A new, benchtop molecular analyser allows renal transplant centres to monitor urine and serum BK viral loads in house, permitting earlier diagnosis and management of BK virus associated nephropathy (BKVAN) in renal transplant recipients.
BK virus infection
Named after the renal transplant patient (with initials B.K.) that it was first isolated from in 1971, BK virus (BKV) is a Polyomavirus, characterized by its nonenveloped, icosahedral capsid and its circular, double-stranded DNA genome. Although BKV is prevalent around the world, estimated to have infected more than 80% of the global population, infection with the virus is usually asymptomatic or associated with only mild respiratory tract symptoms in healthy individuals [1]. Following primary infection, which typically occurs in early childhood, the virus persists in a latent form in the kidneys and urinary tract of its host [2].
Reactivation of BKV can occur in immunocompromised and immunosuppressed individuals [3]. In most cases reactivation of the virus is benign but it can pose a particular challenge for renal transplant patients. In such cases immunosuppression can cause a lytic BKV infection that results in viruria in 30-50% and viremia in 13-22% of renal transplant recipients [4]. BKV infection is one of the most common viral complications to affect renal allografts [5]. It can lead to BKV associated nephropathy (BKVAN) in up to 10% of renal transplant recipients, and is associated with graft failure in 15-80% of affected patients [3,6-9].
Not every latent infection leads to viral reactivation and BKVAN in renal transplant patients. In addition to immunosuppression, other risk factors, such as intragraft inflammation and host-specific immunity have been suggested [3]. The progression of BKVAN may occur without obvious signs or symptoms, other than raised serum creatinine, and so it is often misdiagnosed [9].
Management of BKVAN
Treatment of BKV infection and BKVAN in renal transplant patients usually involves a reduction or modification of immunosuppressive therapy. It is generally agreed that early diagnosis and treatment are extremely important to prevent damage to the allograft [3,9]. At a later stage of infection, when intragraft inflammation has developed, reduction of immunosuppression may not be effective and may even be detrimental to the allograft [3]. For this reason, frequent monitoring of BKV in renal transplant patients, to detect early onset BKV infection, is recommended to ensure timely intervention [3].
Confirmation of BKVAN is performed by histological examination of an allograft biopsy sample. However, clinical intervention is often based on the presence of viral replication as a surrogate marker and early indicator of BKV infection. For this reason, non-invasive urine and blood tests have value in screening for BKV reactivation, monitoring the clinical course of infection, or monitoring response to therapy [3].
Urine cytology has been used to screen for BKV reactivation in renal transplant recipients. However, since the virus may be shed in the urine of healthy individuals, quantitative results are required for this method to have diagnostic value [3]. Furthermore, accurate interpretation of cytology results requires training and expertise as it is often difficult to distinguish BKV from other viral infections [9].
Recently, molecular techniques for the detection and quantification of BKV in blood and urine have become available. Such methods offer greater specificity for BKV and provide a valuable tool for identifying patients at risk of BKVAN before renal function deteriorates.
Monitoring BK viral loads
Quantitative measurements of BK viral load in urine and blood by molecular techniques are useful for monitoring the course of BKV infection [9] and for predicting the development of BKVAN [4,7,10,11]. Viral reactivation can be detected in the urine several weeks before the virus is detected in the blood, and viremia can be detected months before histological evidence of BKVAN is present [3]. Monitoring BKV loads in the urine and serum or plasma of renal transplant recipients, therefore, may be valuable in identifying those at risk of developing BKVAN, allowing further investigation and early intervention if necessary [3,9]. Such measurements are also valuable in monitoring response to therapy [3,9].
Although suggested BKV load thresholds for quantitative molecular measurements vary, and laboratories are encouraged to establish their own cut off values for the purpose of clinical management [9], BK viral loads of greater than 10,000 copies/mL in blood [6,11-14] and greater than 10 million copies/mL in urine are considered predictive for BKVAN [6,11,12].
Current guidelines recommend screening for BKV in the serum or plasma of kidney transplant patients monthly for the first 3-6 months after transplantation, and then every 3 months up to one year post-transplantation [15]. These guidelines also recommend that patients are screened for BKV if there is an unexplained rise in serum creatinine or following treatment for acute rejection [15].
Faster quantification of BKV
Due to the specialist nature of BKV testing and the resources and expertise required to perform BKV measurements by urine cytology or nucleic acid testing, many centres are required to send samples to a reference laboratory for analysis. Some laboratories have adopted in-house polymerase chain reaction (PCR) BKV assays. These can be labour intensive, variable in terms of specificity for BKV, and may require further confirmatory testing on positive samples, which can cause significant delays and can potentially impact patient management.
A new molecular method is now available that can reduce the turnaround time for quantitative BKV results significantly and provide the high specificity required for making important clinical decisions about the management of renal transplant patients. The Diasorin Liaison® Iam benchtop instrument, with its small footprint and ease of operation, offers a cost effective and scalable solution for laboratories servicing renal transplant centres. Demonstrating no cross reactivity with other significant pathogens, including JCV, the Diasorin Iam BKV assay provides reliable, quantitative results on the same day as sample receipt [16].
The Iam BKV assay uses loop-mediated isothermal amplification (Diasorin Q-LAMP) to measure BKV DNA in urine, plasma or serum. Unlike conventional LAMP technology, Diasorin Q-LAMP is a rapid, real-time, fluorescent technique that allows quantitative analysis of individual or multiple targets in a single reaction [16].
Q-LAMP is based on the recognition of multiple primer binding regions on the target nucleic acid and amplification of the target sequence, which is facilitated by polymerase with strand displacement activity. Quantification is achieved through the use of fluorophore-labelled primers and an observed decrease in fluorescence during amplification of the target sequence, together with known calibrators. The Diasorin Iam BKV assay is a duplex Q-LAMP assay, designed to recognize a consensus sequence common to all known BKV subtypes. Integral controls provide verification of the efficiency of the extraction process and demonstrate the absence of inhibitors [16].
The Iam BKV Q-LAMP assay fits easily into daily laboratory routines. Once samples are prepared and loaded onto the Liaison® Iam instrument, no operator intervention is required during an assay run, allowing staff to walk away until the routine is completed and the result is displayed. The Iam BKV assay is extremely sensitive, with a limit of detection (defined as that concentration of BK virus with a 95% probability of detection by probit analysis) of 450 cps/mL in plasma (95% Confidence Interval 350 – 770 cps/mL) and 540 cps/mL in urine (95% CI 440 – 780 cps/mL) [16]. The BKV primers used represent all known BKV subtypes (Ia, Ib-1, IB-2, Ic, II,III and IV) and show no significant homology with a range of pathogens, including SV-40 virus and Herpes viruses, or cross reactivity with the closely-related Polyomavirus, JCV [16].
Improved management of renal transplant patients
The DiaSorin Iam BKV assay for the detection and quantification of BKV has been in use at the 975-bed Westmead Hospital in Sydney since July, 2013. Westmead Hospital is a major teaching hospital for Sydney University and one of Australia’s largest centres for post-graduate training to specialist level in all fields. The Department of Renal Medicine and Transplantation and the Centre for Transplant and Renal Research work closely with the Centre for Infectious Diseases and Microbiology (CIDM), which is part of Pathology West, a leading public pathology service in New South Wales. The focus of the Transplant and Renal Research Group is to improve the lives of people with end-organ failure through transplantation. It also aims to reduce the number of people requiring dialysis by preventing the progression of chronic renal disease.
Senior Hospital Scientist at the Westmead CIDM laboratory, Dr Neisha Jeoffreys, comments, “BKV is an important pathogen in renal transplant patients. It can cause serious complications and so early detection of viral reactivation and accurate monitoring of viral loads is a vital aspect of patient management.”
The Westmead CIDM laboratory provides a BKV testing service to the hospital’s renal transplant outpatient clinics as well as other specialist clinics associated with the centre. They also test samples from other pathology groups in their reference capacity. Dr Jeoffreys explains, “Renal transplant patients are tested routinely using the Iam BKV assay at 1, 2, 3, 6, 9 and 12 months post transplantation. Patients that test positive for BKV are tested more frequently, every 2-4 weeks.”
“”The role of the quantitative Iam BKV assay is to determine if the patient is likely to develop BKVAN, which may lead to premature graft loss. Patients with high BKV levels will have their immunosuppression regime modified in order to reduce BKV levels while preventing graft rejection. Ongoing monitoring of BK viral load then assists the renal physicians to ensure the right amount of immunosuppression is delivered to reduce the risk of BKVAN and maintain a healthy graft. Quantitative results allow the physicians to determine the appropriate point at which to modify the treatment.”
Previously, the laboratory used a qualitative in-house conventional PCR method for the detection of BKV followed by monthly quantification of viral loads in BKV-positive patients using a commercially available real-time PCR assay.
“We feel that the Iam BKV assay enables us to provide a better service for our renal specialists,” Dr Jeoffreys continues. “We like the scalability of the Liaison® Iam instrument. We have 3 instruments, which provide the flexibility to perform 1 or up to 21 samples at the same time, optimizing reagent usage. This has allowed us to provide faster turnaround of results as we can now perform quantitative assays immediately and several times a week. The Liaison® Iam method has also helped to improve workflows as it is fast and easy to perform, with less hands-on time than our previous methods, which makes it more cost effective.”
“It is hoped that the rapid quantitative results provided by the Iam BKV assay will allow our renal physicians to respond more quickly to high or escalating BK viral loads,” Dr Jeoffreys concludes. “This will ultimately reduce the rate of graft loss due to BKVAN and allow for better patient management with reduced immunosuppression.”
The Iam BKV assay was the first assay to become available on the Diasorin Liaison® Iam instrument. The growing portfolio of tests available on this platform includes assays for Varicella zoster virus, Parvovirus B19 and Toxoplasma gondii.
Dr Neisha Jeoffreys is Senior Hospital Scientist at the Centre for Infectious Diseases and Microbiology (CIDM) based at Westmead Hospital, part of the Pathology West Institute of Clinical Pathology and Medical Research (ICPMR).
For further information, please contact Tiffany Page, Global Marketing Manager, Molecular Infectious Disease, DiaSorin, tiffany.page@ie.diasorin.com
www.diasorin.com.
References
1. Goudsmit, J. et al. J. Med. Virol. 10, 91–99 (1982).
2. Shinohara T, Matsuda M, Cheng SH, et al. J Med Virol. 1993;41:301-305.
3. Babel, N, Volk, H and Reinke, P (2011). Nat. Rev. Nephrol. 7: 399–406
4. Hirsch, H. H. et al. J. Med. 347, 488–496 (2002).
5. Ramos, E., Drachenberg, C. B., Wali, R. & Hirsch, H. H. Transplantation 87, 621–630 (2009).
6. Hirsch, H. H. et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation 79, 1277–1286 (2005).
7. Brennan, D. C. et al. Am. J. Transplant. 5, 582–594 (2005).
8. Hirsch HH. Clin Infect Dis. 2005;41:354-360.
9. Bechert, CJ, Schnadig, VJ, Payne, DA and Dong, J. (2010) Monitoring of BK Viral Load in Renal Allograft Recipients by Real-Time PCR Assays. Am J Clin Pathol 133:242-250.
10. Babel, N. et al. Transplantation 88, 89–95 (2009).
11. Dadhania, D. et al. Transplantation 86, 521–528 (2008).
12. Costa, C. et al. Nephrol. Dial. Transplant. 23, 3333–3336 (2008).
13. Hirsch HH, Steiger J. Polyomavirus BK. Lancet Infect Dis. 2003;3:611-623.
14. Ding R, Medeiros M, Dadhania D, et al. Transplantation. 2002;74:987-994.
15. KDIGO, Am. J. Transplant. 9 (Suppl. 3), S44–S58 (2009).
16. Diasorin Iam BKV assay Instructions for Use, BKV-524-02,EN 12/12.
MALDI-TOF mass spectrometry for rapid identification and subtyping of H. cinaedi strains isolated from humans and animals
, /in Featured Articles /by 3wmediaAlthough Helicobacter cinaedi infection is now recognized as an increasingly important emerging disease in humans, it is difficult to identify particular isolates due to their unusual phenotypic profiles and similarity of 16S rRNA sequences among closely related helicobacters. MALDI-TOF MS resolved the present difficulties associated with the identification of H. cinaedi.
by Takako Taniguchi and Prof. Naoaki Misawa
Helicobacter cinaedi infection
Helicobacter cinaedi, was first recognized as a Campylobacter-like organism (CLO), is a Gram-negative, spiral-shaped, motile, microaerobic bacterium, and is now classified into enterohepatic Helicobacter species [1]. This organism was first isolated from homosexual men and was initially recognized as a rectal and intestinal pathogen among members of that population [1]. The first case of H. cinaedi bacteremia in Japan was in an HIV-negative patient, but was receiving immunosuppressive therapy after renal transplantation [2]. Moreover, a few cases of infection with H. cinaedi isolated from feces and blood from an apparently non-immunocompromised child and adult have been reported [3]. Since then, H. cinaedi has become thought of as an opportunistic pathogen that causes bacteremia, cellulitis, septicemia and enteritis in immunocompromised patients [4, 5], immunocompetent patients and even healthy individuals [6]. Kitamura et al. reported an outbreak of nosocomial H. cinaedi infections caused by direct person-to-person transmission [6]. Therefore, healthcare workers need to pay attention to H. cinaedi infection as an increasingly important emerging disease in humans.
Epidemiology
H. cinaedi-like organisms have also been isolated from non-human sources such as dogs, cats, monkeys, hamsters and other rodents [7–10], suggesting that the organism may be widespread in a broad range of animal species. As Gebhart et al. reported that H. cinaedi was found in 75% of the healthy hamsters used in their study [9], it was hypothesized that hamsters might be an important reservoir for human infection [7, 9]. However, no reliable epidemiological evidence of zoonosis has been demonstrated for human cases of H. cinaedi infection [3].
Diagnosis
To isolate H. cinaedi from blood, blood was usually collected in BACTEC culture bottles and incubated in a BACTEC 9050 blood culture system (Becton Dickinson, BD Biosciences) for at least 5 to 7 days. When the incubation time was less than 5 days or other culture systems were used, the organism was not often isolated. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods [2].
We previously isolated at least six different spiral-shaped organisms including H. cinaedi and H. bilis in a puppy with bloody diarrhoea [11]. These organisms were identified based on their morphology, biochemical traits, whole-cell protein profiles, and the results of molecular analysis of their 16S rDNA sequences. However, the biochemical identification of Helicobacter strains based on a limited number of tests is difficult because helicobacters frequently exhibit unusual phenotypic profiles, even in the same species [10, 12]. Furthermore, H. cinaedi cannot be clearly discriminated from H. bilis on the basis of 16S rRNA sequences because of the high level of sequence similarity (greater than 98%) [12].
Application of MALDI-TOF MS for rapid identification and subtyping of H. cinaedi strains
Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has made it possible to analyse the protein composition of a bacterial cell based on intact-cell mass spectrometry (ICMS) profiles as a new technique for species identification. The technique is simple, rapid and accurate for identifying microorganisms regardless of their characteristics, such as Gram-negative and Gram-positive bacteria, mycobacteria, anaerobes and yeast species [13, 14]. MALDI-TOF MS has an advantage in that it has a low-cost performance and is independent of the age of the culture, growth conditions or medium selected, making it applicable for routine bacterial identification in clinical laboratories. Although there is a commercially available library that includes more than three thousand kinds of microorganism such as bacteria, yeast and fungus for identification and phylogenic analysis (MALDI Biotyper Reference Library, Bruker Daltonics), we use a library created in-house.
Therefore, we considered that MALDI-TOF MS might resolve the present difficulties with identification of H. cinaedi. Furthermore, we examined whether H. cinaedi strains isolated from different animals could be differentiated or subtyped by their ICMS profiles [15].
As shown in Figure 1, although common peaks were detected in the H. cinaedi and H. bilis strains examined, the m/z 5200 and 10400 peaks were detected only in strains of H. cinaedi. These peaks showed good reproducibility regardless of the isolate origins, different media and passage numbers on the same medium. Therefore, the ICMS profile of H. cinaedi could be completely differentiated from those of H. bilis. Furthermore, the ICMS profile of H. cinaedi was also distinguishable from those of H. mustelae, H. pylori, H. fennelliae and H. canis, indicating that ICMS profiling using MALDI-TOF MS is applicable for the identification of H. cinaedi.
Cluster analysis of H. cinaedi strains based on the ICMS profiles
Several papers report that direct contact with pets may be a possible route of infection in humans [3–5]; however, details regarding the pathogenesis and epidemiological features, including routes of infection of animal isolates in the context of both humans and animals, are not fully understood. No reliable epidemiological evidence of zoonosis has been demonstrated for human infections caused by H. cinaedi. Therefore, ICMS profiles of H. cinaedi strains isolated from humans and animals were measured, and a phyloproteomic tree was constructed in order to analyse the relationships between the strains. As a result, these H. cinaedi strains were clearly divided into two groups. All of the strains isolated from humans belonged to Cluster 2. All the other animal-derived strains belonged to Cluster 1 (Fig. 2). Interestingly, the ICMS-based phyloproteomic tree agreed with the phylogenetic tree that had been based on the nucleotide sequences of the hsp60 gene. These H. cinaedi strains were also clearly divided into two groups by the hsp60-gene-based phyloproteomic tree. Thus, the data from phyloproteomic and phylogenetic analysis suggest that human strains of H. cinaedi may be distinct from animal strains. Kiehlbauch et al. also reported that there may be subgroups within H. cinaedi isolated from humans, dogs, cats and hamsters that correlate with the host source on the basis of DNA–DNA hybridization and ribotyping analyses [12]. The present study appears to support the hypothesis that H. cinaedi from different host sources may form subgroups, which may prompt a revision of the classification of H. cinaedi.
Conclusion
In conclusion, the construction of ICMS profiles using the MALDI-TOF MS approach may be a useful tool for H. cinaedi identification and subtyping. Further investigations will be required to analyse additional strains from a broader area to confirm whether human strains belong to a distinct subtype of H. cinaedi.
References
1. Quinn TC, Goodell SE, et al. Ann Intern Med. 1984; 101: 187–192.
2. Murakami H, Goto M, et al. J Infect Chemother. 2003; 9: 344–347.
3. Orlicek SL, Welch DF, Kuhls TL. J Clin Microbiol. 1993; 31: 569–571.
4. Kiehlbauch JA, Tauxe RV, et al. Ann Intern Med. 1994; 121: 90–93.
5. Matsumoto T, Goto M, et al. J Clin Microbiol. 2007; 45: 2853–2857.
6. Kitamura T, Kawamura Y, et al. J Clin Microbiol. 2007; 45: 31–38.
7. Comunian LB, Moura SB, et al. Curr Microbiol. 2006; 53: 370–373.
8. Fernandez KR, Hansen LM, et al. J Clin Microbiol. 2002; 40: 1908–1912.
9. Gebhart CJ, Fennell CL, et al. J Clin Microbiol. 1989; 27: 1692–1694.
10. Kiehlbauch JA, Brenner DJ, et al. J Clin Microbiol. 1995; 33: 2940–2947.
11. Misawa N, Kawashima K, et al. Vet Microbiol. 2002; 87: 353–364.
12. Vandamme P, Harrington CS, et al. J Clin Microbiol. 2000; 38:.2261–2266.
13. Saffert RT, Cunningham SA, et al. J Clin Microbiol. 2011; 49: 887–892.
14. Stevenson LG, Drake SK, et al. J Clin Microbiol. 2010; 48: 3482–3486.
15. Taniguchi T, Sekiya A, et al. J Clin Microbiol. 2014; 52: 95–102.
The authors
Takako Taniguchi1 MSc and Naoaki Misawa2* DVM, PhD
1Laboratory of Veterinary Public Health, Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
2Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan
*Corresponding author
E-mail: a0d901u@cc.miyazaki-u.ac.jp
Monitoring BK virus in kidney transplant patients
, /in Featured Articles /by 3wmediaA new, benchtop molecular analyser allows renal transplant centres to monitor urine and serum BK viral loads in house, permitting earlier diagnosis and management of BK virus associated nephropathy (BKVAN) in renal transplant recipients.
BK virus infection
Named after the renal transplant patient (with initials B.K.) that it was first isolated from in 1971, BK virus (BKV) is a Polyomavirus, characterized by its nonenveloped, icosahedral capsid and its circular, double-stranded DNA genome. Although BKV is prevalent around the world, estimated to have infected more than 80% of the global population, infection with the virus is usually asymptomatic or associated with only mild respiratory tract symptoms in healthy individuals [1]. Following primary infection, which typically occurs in early childhood, the virus persists in a latent form in the kidneys and urinary tract of its host [2].
Reactivation of BKV can occur in immunocompromised and immunosuppressed individuals [3]. In most cases reactivation of the virus is benign but it can pose a particular challenge for renal transplant patients. In such cases immunosuppression can cause a lytic BKV infection that results in viruria in 30-50% and viremia in 13-22% of renal transplant recipients [4]. BKV infection is one of the most common viral complications to affect renal allografts [5]. It can lead to BKV associated nephropathy (BKVAN) in up to 10% of renal transplant recipients, and is associated with graft failure in 15-80% of affected patients [3,6-9].
Not every latent infection leads to viral reactivation and BKVAN in renal transplant patients. In addition to immunosuppression, other risk factors, such as intragraft inflammation and host-specific immunity have been suggested [3]. The progression of BKVAN may occur without obvious signs or symptoms, other than raised serum creatinine, and so it is often misdiagnosed [9].
Management of BKVAN
Treatment of BKV infection and BKVAN in renal transplant patients usually involves a reduction or modification of immunosuppressive therapy. It is generally agreed that early diagnosis and treatment are extremely important to prevent damage to the allograft [3,9]. At a later stage of infection, when intragraft inflammation has developed, reduction of immunosuppression may not be effective and may even be detrimental to the allograft [3]. For this reason, frequent monitoring of BKV in renal transplant patients, to detect early onset BKV infection, is recommended to ensure timely intervention [3].
Confirmation of BKVAN is performed by histological examination of an allograft biopsy sample. However, clinical intervention is often based on the presence of viral replication as a surrogate marker and early indicator of BKV infection. For this reason, non-invasive urine and blood tests have value in screening for BKV reactivation, monitoring the clinical course of infection, or monitoring response to therapy [3].
Urine cytology has been used to screen for BKV reactivation in renal transplant recipients. However, since the virus may be shed in the urine of healthy individuals, quantitative results are required for this method to have diagnostic value [3]. Furthermore, accurate interpretation of cytology results requires training and expertise as it is often difficult to distinguish BKV from other viral infections [9].
Recently, molecular techniques for the detection and quantification of BKV in blood and urine have become available. Such methods offer greater specificity for BKV and provide a valuable tool for identifying patients at risk of BKVAN before renal function deteriorates.
Monitoring BK viral loads
Quantitative measurements of BK viral load in urine and blood by molecular techniques are useful for monitoring the course of BKV infection [9] and for predicting the development of BKVAN [4,7,10,11]. Viral reactivation can be detected in the urine several weeks before the virus is detected in the blood, and viremia can be detected months before histological evidence of BKVAN is present [3]. Monitoring BKV loads in the urine and serum or plasma of renal transplant recipients, therefore, may be valuable in identifying those at risk of developing BKVAN, allowing further investigation and early intervention if necessary [3,9]. Such measurements are also valuable in monitoring response to therapy [3,9].
Although suggested BKV load thresholds for quantitative molecular measurements vary, and laboratories are encouraged to establish their own cut off values for the purpose of clinical management [9], BK viral loads of greater than 10,000 copies/mL in blood [6,11-14] and greater than 10 million copies/mL in urine are considered predictive for BKVAN [6,11,12].
Current guidelines recommend screening for BKV in the serum or plasma of kidney transplant patients monthly for the first 3-6 months after transplantation, and then every 3 months up to one year post-transplantation [15]. These guidelines also recommend that patients are screened for BKV if there is an unexplained rise in serum creatinine or following treatment for acute rejection [15].
Faster quantification of BKV
Due to the specialist nature of BKV testing and the resources and expertise required to perform BKV measurements by urine cytology or nucleic acid testing, many centres are required to send samples to a reference laboratory for analysis. Some laboratories have adopted in-house polymerase chain reaction (PCR) BKV assays. These can be labour intensive, variable in terms of specificity for BKV, and may require further confirmatory testing on positive samples, which can cause significant delays and can potentially impact patient management.
A new molecular method is now available that can reduce the turnaround time for quantitative BKV results significantly and provide the high specificity required for making important clinical decisions about the management of renal transplant patients. The Diasorin Liaison® Iam benchtop instrument, with its small footprint and ease of operation, offers a cost effective and scalable solution for laboratories servicing renal transplant centres. Demonstrating no cross reactivity with other significant pathogens, including JCV, the Diasorin Iam BKV assay provides reliable, quantitative results on the same day as sample receipt [16].
The Iam BKV assay uses loop-mediated isothermal amplification (Diasorin Q-LAMP) to measure BKV DNA in urine, plasma or serum. Unlike conventional LAMP technology, Diasorin Q-LAMP is a rapid, real-time, fluorescent technique that allows quantitative analysis of individual or multiple targets in a single reaction [16].
Q-LAMP is based on the recognition of multiple primer binding regions on the target nucleic acid and amplification of the target sequence, which is facilitated by polymerase with strand displacement activity. Quantification is achieved through the use of fluorophore-labelled primers and an observed decrease in fluorescence during amplification of the target sequence, together with known calibrators. The Diasorin Iam BKV assay is a duplex Q-LAMP assay, designed to recognize a consensus sequence common to all known BKV subtypes. Integral controls provide verification of the efficiency of the extraction process and demonstrate the absence of inhibitors [16].
The Iam BKV Q-LAMP assay fits easily into daily laboratory routines. Once samples are prepared and loaded onto the Liaison® Iam instrument, no operator intervention is required during an assay run, allowing staff to walk away until the routine is completed and the result is displayed. The Iam BKV assay is extremely sensitive, with a limit of detection (defined as that concentration of BK virus with a 95% probability of detection by probit analysis) of 450 cps/mL in plasma (95% Confidence Interval 350 – 770 cps/mL) and 540 cps/mL in urine (95% CI 440 – 780 cps/mL) [16]. The BKV primers used represent all known BKV subtypes (Ia, Ib-1, IB-2, Ic, II,III and IV) and show no significant homology with a range of pathogens, including SV-40 virus and Herpes viruses, or cross reactivity with the closely-related Polyomavirus, JCV [16].
Improved management of renal transplant patients
The DiaSorin Iam BKV assay for the detection and quantification of BKV has been in use at the 975-bed Westmead Hospital in Sydney since July, 2013. Westmead Hospital is a major teaching hospital for Sydney University and one of Australia’s largest centres for post-graduate training to specialist level in all fields. The Department of Renal Medicine and Transplantation and the Centre for Transplant and Renal Research work closely with the Centre for Infectious Diseases and Microbiology (CIDM), which is part of Pathology West, a leading public pathology service in New South Wales. The focus of the Transplant and Renal Research Group is to improve the lives of people with end-organ failure through transplantation. It also aims to reduce the number of people requiring dialysis by preventing the progression of chronic renal disease.
Senior Hospital Scientist at the Westmead CIDM laboratory, Dr Neisha Jeoffreys, comments, “BKV is an important pathogen in renal transplant patients. It can cause serious complications and so early detection of viral reactivation and accurate monitoring of viral loads is a vital aspect of patient management.”
The Westmead CIDM laboratory provides a BKV testing service to the hospital’s renal transplant outpatient clinics as well as other specialist clinics associated with the centre. They also test samples from other pathology groups in their reference capacity. Dr Jeoffreys explains, “Renal transplant patients are tested routinely using the Iam BKV assay at 1, 2, 3, 6, 9 and 12 months post transplantation. Patients that test positive for BKV are tested more frequently, every 2-4 weeks.”
“”The role of the quantitative Iam BKV assay is to determine if the patient is likely to develop BKVAN, which may lead to premature graft loss. Patients with high BKV levels will have their immunosuppression regime modified in order to reduce BKV levels while preventing graft rejection. Ongoing monitoring of BK viral load then assists the renal physicians to ensure the right amount of immunosuppression is delivered to reduce the risk of BKVAN and maintain a healthy graft. Quantitative results allow the physicians to determine the appropriate point at which to modify the treatment.”
Previously, the laboratory used a qualitative in-house conventional PCR method for the detection of BKV followed by monthly quantification of viral loads in BKV-positive patients using a commercially available real-time PCR assay.
“We feel that the Iam BKV assay enables us to provide a better service for our renal specialists,” Dr Jeoffreys continues. “We like the scalability of the Liaison® Iam instrument. We have 3 instruments, which provide the flexibility to perform 1 or up to 21 samples at the same time, optimizing reagent usage. This has allowed us to provide faster turnaround of results as we can now perform quantitative assays immediately and several times a week. The Liaison® Iam method has also helped to improve workflows as it is fast and easy to perform, with less hands-on time than our previous methods, which makes it more cost effective.”
“It is hoped that the rapid quantitative results provided by the Iam BKV assay will allow our renal physicians to respond more quickly to high or escalating BK viral loads,” Dr Jeoffreys concludes. “This will ultimately reduce the rate of graft loss due to BKVAN and allow for better patient management with reduced immunosuppression.”
The Iam BKV assay was the first assay to become available on the Diasorin Liaison® Iam instrument. The growing portfolio of tests available on this platform includes assays for Varicella zoster virus, Parvovirus B19 and Toxoplasma gondii.
Dr Neisha Jeoffreys is Senior Hospital Scientist at the Centre for Infectious Diseases and Microbiology (CIDM) based at Westmead Hospital, part of the Pathology West Institute of Clinical Pathology and Medical Research (ICPMR).
For further information, please contact Tiffany Page, Global Marketing Manager, Molecular Infectious Disease, DiaSorin, tiffany.page@ie.diasorin.com
www.diasorin.com.
References
1. Goudsmit, J. et al. J. Med. Virol. 10, 91–99 (1982).
2. Shinohara T, Matsuda M, Cheng SH, et al. J Med Virol. 1993;41:301-305.
3. Babel, N, Volk, H and Reinke, P (2011). Nat. Rev. Nephrol. 7: 399–406
4. Hirsch, H. H. et al. J. Med. 347, 488–496 (2002).
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