM, Nagy E. Laboratory diagnosis of Clostridium difficile infections: there is light at the end of the colon. Clin Infect Dis. 2013; 57(8): 1175–1181.
9. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol. 2010; 31(5): 431–455.
10. Dionne LL, Raymond F, Corbeil J, Longtin J, Gervais P, Longtin Y. Correlation between Clostridium difficile bacterial load, commercial real-time PCR cycle thresholds, and results of diagnostic tests based on enzyme immunoassay and cell culture cytotoxicity assay. J Clin Microbiol. 2013; 51(11): 3624–3630.
11. Su WY, Mercer J, Van Hal SJ, Maley M. Clostridium difficile testing: have we got it right? J Clin Microbiol. 2013; 51(1): 377–378.
12. Leslie JL, Cohen SH, Solnick JV, Polage CR. Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing? Eur J Clin Microbiol Infect Dis. 2012; 31(12): 3295–3299.

The author
Michelle J. Alfa, PhD
Boniface Research Centre, Dept. of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
E-mail: malfa@dsmanitboa.ca

H. cinaedi: infection, detection and diagnosis

, 26 August 2020/in Featured Articles /by 3wmedia

Helicobacter cinaedi is a relatively recently identified bacterium, but it is recognized as an increasingly important cause of disease in humans. This article summarizes methods for its detection and identification as well as routes of infection and treatment.

by Prof. Yoshiaki Kawamura, Dr Tatsuya Okamoto, Dr Shigemoto Fujii and Prof. Takaaki Akaike

What is Helicobacter cinaedi?
Within the genus Helicobacter, 33 species to date have been proposed and validated, but only 7 species have been isolated from human clinical specimens (Table 1). H. pylori, classified as a ‘gastric Helicobacter species’, is a well-known member of Helicobacter, but some less well-known ‘enterohepatic Helicobacter species’, such as H. cinaedi, H. bilis, H. canadensis, H. canis, H. fennelliae, and H. pullorum, have also been isolated from human clinical specimens.

H. cinaedi was first isolated as a Campylobacter-like organism type 1 (CLO-1) in 1984 from rectal swabs from homosexual men displaying intestinal symptoms [1] and the following year the organism was named ‘Campylobacter cinaedi’; however, it was subsequently reclassified as Helicobacter [2]. During the last two decades, there have been many reports of the isolation of H. cinaedi from blood or intestinal tract of human immunodeficiency virus-infected or immunocompromised patients, but, recently, increasing numbers of infections have also been reported in immunocompetent patients.

In Japan, the isolation of H. cinaedi was first reported in 2003; since then, its isolation has been reported from patients regardless of gender and within a wide age range, from the newborn to the elderly, in many hospitals throughout the country. We have experienced many cases of H. cinaedi cellulitis and bacteremia in both immunocompromised and immunocompetent subjects in hospitals. This microorganism should, therefore, be considered a causative agent of nosocomial infection [3].

Illnesses caused by H. cinaedi
Clinical symptoms of H. cinaedi infection include fever, diarrhoea, abdominal pain, gastroenteritis, proctitis, cellulitis, erysipelas, arthritis, meningitis, and bacteremia. In contrast to other Helicobacter species, numerous reports have causally linked H. cinaedi infection with bacteremia, which contributes to this organism’s strong vascular invasion ability. In many cases of H. cinaedi bacteremia, the main symptom is fever accompanied by arthritis and cellulitis at various sites. In addition to these sites providing a source of primary infection, the resultant bacteremia can serve as a source of secondary infections; thus, all these various symptoms are clinically important.

In our experience, at various times after orthopedic surgery (range, 8–113 days; mean, 29 days), some patients had a sudden onset of local flat cellulitis (salmon-pink in colour) at different sites on the operated side, along with fever and an increase in C-reactive protein level (Fig. 1) [3]. Cellulitis was often multifocal without wound infection. Many of these patients had been treated for fracture and were immunocompetent.

In recent years, we have demonstrated the potential association of H. cinaedi with atrial arrhythmias and atherosclerosis [4]. This could be through bacterial translocation of H. cinaedi from the intestinal tract into the blood stream. The possible cause-and-effect relationship between H. cinaedi and vascular diseases may warrant further epidemiological study on proatherosclerotic effect of H. cinaedi infection.

The virulence factor of H. cinaedi is largely unknown. The complete genome sequence of a human clinical isolate was announced in 2012 [5] and revealed that the organism holds a Type VI secretion system, which is expected to be related to its virulence, together with two known virulence factors, cytolethal distending toxin and alkyl hydroperoxide reductase.

Detection, cultivation and identification of H. cinaedi
It is well known that H. cinaedi is a fastidious and slow-growing organism, and that detection and cultivation are extremely difficult. In many cases, H. cinaedi is first detected from a blood culture using an automatic blood culture system. It is generally noted that 4–10 days (average 5.6 days, in our experience) are needed for a positive result in the culture bottle of an automatic blood culture system, such as the BACTEC system (Becton Dickinson) using an aerobic bottle. Therefore, when the culture test using this system is terminated within 3–4 days, the bacterial growth may be still below its detection limit. Information on the detection of H. cinaedi using the BacT/ALERT system (Biomérieux) is scanty. In our experience, the VersaTREK system (Thermo Scientific) is superior for the detection of this microorganism.

Both H. cinaedi and H. pylori are members of the genus Helicobacter; however, the former is extremely difficult to culture. H. cinaedi isolates essentially require microaerobic conditions (5–10% O₂) and a high level of humidity. Often a blood agar plate stored in a refrigerator for a few days may fail to support the growth of H. cinaedi because of low water content. Use of fresh medium is strongly recommended. It is established that H. cinaedi growth is accelerated by adding hydrogen gas (5–10%) to microaerobic conditions. The culture success rate can be improved by using a gas mixture such as 6% O₂, 7% H₂, 7% CO₂, and 80% N2 at the initial culture from the clinical specimen or in the culture bottle. Unfortunately, many of the commercially available microaerobic gas-generating packs, such as the GasPak system (Becton Dickinson), deoxidize and generate CO₂ but do not generate hydrogen gas; therefore, in some cases H. cinaedi does not grow, or growth is inadequate.

H. cinaedi cultured on an agar plate grows in a film, which is difficult to identify visually. Therefore, the culture should be carefully checked on the plate.

The biochemical identification of this organism is problematic owing to unstable phenotypic reactions. In many cases commercially available identification kits do not produce reliable results. Therefore, identification based on nucleotide sequence or species-specific polymerase chain reaction (PCR) has been used. We have developed a nested PCR system with high specificity and sensitivity (approximately 10² CFU/ml) for detecting H. cinaedi [6]. We have also established an immunological diagnosis method (antibody detecting test) with high specificity to detect the exposure history of H. cinaedi [7].

Antimicrobial therapy and prognosis
To date, antimicrobial susceptibility testing for H. cinaedi has mainly used the agar-dilution method, but this method is too cumbersome for routine use in hospital laboratories. A broth microdilution method for antimicrobial susceptibility testing of H. cinaedi, which can be performed easily, has been developed by our research group [8].

H. cinaedi strains generally show low minimum inhibitory concentration (MIC) values for carbapenems, aminoglycosides, and tetracycline (MIC₉₀ = 1 µg/ml for imipenem/cilastatin, gentamicin, and tetracycline). In contrast, H. cinaedi has well-known resistance to macrolides, with especially high MIC values (MIC₉₀ >64 µg/ml for erythromycin). Recently in Japan and elsewhere, H. cinaedi isolates have shown high resistance to quinolones (MIC₉₀ = 64 µg/ml for ciprofloxacin and levofloxacin) due to point mutation(s) of DNA gyrase genes [8].

Symptoms caused by H. cinaedi, such as fever or cellulitis, usually resolve after 2 to 3 days of drug therapy, but the Centers for Disease Control and Prevention recommended long-term therapy for about 2 to 6 weeks, rather than short-term therapy for only 10 days [9]. Prognosis is generally good, but it is noteworthy that, depending on the study, about 30–60% of patients have recurrent symptoms. Unfortunately, there are no guidelines for the treatment of H. cinaedi infections, including the clinical breakpoints of antimicrobial agents. The MIC values described above are based on our data.

Infection route
H. cinaedi has been found in a wide range of animals including cats, dogs, hamsters, rats, and foxes. There have been many reports of zoonotic transmission vectors, but no reports of the simultaneous isolation of H. cinaedi from human patients and the animals that they have been in close contact with. It is noteworthy that H. cinaedi isolates from human, dog, and hamster formed a distinct ribotype pattern group by host source [10].

Epidemiological analysis methods, such as pulse-field gel electrophoresis, randomly amplified polymorphic DNA, and multilocus sequence typing, have been proposed for H. cinaedi isolates [3, 11]. As described above, we developed a nested PCR system and immunological diagnosis method. Using these methods, we tested many healthy hospital employees (doctors, nurses, staff members, etc.) and found that some currently uninfected individuals had previously had H. cinaedi infections, indicating that there could be asymptomatic carriers with intestinal colonization of H. cinaedi. Our study also suggested that occurrence of such asymptomatic carriers may be related to nosocomial infection.

However, the complete route of infection route, including nosocomial transmission, of H. cinaedi remains unclear.

Summary
A full understanding of H. cinaedi infection remains elusive; however, some features and the clinical relevance of this infection have become increasingly recognized recently. To detect and isolate H. cinaedi from human blood samples using an automated blood culture system, a long-term incubation (up to 10 days) is needed and further skillful culture techniques are required. In many clinical laboratories, however, appropriate culture for isolation of this bacteria might not performed, which may lead to false-negative findings for H. cinaedi. As H. cinaedi was considered not to cause acute severe disease, it seems that its importance may have not been recognized clinically. However, we now know that this microorganism likely causes nosocomial infections that are difficult to eradicate and have a high incidence of recurrence. In recent years, a possible association with chronic illnesses such as arrhythmia and arteriosclerosis has been reported, and therefore we will need to carefully monitor and ascertain trends in H. cinaedi infections.

References
1. Fennell CL, et al. E. Characterization of Campylobacter-like organisms isolated from homosexual men. J Infect Dis. 1984; 149: 58–66.
2. Vandamme P, et al. Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int J Syst Bacteriol. 1991; 41: 88–103.
3. Kitamura T, et al. Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery. J Clin Microbiol. 2007; 45: 31–38.
4. Khan S, et al. Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses. Sci Reports 2014; In press.
5. Goto T, et al. Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia. J Bacteriol. 2012; 194: 3744–3745.
6. Oyama K, et al. Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR. J Clin Microbiol. 2012; 50: 3893–3900.
7. Iwashita H, et al. Identification of the major antigenic protein of Helicobacter cinaedi and its immunogenicity in humans with H. cinaedi infections. Clin Vaccine Immunol. 2008; 15: 513–521.
8. Tomida J, et al. Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi. Microbiol Immunol. 2013; 57: 353–358.
9. Kiehlbauch JA, et al. Helicobacter cinaedi-associated bacteremia and cellulitis in immunocompromised patients. Ann Intern Med. 1994; 121: 90–93.
10. Kiehlbauch JA, et al. Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals. J Clin Microbiol. 1995; 33: 2940–2947.
11. Rimbara E, et al. Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan. J Clin Microbiol. 2012; 50: 2553–2560.
12. Solnick JV, Schauer DB. Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases Clin Microbiol Rev. 2001; 14: 59–97.

The authors
Yoshiaki Kawamura¹ PhD; Tatsuya Okamoto² MD, PhD; Shigemoto Fujii³ PhD; Takaaki Akaike³* MD, PhD
¹Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Japan
²Intensive Care Unit, National Center for Global Health and Medicine, Tokyo, Japan
³Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Japan
*Corresponding author
E-mail: takaike@med.tohoku.ac.jp

TNF Receptors – powerful biomarkers for detecting diabetic kidney disease a decade in advance

, 26 August 2020/in Featured Articles /by 3wmedia

Kidney disease is one of the most life-threatening complications of diabetes and as the global incidence of diabetes soars, largely due to the dramatic increase in type 2 diabetes (T2DM), there will be a seismic shift in the number of patients in need of treatment through dialysis or transplant. Since up to 40% of diabetic patients develop symptoms of diabetic kidney disease (DKD), accurate and early identification of which patients are at the highest risk of progression from DKD to end stage renal disease (ESRD) will enable early initiation of protective renal therapies with subsequent reduction in healthcare costs and improved patient outcomes.

The cytokine TNFα, part of the Tumour Necrosis Factor (TNF) superfamily that plays a key role in homeostasis, has been implicated in the pathogenesis of diabetic kidney disease for over 20 years [1]. Researchers conclude that the elevated levels seen in diabetic patients could be the result of a TNFα driven dysregulation of the inflammatory/apoptotic pathways, which leads to kidney injury. The spotlight has recently shifted onto the TNF α receptors, Tumour Necrosis Factor Receptor 1 (TNFR1) and Tumour Necrosis Factor Receptor 2 (TNFR2), after a number of studies showed how elevated levels of these proteins were a predictor of progressive kidney disease.

In this article we look at the development of an In-Vitro Diagnostic test (IVD), the ‘EKF sTNFR1 Test’. This has been developed by EKF Diagnostics to measure levels of TNFR1 in plasma or serum in light of scientific evidence that this robust biomarker provides valuable prognostic information for diabetic patients at risk of progressive renal decline and ESRD.

The scientific evidence for the involvement of TNF receptors in kidney disease
Cytokine TNFα is a transmembrane protein generated by many cells, including lipocytes, endothelial cells and leukocytes. After processing by TNFα-converting enzyme (TACE), the soluble form of TNFα is cleaved from transmembrane TNFα and mediates its biological activities through binding the receptors TNFR1 and TNFR2 either in their transmembrane or soluble forms to activate inﬂammatory and stress response pathways (Figure 1). Transmembrane TNF-α also binds to TNFR1 and TNFR2 so that both transmembrane and soluble TNF-α can mediate downstream signalling events (apoptosis, cell proliferation and cytokine production).

In 2009, at the Joslin Diabetes Center, USA (the world’s largest diabetes research centre and an affiliate of the Harvard Medical School), researchers found that the presence of circulating soluble TNF receptors (sTNFR1 and sTNFR2) were strongly correlated with decreased renal function, or glomerular filtration rate (GFR). The research threw up questions about why these soluble receptors were indicative of renal disease. Were they playing an active part in causing disease, or were they just the by-product of the process? Elevations in circulating sTNFR1 have previously been reported in a wide variety of clinical conditions including cancer, congestive heart failure, rheumatoid arthritis, neurological diseases and infection; so what was their role in kidney disease?

Interestingly, as Niewczas et al. [2] pointed out, the decline of renal function was occurring in T1DM patients who had normal albumin excretion levels. This gave a clue to the researchers that the concentrations of these receptors were not merely markers of the injury leading to ESRD but were also involved in the inception of renal function decline, playing a part in inflammation and apoptosis.

1n 2012, the Joslin researchers published two further studies, on Type 1 and Type 2 diabetes cohorts, [3,4] and found that TNF receptor levels were robust predictors of progressive decline in GFR. The results showed that Type 1 Diabetes patients who had normal renal function at the onset, but TNFR2 levels in the highest quartile had a 60% cumulative incidence of reaching stage 3 Chronic Kidney Disease (CKD) with subsequent risk of progression to ESRD (compared to less than 20% in the lowest three quartiles) (Figure 2).

Most significantly, in Type 2 Diabetes patients with evidence of overt Kidney Disease (as evidence by elevated levels of albumin excretion levels) at the onset of the study, those with levels of TNFR1 in the fourth quartile had an 80% chance of developing renal disease over the twelve year period (compared to less than 20% of those in the lower three quartiles) (Figure 3).

These studies revealed that elevated TNF Receptor levels were a robust predictor of progressive disease in both Type 1 diabetes and Type 2 diabetes. In both studies, the levels of the TNFα levels also tended to predict progressive kidney disease, but less strongly than the TNF receptor levels. The data provided further evidence that inflammation in general, and the TNFα signalling pathway in particular, plays a role in kidney disease.

TNF receptors (TNFR1 and TNFR2) and their role in the disease process
So how are circulating TNFR receptors associated with early GFR reduction and kidney damage? It is known that the 55 kD TNFR1 and 75 kD TNFR2 receptors play a crucial part in apoptosis, survival and key aspects of the inflammatory and immune response. TNFR1 is abundant on all nucleated cells, but TNFR2 expression is restricted mainly to endothelial cells and leukocytes although this varies between normal and diseased tissues. Circulating TNFR1 in the plasma is released by two mechanisms: the inducible cleavage of the 34 kD TNFR1 extracellular domain by an enzyme known as ADAM17 and the constitutive release of a full-length 55 kD TNFR1 within exosome-like vesicles.

It is not-well understood whether the same mechanisms apply to TNFR2 release, or how this process is regulated and the biology of the soluble forms remain largely undiscovered. What is understood, however, is that in plasma, TNF receptors block TNFα from binding its target cell surface receptor and can therefore cause a prolonged and delayed effect of the cytokine. How subsequent damage occurs to the kidney is not well known, however sTNFRs have been shown to be involved in tubulointerstitial ﬁbrosis, the characteristic tissue scaring that leads to kidney disease [5].

Seeing into the future: a powerful diagnostic test for DKD
The diagnosis of DKD is conventionally made by assessment of overall GFR and the presence of kidney damage is ascertained by either kidney biopsy or other markers of kidney damage such as microalbuminuria or proteinuria (collectively known as albuminuria – a condition where protein is lost in the urine). GFR is estimated in clinical practice using readily calculated equations that adjust serum creatinine values (measurement of the by-product of muscle metabolism cleared by the kidneys) to age, sex, and ethnicity. However, while laboratory tests which assess both serum creatinine and albuminuria are inexpensive and readily available, these parameters have a low predictive value.

In 2012, EKF Diagnostics signed an exclusive licence agreement for novel kidney biomarker technology that focused on sTNFR1 and sTNFR2. This was developed by a team led by Prof. Andrzej Krolewski, MD, PhD, Head of Section on Genetics and Epidemiology at the Joslin Diabetes Center, Professor of Medicine at Harvard Medical School. Prof. Krowlewski was recently awarded the American Diabetes Association’s 2014 Kelly West Award in Epidemiology for services to diabetes epidemiology.

EKF Diagnostics has worked in partnership with Joslin and other key diabetes research centres to further validate the clinical utility of the markers and develop its first IVD product, the sTNFR1 test kit. The sTNFR1 test is an easy-to-use, microtitre plate ELISA-based assay requiring minimal training, which uses standard laboratory equipment and monoclonal antibodies to analyse just 50 µL of blood serum or plasma. Accurate and reliable results are obtained in a few hours and the standard assay format means that the test requires minimal training.

Julian Baines, Group Chief Executive Officer of EKF Diagnostics highlights the benefits of the test, “Our new sTNFR1 test adds greatly to information provided by standard clinical tests and provides valuable long term prognostic information for progressive renal decline to ESRD with the potential to streamline diabetic patient management, reduce time and costs and improve patient outcomes.”

Further evidence for the use of sTNFRs for the early prediction of DKD
A number of high impact studies published this year have independently corroborated the original research by the Joslin Diabetes Center. This newly published data from eminent European research centres in France (SURDIAGNE Study Group) and Finland (FinnDiane Study Group) add to the expanding data set underpinning the value of sTNFR1/2 biomarkers.

In the FinnDiane cohort study of over 400 subjects with Type 1 Diabetes followed over an average of 9 years, researchers found that, “Circulating levels of sTNFR1 were independently associated with incidence of ESRD. This association was reported as both significant and biologically plausible and demonstrated added value of sTNFR1 as a biomarker” [6].

In France, Saulnier et al. [7] found results from a study of n=522 Type 2 Diabetes patients with DKD were in accordance with published data, showing a deleterious effect of TNFR1 serum concentrations on renal outcomes.

Further evidence continues to mount for how TNFR biomarkers could be used to improve diabetic patient management and outcomes through early intervention. Lopes-Virella et al. [8] have shown in a large cohort of type 1 diabetes patients, followed for six years, how high levels of sTNFR1 and sTNFR2 can predict progression to macroalbuminuria in patients completely free of disease at baseline. TNFR biomarkers can also help doctors to stratify patients with early kidney disease according to the risk of ESRD. Skupien et al [9] show a strong association between a single baseline measurement of TNFR2 serum concentration combined with measurement of HbA1c levels and the future rate of renal function decline in T1DM patients with proteinuria. Identifying patients at highest risk can ensure they are enrolled in therapeutic programmes to delay the rapid decline in renal function.

The future management of kidney disease
Recent statistics show that 25-40% of patients with diabetes are at significant risk of progression to ESRD and cardiovascular morbidity and mortality [10]. The global increase in the incidence in Type 2 diabetes will put more pressure on healthcare systems making it imperative to identify patients at risk of progressive diabetic kidney disease, and initiate protective renal and cardiovascular therapies. Improving outcomes for chronic kidney disease in diabetic patients also has an important impact on mortality; for example, compared with non-diabetic individuals, patients with Type 1 diabetes have no increase in mortality in absence of DKD [11]. There is now solid evidence that sTNFR1 and sTNFR2 can be useful as biomarkers to predict the progression of kidney disease – and not just in patients with diabetes: recent research in Sweden has shown how circulating sTNFRs are relevant biomarkers for kidney damage and dysfunction in elderly individuals in a community setting [12].

Current treatments for CKD, such as control of hypertension and lifestyle interventions (weight loss, diet control, smoking cessation), can reduce the risk of progression to ESRD; therefore, an advanced knowledge of disease risk up to 10 years in advance that the sTNFR1 test kit can provide would be an extremely valuable tool to effectively prevent or reduce morbidity and mortality. Significantly, the sTNFR1 test is also contributing to the development of new targeted therapies aimed at delaying or halting decline in renal function.

References
1. Hasegawa G et al. Possible role of tumor necrosis factor and interleukin-1 in the development of diabetic nephropathy. Kidney Int. 1991; 40: 1007 –1012.
2. Niewczas MA et al. Serum concentrations of markers of TNF alpha and Fas-mediated pathways and renal function in nonproteinuric patients with type 1 diabetes. Clin J Am Soc Nephrol. 2009; 4: 62-70.
3. Ghoda T et al. Circulating TNF receptors 1 and 2 predict stage 3 CKD in Type 1 diabetes. J Am Soc Nephrol. 2012; 23: 516-24.
4. Niewczas MA et al. Circulating TNF receptors 1 and 2 predict ESRD in Type 2 Diabetes. J Am Soc Nephrol. 2012; 23: 507-15.
5. Guo G et al. Role of TNFR1 and TNFR2 receptors in tubulointerstitial ﬁbrosis of obstructive nephropathy. Am J Physiol. 1999; 277: F766–F772.
6. Forsblom C et al. Added Value of Soluble Tumor Necrosis Factor Alpha Receptor-1 as a Biomarker of ESRD Risk in Patients With Type 1 Diabetes. Diabetes Care 2014; 37: 1–9.
7. Saulnier et al. Association of Serum Concentration of TNFR1 With All-Cause Mortality in Patients With Type 2 Diabetes and Chronic Kidney Disease: Follow-up of the SURDIAGENE Cohort Published online before print March 12, 2014, doi: 10.2337/dc13-2580.
8. Lopes-Virella MF et al. Baseline markers of inflammation are associated with progression to macroalbuminuria in type 1 diabetic subjects. Diabetes Care 2013; 36: 2317-23. doi: 10.2337/dc12-2521.
9. Skupien et al. Synergism between circulating tumor necrosis factor receptor 2 and HbA1c in determining renal decline during 5-18 years of follow-up in patients with type 1 diabetes and proteinuria. In press: Accepted for publication in Diabetes Care, April 22, 2014.
10. MacIsaac RJ. Markers of and Risk Factors for the Development and Progression of Diabetic Kidney Disease.American Journal of Kidney Diseases 2014; 63: S39–S62.
11. Orchard TJ et al. In the absence of renal disease, 20 year mortality risk in type 1 diabetes is comparable to that of the general population: a report from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetologia 2010; 53: 2312– 2319.
12. Carlsson AC et al. Soluble TNF Receptors and Kidney Dysfunction in the Elderly. J Am Soc Nephrol. 2014; 25: 1313-1320.