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GENEPROOF PCR KITS FOR THE DETECTION OF INFECTIONS IN TRANSPLANTATION

GeneProof is a biotechnology company operating in the field of in vitro molecular diagnostics of serious infections and genetic diseases.

GENEPROOF PCR KITS FOR THE DETECTION OF INFECTIONS IN TRANSPLANTATION

Organ transplantation is one of the greatest medical achievements of the twentieth century, extending the lives and improving the quality of life for hundreds of thousands of patients worldwide. Numerous acts of self-sacrifice on the part of organ donors and their families, as well as numerous significant scientific and clinical advances made by physicians, have turned transplantation into not only a way to save lives, but also a symbol of human solidarity [1].

 

Every year in Ukraine, about 5,000 people need organ transplants. For them, this operation is the only chance for life. For decades after independence, the issue of saving people in need of transplants was “up in the air”. Only a few posthumous organ transplants were performed. At the same time, only a few dozen transplants were performed from living donors. 

 

The turning point in transplantation was the adoption of amendments to the Law “On the Use of Transplantation of Anatomical Materials to Humans” of December 20, 2019. At the same time, after a significant break, a heart and kidney transplant from a deceased donor was performed at the Kovel District Hospital with the participation of doctors from the capital [2].

(Source — https://media.istockphoto.com/)

Immunocompromised patients are particularly vulnerable to common or opportunistic viral infections. They often occur after solid organ or hematopoietic cell transplantation, with morbidity and mortality rates of up to 40%. Such infections can originate from the donor, be out-of-hospital acquired, or correspond to the reactivation of existing latent endogenous viruses in the patient.

 

Successful prevention and early detection of viral infections, including reactivation, are the basic principles of transplant patient management. For effective strategies of preventive and therapeutic treatment, accurate quantification of viral load is important. As a rule, in immunocompromised organisms, the reactivation of several viruses can occur simultaneously, making a comprehensive identification of replicating pathogenic viruses extremely important. Monitoring of opportunistic viral infections in patients after transplantation is most often performed using several quantitative PCR assays.

 

Among the manufacturers of molecular diagnostic kits for testing immunocompromised patients or transplanted organs for PCR is the world-renowned company GeneProof.

GeneProof is a biotechnology company operating in the field of in vitro molecular diagnostics of serious infections and genetic diseases [3].

 

GeneProof's portfolio is primarily focused on infectious diseases of viral and bacterial origin. With a strong focus on quality, GeneProof offers technologically advanced real-time PCR kits [4].

GeneProof's solutions for diagnosing immunocompromised patients or organ transplant recipients include the following kits:

 

Cytomegalovirus (CMV)

Human cytomegalovirus is a common opportunistic infection among immunocompromised individuals. The immunity of such individuals is maximally weakened using immunosuppression during solid organ transplantation or hematopoietic stem cell transplantation (HSCT), and thus they are prone to reactivation of human CMV (latent virus), primary infection, and reinfection. CMV infections can cause severe morbidity and transplant failure, often resulting in prolonged hospitalization and significantly higher treatment costs. Transplantation from a seropositive to a seronegative (R-/D+) individual poses the greatest risk of CMV-associated disease in recipient patients. Therefore, determination of the serologic status of the recipient and donor is important for assessing the risk of developing CMV-associated disease. However, it can be difficult to find a donor and recipient with the same serostatus, and even a matching serostatus does not completely eliminate the risk of CMV-associated morbidity.

 

Coordinated innate and adaptive immune response is crucial for control of HCMV infection in immunocompromised transplant recipients. Whereas innate interferon (IFN) and natural killer (NK) cell responses are important in immediate control of CMV infection, adaptive T cell immune responses are important in both active infection and reactivation control phases [5].

 

Epstein-Barr Virus (EBV)

Epstein-Barr virus (EBV) is a ubiquitous herpes virus that infects the majority of the population worldwide. The virus can establish a lifelong latent infection in host B-lymphocytes. In the setting of immunocompromise as is the case post transplantation, the virus can reactivate and cause one of the deadliest complications post hematopoietic stem cell transplantation (HSCT), post-lymphoproliferative disease (PTLD), the incidence of which has been increasing. Multiple risk factors have been associated with the onset of PTLD such as age, reduced intensity conditioning, EBV serology mismatch and cytomegalovirus (CMV) reactivation. The rarity of clinical trials involving PTLD and the lack of approved treatment modalities renders the management of PTLD challenging. While the first-line treatment involves weekly administration of rituximab, there is no consensus when treating rituximab-refractory PTLD [6].

 

BK/JC Virus (BK/JC)

Polyoma viruses are ubiquitous infecting many different mammalian species including humans. There are five known human polyoma viruses. Most human polyoma disease is caused by BK and JC viruses which are usually acquired in childhood.

 

Approximately 50–80% of humans have seropositivity to these viruses. Clinically apparent diseases in immunocompetent hosts are extremely rare. These viruses remain latent possibly in the lymphoid organs, neuronal tissue, and kidney and under the circumstances of severe immunosuppression both these viruses reactivate. Neurotropic JC virus reaches the brain and causes progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system with a high mortality rate. BK virus is urotheliotropic and its reactivation causes a form of interstitial nephritis, known as BK or polyoma virus associated nephropathy which is associated with high graft loss if not recognized early. There are no known effective antiviral agents for any of the polyoma viruses [7].

 

BK virus, first isolated in 1971, is a significant risk factor for renal transplant dysfunction and allograft loss. Unfortunately, treatment options for BK virus infection are limited, and there is no effective prophylaxis. Although overimmunosuppression remains the primary risk factor for BK infection after transplantation, male gender, older recipient age, prior rejection episodes, degree of human leukocyte antigen mismatching, prolonged cold ischemia time, BK serostatus and ureteral stent placement have all been implicated as risk factors. Routine screening for BK has been shown to be effective in preventing allograft loss in patients with BK viruria or viremia. Reduction of immunosuppression remains the mainstay of BK nephropathy treatment and is the best studied intervention. Laboratory-based methods such as ELISPOT assays have provided new insights into the immune response to BK and may help guide therapy in the future.

 

BK virus can be detected in both blood and urine. After reactivation of BKV, the virus is first detected in the urine, and viremia develops in a few weeks. The viral load of BKV is measured by real-time PCR [8].

 

Adenovirus

Adenoviruses, non‐enveloped, lytic double‐stranded DNA viruses are causing mostly self‐limited respiratory, gastrointestinal, or conjunctival disease in immunocompetent patients throughout the year.

 

Although there is no consensus on the definitions of adenovirus infection and disease, we propose to maintain the same definitions published in the previous guidelines. Asymptomatic adenovirus infection is defined as detection of adenovirus in patients from stool, blood, urine, or upper airway specimens (by viral culture, antigen tests, or PCR) in the absence of signs and symptoms associated with adenovirus disease.

 

Adenovirus disease is defined as the presence of attributable organ signs and symptoms combined with adenovirus detection in the biopsy specimens (immunohistochemical stain) or from bronchoalveolar lavage or cerebrospinal fluid (culture, antigen detection, or PCR), in the absence of another diagnosis. A positive adenovirus PCR from tissue or fluid and a negative immunohistochemical stain is not consistent with invasive disease. A positive adenovirus PCR test result from tissue or fluid is difficult to interpret in the absence of immunohistochemical confirmation. Adenovirus disease is considered disseminated if two or more organs are involved, not including viremia. The ability of adenovirus to establish latency may lead to challenges in the interpretation of the presence of DNA in clinical specimens.

 

The available diagnostic methods for adenovirus infections are as follows: viral culture, direct antigen detection, molecular methods, and histopathology. Serology and electron microscopy are available, but not routinely used in clinical practice [9].

 

Aspergillus

The number of patients undergoing transplantation has increased exponentially in recent years. Transplant recipients are among the most significant subgroups of immunosuppressed hosts at risk for invasive aspergillosis [10].

 

Aspergillus species are the major cause of health concern worldwide in immunocompromised individuals. Opportunistic Aspergilli cause invasive to allergic aspergillosis, whereas non-infectious Aspergilli have contributed to understand the biology of eukaryotic organisms and serve as a model organism. Morphotypes of Aspergilli such as conidia or mycelia/hyphae helped them to survive in favorable or unfavorable environmental conditions. These morphotypes contribute to virulence, pathogenicity and invasion into hosts by excreting proteins, enzymes or toxins [11].

 

Aspergillosis can only be proven either by histology or by culture from a physiologically sterile source. In theory, this could be a blood culture but in clinical practice Aspergillus fungal growth from the scanblood is extraordinarily infrequent.

 

Apart from antigen detection from blood and other clinical samples, the detection of specific sequences of the fungal genome is another method promising earlier diagnosis. Since the 1990s, different polymerase chain reaction techniques have been used to detect circulating fungal DNA [12].

 

Herpes Simplex Virus (HSV-1/2)

Herpes simplex viruses are among the most ubiquitous in human infections. In most cases, HSV infections in individuals with an intact immune system are relatively mild and can cause discomfort but often go unnoticed. In rare instances, HSV can enter the bloodstream, leading to more severe manifestations affecting multiple regions of the skin, the viscera, and the central nervous system. This is especially true for neonates and individuals with an immunocompromised status, for whom HSV infections or reactivations can be life-threatening.

 

In solid organ transplantation recipients, HSV infection or reactivation can also lead to more severe manifestations, including esophagitis, hepatitis, pneumonitis, and potential graft loss. Therefore, achieving a rapid, accurate, and definitive diagnosis of HSV bloodstream infections is crucial in clinical settings. Rapid diagnosis enables timely administration of antiviral treatment, influences patient management decisions for those at high risk, and can contribute to shorter hospital stays, thereby reducing healthcare costs.

 

Various methods have been employed for the diagnosis of HSV-1 and HSV-2 infections including conventional viral culture, serological tests, and nucleic acid amplification tests (NAAT). Among these diagnostic methods, polymerase chain reaction (PCR) is widely utilized due to its high sensitivity and specificity, as well as its short turnaround time for detecting viral nucleic acid [13].

 

Herpesvirus 6/7 (HHV-6/7)

In recent years, there has been growing interest in the role of human herpes virus type 6 and 7 as emerging pathogens or copathogens in transplant recipients. HHV-6 and HHV-7 belong to the β-herpesvirus family and are closely related to another member of this family, cytomegalovirus. After primary infection, these viruses remain latent in the human body and can reactivate after transplantation. Various clinical processes, such as fever, rash, pneumonitis, encephalitis, hepatitis and myelosuppression, have been described in association with herpesvirus. Furthermore, there is growing evidence that the main impact of HHV-6 and HHV-7 reactivation in transplantation is related to indirect effects, such as their association with cytomegalovirus disease, increased opportunistic infections, and graft dysfunction and rejection. The pathogenesis of HHV-6 and HHV-7 in the post-transplant period, methods of their diagnosis, as well as the evaluation of antiviral drugs and strategies for their prevention and treatment are currently the subject of extensive research [14].

 

Herpesvirus 6/7 (HHV-6/7)

In recent years, there has been growing interest in the role of human herpes virus type 6 and 7 as emerging pathogens or copathogens in transplant recipients. HHV-6 and HHV-7 belong to the β-herpesvirus family and are closely related to another member of this family, cytomegalovirus. After primary infection, these viruses remain latent in the human body and can reactivate after transplantation. Various clinical processes, such as fever, rash, pneumonitis, encephalitis, hepatitis and myelosuppression, have been described in association with herpesvirus. Furthermore, there is growing evidence that the main impact of HHV-6 and HHV-7 reactivation in transplantation is related to indirect effects, such as their association with cytomegalovirus disease, increased opportunistic infections, and graft dysfunction and rejection. The pathogenesis of HHV-6 and HHV-7 in the post-transplant period, methods of their diagnosis, as well as the evaluation of antiviral drugs and strategies for their prevention and treatment are currently the subject of extensive research.

 

Recent years have witnessed a growing interest in the role of human herpesvirus (HHV) type 6 and type 7 as emerging pathogens or copathogens in transplant recipients. Both HHV-6 and HHV-7 belong to the beta-herpesvirus family and are closely related to another member of the family, cytomegalovirus. After the primary infection, these viruses remain latent in the human host and can reactivate after transplantation. Various clinical processes such as fever, rash, pneumonitis, encephalitis, hepatitis, and myelosuppression have been described in association with herpesvirus. Moreover, a growing body of evidence suggests that the major impact of HHV-6 and HHV-7 reactivation in transplantation is related to indirect effects, such as their association with cytomegalovirus disease, increased opportunistic infections, and graft dysfunction and rejection. The pathogenesis of HHV-6 and HHV-7 during the post-transplantation period, the methods used for their diagnosis, and the evaluation of antiviral drugs and strategies for their prevention and treatment are now the subject of extensive research.

 

Herpesvirus 8 (HHV-8)

Human herpes virus 8 (HHV-8) is a geographically limited virus that causes neoplastic and nonneoplastic diseases predominantly in endemic regions. Primary HHV-8 infection, which is usually asymptomatic in immunocompetent individuals, result in lifelong latency. When the equilibrium between virus and host immunity is disturbed, such as after organ transplantation, HHV-8 may activate molecular pathways that drive oncogenesis.

 

Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease are the major malignancies associated with HHV-8. The incidences of these neoplastic pathologies mirror the geographic HHV-8 seroprevalence, and certain groups of patients are at higher risk. In this context, the risk of HHV-8 and its clinical disease is highest in immunocompromised patients, including transplant recipients. 

 

Solid organ transplant recipients from endemic regions may develop HHV-8 reactivation or primary infection, manifesting as Kaposi's sarcoma or, less commonly, primary effusion lymphoma (PEL) and Castleman's disease; these neoplastic diseases are much less common in regions with low virus prevalence. Currently, there is no standardised method for screening for HHV-8 infection in transplantation, although an HHV-8 PCR assay is available to confirm clinical suspicion of infection [15].

 

Parvovirus B19

Parvovirus B19 is a common, ubiquitous human pathogen. It is a small, single-stranded linear, nonenveloped DNA virus. Its worldwide genetic variability is low, and there is no clear correlation between genotype and distinctive clinical manifestation. Parvovirus B19 replicates most efficiently and preferentially in human erythrocyte precursors. Most people are infected between the ages of 5 and 15 years; by adulthood, up to 80% are seropositive.

 

Infection appears to confer lifelong immunity to immunocompetent hosts, but reinfection is possible in a minority of cases. It has been hypothesized that Parvovirus B19 can persist in the bone marrow and other tissues, supporting possible reactivation rather than reinfection in certain seropositive patients. Parvovirus B19 infection can be either symptomatic or asymptomatic, depending on the age, hematologic and immunologic status of the host. While most patients recollect only nonspecific flu-like symptoms, distinctive clinical entities have been associated with Parvovirus B19 infection in both immunocompetent hosts and solid organ transplant (SOT) recipients.

 

The current use of polymerase chain reaction assays significantly improved the detection of viral DNA. However, some PCR assays are unable to detect non-B19 strains (genotypes 2 and 3). Furthermore, Parvovirus B19 DNA can be detected by PCR in the serum of some patients for a long time after the acute phase of infection. Thus, a positive PCR for Parvovirus B19 must be carefully interpreted in the context of the clinical setting and other laboratory data [16].

 

Varicella-Zoster Virus (VZV)

Varicella-zoster virus (VZV) is a ubiquitous, highly neurotropic, exclusively human α-herpesvirus. Primary infection usually results in varicella (chickenpox), after which VZV becomes latent in neurons of cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis. As humans undergo a natural decline in cell-mediated immunity (CMI) to VZV with age, VZV frequently reactivates to produce zoster, characterized by maculopapular or vesicular rash and dermatomal-distribution pain. Pain and rash usually occur within days of each other [17].

 

Definitive laboratory testing can be used for atypical cases of VZV or herpes zoster (HZ) and should routinely be used for suspected disseminated or visceral disease. Rapid diagnostic methods, including polymerase chain reaction and direct fluorescent assays (DFA), are the methods of choice. PCR testing, the most sensitive test for VZV, can be used for detecting invasive disease, and detects VZV in vesicle fluid, serum, spinal fluid, and other tissues [18].

 

CONCLUSIONS

So, due to the increasing rate of transplants in Ukraine since the end of 2019, the number of PCR tests is only growing for both donors and recipients. PCR diagnostics are often performed proactively to prevent disease in transplant recipients, as well as to diagnose acute or reactivated disease and monitor response to therapy. GeneProof kits will help the researcher in these tasks. The high quality of the kits and the ease of performing the tests include the following key points:

  • one workflow allows combining PCR kits from different diagnostic groups in a single run using Universal PCR Profile and universal PCR Profile enables simultaneous detection of multiple parameters in one run, even DNA and RNA pathogens at the same time;
  • Universal Internal Control is another step how to simplify the lab workflow, especially when multiple assays are combined in a single run; use just one Universal Internal Control at the beginning of the process instead of adding multiple specific Internal Controls and achieve the best efficiency;
  • Dual Target Detection ensures correct pathogen identification and high sensitivity by detecting two independent gene regions of DNA/RNA simultaneously.

 

REFERENCES

1. utcc.gov.ua/transplantatsiya/pytannya-shho-stavlyatsya-najchastishe/

2. moz.gov.ua/uk/transplantacija-v-ukraini-jak-zminilas-galuz-za-ostanni-4-roki

3. www.geneproof.com/about-geneproof/t1054

4. www.geneproof.com/benefits-of-geneproof-pcr-kits/t1105

5. Sezgin, E., An, P., & Winkler, C. A. (2019). Host Genetics of Cytomegalovirus Pathogenesis. Frontiers in Genetics, 10. doi:10.3389/fgene.2019.00616.

6. Al Hamed, R., Bazarbachi, A. & Mohty, M. Epstein-Barr virus-related post-transplant lymphoproliferative disease (EBV-PTLD) in the setting of allogeneic stem cell transplantation: a comprehensive review from pathogenesis to forthcoming treatment modalities. Bone Marrow Transplant 55, 25–39 (2020). https://doi.org/10.1038/s41409-019-0548-7. 

7. Boothpur R, Brennan D.C. Human polyoma viruses and disease with emphasis on clinical BK and JC. J Clin Virol. 2010 Apr;47(4):306-12. doi: 10.1016/j.jcv.2009.12.006.

8. Deirdre Sawinski, Simin Goral, BK virus infection: an update on diagnosis and treatment, Nephrology Dialysis Transplantation, Volume 30, Issue 2, February 2015, Pages 209–217, https://doi.org/10.1093/ndt/gfu023. 

9. Florescu DF, Schaenman J. M; AST Infectious Diseases Community of Practice. Adenovirus in solid organ transplant recipients: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019 Sep;33(9):e13527. doi: 10.1111/ctr.13527.

10. Singh N, Paterson DL. 2005. Aspergillus Infections in Transplant Recipients. Clin Microbiol Rev 18: https://doi.org/10.1128/cmr.18.1.44-69.2005. 

11. Shankar, J., Tiwari, S., Shishodia, S. K., Gangwar, M., Hoda, S., Thakur, R., & Vijayaraghavan, P. (2018). Molecular Insights Into Development and Virulence Determinants of Aspergilli: A Proteomic Perspective. Frontiers in Cellular and Infection Microbiology, 8. doi:10.3389/fcimb.2018.00180.

12. Maschmeyer, G., Haas, A., & Cornely, O. A. (2007). Invasive Aspergillosis. Drugs, 67(11), 1567–1601. doi:10.2165/00003495-200767110-00004.

13. Zhen W, Sheikh F, Breining DA, Berry GJ. 2024. Rapid diagnosis of herpes simplex virus 1 and 2 bloodstream infections utilizing a sample-to-answer platform. J Clin Microbiol 62:e00131-24. https://doi.org/10.1128/jcm.00131-24. 

14. Natividad Benito, Asunción Moreno, Tomás Pumarola, M.ª Ángeles Marcos, Virus del herpes humano tipo 6 y tipo 7 en receptores de trasplantes, Enfermedades Infecciosas y Microbiología Clínica, Volume 21, Issue 8, 2003, Pages 424-432, ISSN 0213-005X, https://doi.org/10.1016/S0213-005X(03)72980-2.

15. Ariza-Heredia, Ella J.; Razonable, Raymund R. Human Herpes Virus 8 in Solid Organ Transplantation. Transplantation 92(8): p. 837-844, Oct 27, 2011. | DOI: 0.1097/TP.0b013e31823104ec. 

16. Eid, A. J., & Posfay-Barbe, K. M. (2009). Parvovirus B19 in Solid Organ Transplant Recipients. American Journal of Transplantation, 9, S147–S150. doi:10.1111/j.1600-6143.2009.02905.x. 

17. Yawn BP, Gilden D. The global epidemiology of herpes zoster. Neurology. 2013 Sep 3;81(10):928-30. doi: 10.1212/WNL.0b013e3182a3516e.

18. Pergam SA, Limaye AP; AST Infectious Diseases Community of Practice. Varicella zoster virus in solid organ transplantation. Am J Transplant. 2013 Mar;13 Suppl 4(Suppl 4):138-46. doi: 10.1111/ajt.12107.

Published

30 січня 2025