Prevalence of Torque Teno Virus in Blood Donors and its Implication on Blood Safety in Pakistan

Nosheen Basharat1, Usman Waheed2,3*, Muhammad Arshad1, Noor e Saba4, Iram Masood1, Akhlaaq Wazeer1, Ahmad Farooq1, Sadaf Moneeba1, Abdul Rauf5 and Hasan Abbas Zaheer2,3 1Department of Biological Sciences, International Islamic University, Islamabad 2Department of Pathology and Transfusion Medicine, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad 3Safe Blood Transfusion Programme, Ministry of National Health Services, Government of Pakistan 4Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 5Department of Zoology, University of AJK, Muzaffarabad, AJK Article Information Received 18 July 2017 Revised 22 July 2018 Accepted 28 January 2020 Available online 03 August 2020

B lood transfusions have become a routine process in the hospitals globally. (https://www.nhlbi.nih.gov/health/ health-topics/topics/bt). Presently, transfusion practices are critical part of the health practices. Blood transfusion is required in cases of severe anaemia, surgery, complication of pregnancy and trauma among others. Therefore, proper screening of the donor's blood is pivotal to reduce the risk of transfusion transmitted infections. All donated blood is screened for hepatitis C, hepatitis B, HIV/AIDS, malaria, and syphilis, as these markers can be transmitted to the recipient at the time of transfusion (Karimi et al., 2013).
In 1997, Torque teno virus (TTV) isolate was first discovered in a Japanese patient with post-transfusion hepatitis whose liver had a higher level of enzyme alanine aminotransferase (ALT) (Nishizawa et al., 1997). In 2005, the International Committee on Taxonomy of Viruses (ICTV) proposed to classify TTV into the new genus Anellovirus, of the family circoviridae and to assign to the acronym from the Latin torque (necklace) and tenuis (narrow), referring to the characteristics of their genome (Manzin et al., 2015).
The TTV virion size is 30-50 nm. TTV DNA genome is a non-enveloped, single stranded and covalently circular O n l i n e F i r s t A r t i c l e having size of 3.8 kb (Magua et al., 2015). The virus uses host machinery for replication (Spandole et al., 2015). It has untranslated regions (UTR) and open reading frame (ORF) regions which are highly specific and also used to locate the presence of TTV DNA in samples (de Oliveira, 2015). GC rich region, TATA box and mRNA were also discovered in TTV genome. (Bostan et al., 2013). The TTV is asymptomatic when present alone in the patients (Mankotia and Irshad, 2014). TTV occurrence was reported not only in liver but also in other body organs and tissues like mammary glands, brain, kidney, and bone cells. (Ishimura et al., 2010). Different routes of transmission were observed in TTV transmission into populations, such as parenteral, bile, faeces, etc (Shaheli et al., 2015).
The TTV is not a seasonal or epidemic infection. It can cause temporary and permanent infections. TTV was also believed to be responsible for high level of ALT in infected peoples (Bostan et al., 2013).
Hepatitis viruses (HBV and HCV) and TTV synchronized infection has been widely studied (Spandole et al., 2015). Patients with chronic hepatitis C who develop hepatocellular carcinoma, have elevated Torque Teno-virions (Tokita et al., 2002). Death rate of patients with HBV-TTV co-infection is higher as compared to HBV infection alone (Desai et al., 2005). Although TTV is frequently detected in patients chronically infected with HBV and HCV (Tokita et al., 2002), patient's biochemical and clinical profiles with chronic HBV and HCV coinfected with TTV were not considerably dissimilar from the patients without TTV (Chattopadhyay et al., 2005). However, the possibility that TTV contributes to the progression of liver disease in people infected with HBV or HCV cannot be totally excluded (Spandole et al., 2015).
Global prevalence of TTV infection among healthy donors is observed in many studies. TTV prevalence knowledge in specific regions assist in formulation of a strategy to curtail viral transmission in healthy population. There is complete lack of studies showing prevalence of TTV in healthy blood donors in Pakistan. The current study was conducted to determine the prevalence of TTV in healthy blood donors in Islamabad, Pakistan and to assess the relationship of TTV with hepatitis B and hepatitis C seropositivity.

Materials and methods
The Ethical Review Board (ERB) of Shaheed Zulfiqar Ali Bhutto Medical University (SZABMU) endorsed the study. Samples were collected from Department of Transfusion Medicine, SZABMU. The donors belonged to different geographical areas of Pakistan.
A total of 9,000 samples were collected from November 2016 to June 2017 from healthy blood donors. Out of these, 75 HBV positive (asymptomatic), 75 HCV positive (asymptomatic) and 132 healthy donor samples (non-reactive for HBV, HCV) were included in the study. The sample tubes were centrifuged at 14,000 rpm for 5 min to separate serum/plasma from whole blood. The resulting supernatant was transferred to 1.5ml eppendorf that was used for further serological testing of HBV, HCV and TTV. For HBV and HCV screening, the samples were tested on Abbott's Architect analyzer through chemiluminescence immunoassay (CLIA) as per manufacturer's instructions. Hepatitis B was confirmed by testing for HBsAg while in the case of HCV, testing was performed for anti-HCV antibodies.
All collected samples were tested for ALT level with Chemwell analyzer, (Awareness Technologies, USA). For the qualitative detection of TTV antigen, automated ELISA kits (Abbexaabx053944, Abbexa Ltd., Cambridge Science Park, Cambridge, CB4 0FN, UK) were used, according to manufacturer's instruction.
For PCR amplification, DNA isolation was carried out by using Sambrook and Russel (2001) DNA isolation protocol from serum samples. The isolated DNA was run on 1% agarose gel. Primers shown below and PCR protocol was taken from Ninomiya et al. (2008).

Results
The average of ALT level found in this study was 35 IU/L. Samples were run on 96 well ELISA kits. Collectively ELISA showed 78/282 positive samples from which 15/75 were from HCV, 27/75 from HBV and 36/132 positive results from healthy donors samples. The percentage of TTV infection detected on ELISA among HCV, HBV and healthy donor's samples was 20%, 36% and 27.27%, respectively as shown in the Table I. Samples were then run on PCR thermocycler. Collectively PCR showed 78/282 positive samples of which 18/75 were from HCV, 30/75 from HBV and 30/132 positive results from healthy donor's samples. After PCR samples were checked on 2% agarose gel.
The percentage of TTV infection detected by PCR by counting gel bands on agarose gel electrophoresis among HCV, HBV and healthy donors samples was 24%, 40% and 22.7%, respectively (Table II).
p-value was calculated using SPSS version 20 by 1 sample t-test. P-value of the results found equal to 1.00. The results showed both ELISA and PCR of equal significance. Comparison of both the test (ELISA and PCR) is shown in Figure 1.

Discussion
Screening for transfusion-transmissible infections (TTIs) is a critical part of the process of ensuring that transfusion is as safe as possible. Unsafe blood transfusion is very costly both from a human and an economic point of view. These TTIs include HBV, HCV, HIV, syphilis and malaria. In addition, dengue and HEV are yet to be documented as important TTIs.
TTV is capable of transmission through blood and blood products (El-Taher et al., 2015). Frequency of TTV has been reported as high as 90% in Hong Kong (Niel et al., 1999) and the lowest reported was 29.7% from Belgium blood donors (Ali et al., 2004). Other studies have reported different percentages, e.g. in Brazil the prevalence of TTV was 60%, and 62% in northern and southern region of Brazil, respectively (Pinto et al., 2007). In France, 66% prevalence of TTV was reported in 2006 (Biagini et al., 2006). Chattopadhyay and fellows in 2005 and Magua and group in 2015 reported TTV prevalence of Indian population as 43% and 72%, respectively (Chattopadhyay et al., 2005;Magua et al., 2015). In comparison to all these reports, the TTV prevalence in our studied population was found to be the lowest, i.e. 25%. The results, therefore, highlights the prevalent nature TTV globally but less in Pakistani population.
A study from UAE on TTV prevalence reported 97.9% and 95.7% TTV prevalence in patients with HBV and HCV which are greater than our findings of 38% and 22% (Alfaresi, 2006). Similarly, in India 87.6% and 77% TTV positive viraemia was reported in HBV and HCV which is much higher than our findings (Magua et al., 2015).
In the present study, most of the TTV samples had normal level of ALT, i.e. 35 IU/L. Hence, it was observed that the level of ALT does not have any effect on pathogenicity of TTV. The same findings were reported in Saudi blood donors (Al-Mozaini et al., 2006).

Conclusion
TTV can be transmitted by blood and blood products similar to other TTIs. Therefore, safe blood supply is an uphill task for the national blood transfusion system. TTV prevalence has been reported globally and its prevalence information in Pakistan will help to formulate a strategy for virus control in the healthy population. The study provides sufficient evidence on the significance of TTV for blood safety purposes. Further studies are required to consider inclusion of TTV in routine mandatory screening of blood in blood centers.