Determination of TTV viral load in biological material of target patient groups using a molecular genetic method

BACKGROUND AND AIMS The apathogenic and torque teno virus (TTV) is associated with the state of immunosuppression in solid organ transplant recipients. After kidney transplantation, quantification of TTV viral load may serve as a risk stratification tool for allograft rejection and infectious events. Besides a robust and independent association between recipient age, sex and TTV load, respectively, limited data exists on other potential determinants of TTV load. This trial was designed to analyse the association between TTV and detailed recipient and donor baseline characteristics and clinical follow-up parameters. METHOD This retrospective analysis included all 386 consecutive adult recipients of a kidney allograft transplanted between 1 January 2016 and 30 June 2018 at the Medical University Vienna from the prospective observational TTV-POET trial (institutional review board approval number: 1785/2016; German Clinical Trials Registry number: DRKS00012335). For the present analysis, we included TTV measures before transplantation (d0), at month 1 (m1), at month 3 (m3) and at month 12 (m12) after transplantation. TTV DNA was extracted from patient plasma and assessed by real-time PCR with laboratory-developed primers. TTV load was associated with recipient and donor baseline characteristics and follow-up data. RESULTS The median recipient and donor age at transplantation was 55 years. A total of 135 (35%) kidney allograft recipients were female; 74 patients (19%) had a history of prior kidney transplantation; and 318 patients (82%) received a deceased donor kidney transplant. A total of 34 patients (9%) had pre-formed donor-specific antibodies (DSA); 19 patients (5%) received an AB0-incompatible transplant. For the whole cohort, 1-year patient survival was 95.6% and 1-year death censored graft survival was 94.8%. Baseline TTV load (d0) was lower in female recipients (P = 0.003) and higher in older recipients {β 0.033, [95% confidence interval (95% CI) 0.002–0.066]; P = 0.039}. Baseline TTV load was not associated with recipient body mass index and a history of immunological causes of end-stage renal disease and diabetes mellitus, respectively. TTV load within the first year after kidney transplantation did not show an association with the prevalence of preformed DSA, induction treatment, AB0-incompatibility and HLA-mismatch, respectively. Our trial detected an association between donor age and TTV load. Recipients from older donors showed higher TTV loads (m1, β 0.037, 95% CI 0.008–0.065, P = .01; m3, β 0.04, 95% CI 0.012–0.068, P = .005; m12, β 0.065, 95% CI 0.028–0.01, P = .001). There was no association between TTV load during the first year after transplantation and donor sex. When investigating the association between TTV load and clinical follow-up data, TTV levels showed a negative correlation with peripheral blood leukocyte and lymphocyte counts during the first year after transplantation [lymphocytes: m1, β –0.867, 95% CI –1.367–0.364, P = .01; m3, β –0.985, 95% CI –1.709–0.260, P = .008; m12, β –1.304, 95% CI –2.156–0.452, P = .003; data on leukocytes not shown). These findings might reflect the role of lymphocytes for TTV control. Additionally, TTV viral load negatively correlated with the estimated glomerular filtration rate (MDRD) within the first year after kidney transplantation (m1, β –0.043, 95% CI –0.066–0.02, P < .001; m3, β –0.024, 95% CI –0.049–0.001, P = .06; m12, β –0.078, 95% CI –0.111–0.045, P < .001). One might speculate that a higher eGFR might be a proxy for a healthier donor being more capable of controlling TTV infection. TTV viral load was associated with tacrolimus trough levels in month 12 after transplantation (β 1.262, 95% CI 0.0221–0.250; P = .019), but not with the amount of mycophenolic acid dosage. CONCLUSION This study revealed an association between TTV viral load and distinct baseline donor and recipient characteristics and clinical follow-up data, including kidney function. Further analysis, especially longitudinal assessment of TTV levels, may help to further dissect the interplay between TTV viral load and the state of immunosuppression in patients undergoing kidney transplantation.

Prospective cohort study of Torque Teno Virus (TTV) viral load kinetics and the association with graft rejection in renal transplant patients.

Renal impairment is a common complication after liver transplantation (LT). While BK polyomavirus (BKV) has been linked to renal failure in kidney transplant recipients, Torque teno virus (TTV) is a surrogate marker for immunosuppression that does not have a clear association with any human disease. The impact of BKV and TTV on renal impairment after LT is unknown. In this retrospective study, urine and serum samples from 136 liver transplant recipients were screened for BKV and TTV by quantitative PCR. In addition, serum was screened for BKV-specific antibodies and the VP1 typing region was sequenced for BKV genotyping. All parameters were correlated with clinical data.Results/Key findings. BK viruria was detected up to 21 years after transplantation in 16.9 % of cases. BK viraemia was detected in 8.7 % of patients with BK viruria up to 4 years after LT. BKV-specific antibodies were detected in 93.6 % of all LT recipients and correlated with BKV viral load in urine. There was no correlation between renal impairment and the detection of BK DNA in urine (OR 0.983). TTV DNA was detected in 84.6 % of serum samples and in 66.6 % of urine samples. The TTV viral load in serum correlated with the BKV viral load but had no impact on renal impairment. Our data indicate that the detection of BKV and TTV is not a risk factor for renal impairment after LT. A correlation of TTV and BKV viral load seems to be an indicator for the immune status of the host.

Torque teno virus (TTV) is an unenveloped, circular, single stranded DNA virus with a genome size of approximately 3.8 kb. Previous studies have demonstrated varying grades of association between TTV DNA levels and immune deficiencies related to age, chronic infections and cancer. Alzheimer's disease (AD) has been related to persistent viral infections such as HSV-1 and CMV, but it is not known whether TTV viral load could serve as a functional biomarker of cellular immunity in this setting. Therefore, the objective of this study was to investigate whether TTV infection and viral load is related to AD status, CMV immunity, systemic inflammation or HLA types connected to anti-viral immunity. A total of 50 AD subjects and 51 non-demented controls were included in the study. AD subjects were diagnosed according to NINCDS-ADRDA and DSM-IV criteria and neuroradiologic findings were consistent with the diagnosis. TTV viral load was analyzed in plasma samples using a quantitative real-time PCR. Using a cut-off for TTV status at 200 copies/ml, 88% (89/101) of the study subjects were classified as TTV positive. TTV viral load significantly increased with age (beta 0.049 per year, p<0.001) but significantly decreased in relation to CMV IgG levels (beta -0.022 per 1000 units, p = 0.005) and HLA-B27 positivity (beta -0.53, p = 0.023). In conclusion, TTV immune control is not significantly affected by AD status, but appears related to age, CMV humoral immune response and HLA type.

HIV-1 infection induces a general status of perturbation on immune system cells leading patients to be susceptible to opportunistic infections and tumours. Immune cells of HIV-1-infected patients show several functional defects associated to the chronic cell-hyperactivation and cell-exhaustion. Most of these impairments characterize advanced stage of HIV-1 disease (CD4+ T cell lymphopenia and ongoing HIV-1 replication). To investigate if signs of B cells exhaustion and impaired viral immune-surveillance were present in asymptomatic HIV-1-infected patients with preserved CD4+ T cell counts and Highly Active Antiretroviral Therapy (HAART)-untreated. Forty-three asymptomatic HAART-untreated HIV-1-infected patients were evaluated for immunological parameters defining immune exhaustion of B cells, and for Torque Teno Virus (TTV) viral load. Twenty aviremic HAART-treated patients and 34 healthy individuals were used as control groups. Peripheral blood samples were analyzed by flow cytometry for the identification of B cell subpopulations. IL-7 serum levels were quantified by ELISA. TTV viral load was determined by real-time PCR. Mann-Whitney U-test and Spearman rank correlation coefficient test were used for statistical analysis. Asymptomatic HIV-1-infected patients showed dramatic expansion of exhausted tissue-like memory B cells, which correlated with HIV-1 and TTV viral loads. They also displayed increased IL-7 levels. Normal B cell subsets, IL-7 levels and TTV viral load were observed in aviremic HAART-treated patients. Taken together these results show that asymptomatic HIV-1-infected patients showed the emergence of exhausted B cell elements associated with an impaired control of TTV replication. Successfully HAART-treated patients showed normal B cell subpopulations frequency and TTV viral load. Early application of HAART may prevent the loss of immune functions.

Torque Teno Virus Monitoring in Pediatric Hematopoietic Stem Cell Transplantation

Torque Teno Virus DNA Load after Heart Transplantation and Its Association with the Strength of Immunosuppression: Preliminary Data of a Prospective Single Center Study

No correlation between Torque Teno virus viral load and BK virus replication after kidney transplantation

Torque teno virus (TTV) replication is tightly modulated by the host immune response. As such, TTV viremia has been investigated as a biomarker of immune competence for monitoring the risks of allograft rejection and opportunistic infections in solid organ transplant recipients. We aimed to investigate the associations between TTV levels in bronchoalveolar lavage fluid samples (BALF) and pathogen detection in patients with hematological diseases with suspicion of lower respiratory tract infection (LRTI). TTV DNA was quantified in BALF using qPCR. Linear and logistic regressions were used to study associations between TTV levels in BALF and patients' characteristics, cytological patterns and pathogen detection. A total of 330 BALF from 272 patients with hematological malignancies were included. TTV DNA was detected in 44.5% of samples. The median TTV viral load (VL) in positive samples was 6.24 log copies/mL (IQR: 4.68-7.66). Strong positive associations were found between TTV levels in BALF and a diagnosis of lymphoma, a neutrophil fraction > 3% of BALF cells, and the detection of at least one respiratory virus. To a lesser extent, allogeneic hematopoietic stem cell transplantation, significant bacterial growth in BALF culture, and cytomegalovirus detection in BALF were also associated with TTV levels. We identified in this study multiple associations with TTV VL in BALF. These findings suggest that high TTV levels in BALF could reflect an impaired local immune status, and correlate with the risk of viral LRTI in immunocompromised patients.

Torque Teno virus (TTV) is a ubiquitous virus that causes chronic infection in humans with unknown clinical consequences. Here, we investigated the influence of TTV infection on HCV direct-acting antiviral (DAA) efficacy in HIV/HCV coinfected and HCV monoinfected patients as controls. Of 92 study patients, 79.3% were TTV DNA positive; untreated patients exhibited a significantly higher proportion of TTV DNA-positivity vs. sustained virological response (SVR) patients (100.0% vs. 65.2%, p < 0.001), while TTV positivity was not significant in DAA failure patients vs. SVR patients despite HIV/HCV coinfection. TTV DNA viral load was higher among HCV monoinfected patients vs. HIV/HCV coinfected, although marginally significant (p = 0.074) and no significant viral load difference was detected between DAA failures and SVR patients, while untreated vs. SVR patients had a significantly higher viral load (19,884, IQR 5977–333,534, vs. 469, IQR 10–4124, p = 0.004). Alpha-genogroup 3 TTV was the most prevalent genetic group, and no specific strain or genogroup was observed in relapser patients. Among HIV/HCV patients with HCV RNA detectable at end of treatment (EOT), TTV DNA was detected in 9/17 treatment responder patients and 3/5 relapser patients, thus, TTV infection does not appear to influence the control HCV viremia after EOT. Levels of IL-6 IL-4, and CD14 were not significantly different between TTV PCR-positive and -negative patients. These results suggest no association between TTV DNA positivity or viral load and HCV DAA failure whether patients were HIV/HCV coinfected or HCV monoinfected.

Studies have shown that Torque teno virus (TTV) viral load predicts antibody response to SARS-CoV-2 mRNA vaccines in kidney transplant recipients (KTRs). However, its correlation with pneumococcal vaccine antibody response remains unknown. We enrolled KTRs receiving the 13-valent pneumococcal conjugate vaccine (PCV13). TTV viral load and 12 pneumococcal serotype-specific (PSS) IgG antibodies were measured at baseline. PSS IgG antibodies again 12 weeks post-vaccination. Vaccine responders were defined as those with a minimum two-fold increase in the geometric mean concentration across all 12 PSS IgG antibodies. Logistic regression was used to identify predictors of vaccine response, and receiver operating characteristic curve analysis assessed the diagnostic performance of TTV viral load. Among 65 vaccinated KTRs, 38% responded to the vaccine. TTV viral load was inversely associated with vaccine response (adjusted odds ratio: 0.64, 95% CI: 0.46-0.88, p = 0.006); no other variables were significantly associated. TTV viral load demonstrated modest diagnostic utility, with an area under the curve of 0.74 (95% CI: 0.61-0.84) and an optimal threshold of 4.71 log₁₀ copies/mL (sensitivity 60%, specificity 85%). These findings suggest that TTV viral load may serve as a predictor of PCV13 vaccine responsiveness and could help optimize immunization strategies in KTRs.

Torque Teno Virus Does Not Predict Cytomegalovirus Infection Post-Lung Transplantation

Among immunocompromised patients with acute respiratory failure, identification of those at higher risk for opportunistic infections is crucial to optimize management. The torque teno virus (TTV) DNA burden in the blood has been identified as a surrogate marker of functional immunity in solid organ transplant recipients. This study investigates the clinical relevance of the presence of TTV DNA in nasopharyngeal swabs of immunocompromised patients with acute respiratory failure (ARF). We enrolled immunocompromised patients with ARF admitted to 32 intensive care units. Nasopharyngeal swabs collected on admission were tested for TTV DNA. Causes of ARF were reviewed by three expert investigators blinded to TTV results, with specific attention to the presence of opportunistic infections. The primary endpoint was the association between TTV DNA burden in nasopharyngeal swabs and the rate of opportunistic infections causing the ARF. Of the 505 patients, respiratory TTV DNA was detected in 304 of 505 (60%), with TTV burden ≥2.9 log10 copies/mL in 184 of 305 (36%). TTV burden ≥2.9 log10 copies/mL was significantly associated with a higher prevalence of opportunistic infections (20% [36/178] vs. 11% [33/307]; adjusted odds ratio, 2.41; 95% CI, 1.35-4.28; p 0.002). High TTV burden ≥2.9 log10 copies/mL was also associated with a higher rate of all cause pulmonary infections (67% [119/178] vs. 56% [108/192] when not detected), microbiologically documented bacterial infections (35% [62/178] vs. 23% [45/192]), and with a higher rate of influenza-like respiratory virus detection in nasopharyngeal swabs (15% [27/184] vs. 6% [12/201] when not detected). Furthermore, TTV detection was associated with a higher rate of mechanical ventilation or death at day 28 (59% [179/304] vs. 48% [97/201] when not detected). In immunocompromised patients with ARF, high TTV burden in the respiratory tract is associated with higher rates of pulmonary infections due to opportunistic pathogens and with adverse outcomes.

Torque teno virus (TTV) is a recently identified virus that has a wide range of host tropisms from humans to shrews. Human TTV and Torque teno mini virus are distributed worldwide, and their high prevalence in human populations has been reported. Pigs have their own species-specific TTV, and like human TTV, a high prevalence of porcine TTV also has been reported. Despite its high prevalence, the role of TTV-related disease or syndrome in humans and pigs has not been determined. In the swine industry, TTV is thought to be one of the agents that aggravate clinical manifestation of Porcine circovirus-associated disease (PCVAD), a newly emerging, economically devastating disease. The purpose of the current study was to quantify TTV viral load in serum obtained from Porcine circovirus-2-negative pigs and PCVAD-affected pigs with real-time quantitative polymerase chain reaction assays and to compare TTV viral load between these groups. Results of this study indicate that there are no statically remarkable differences in TTV viral load between the 2 groups, which indicates that TTV might not be an agent of aggravation in PCVAD.

Torque Teno Virus (TTV) is a small, non-enveloped, single-stranded and circular DNA virus that infects the majority of the population worldwide. Increased levels of plasma TTV viral load have been observed in various situations of immune deficiency or dysregulation, and several studies have suggested that TTV levels may be inversely correlated with immune competence. The measurement of TTV viremia by qPCR has been proposed as a potential biomarker for the follow-up of functional immune competence in immunosuppressed individuals, particularly hematopoietic stem cell transplant recipients. We hypothesized that TTV viral load could be used as a prognostic marker of immune checkpoint inhibitor (ICI) efficacy, and therefore investigated the TTV viral load in melanoma patients treated with nivolumab or pembrolizumab before and after 6 months of treatment. In the present study, TTV viral load was not different in melanoma patients before anti-PD-1 introduction compared to healthy volunteers, was not modified by ICI treatment and did not allowed to distinguish patients with treatment-sensitive tumor from patients with treatment-resistant tumor.