Genotyping of Torque Teno Virus among Women with Urinary Tract Infection in Diyala Governorate

doi:10.21203/rs.3.rs-3318276/v1

Introduction: Urinary tract infections are a persistent global problem; several types of pathogens associated with this disease Torque Teno Virus is a possible cause that can affect all ages and affect women more than men due to anatomy and physiological functions.

Objectives: To determine the infection rate and genotypes of Torque Teno Virus among women with urinary tract infection and study the correlation between Torque Teno Virus infection and different parameters.

Methods: A cross-sectional study was performed using data obtained from women with urinary tract infections (50 pregnant women and 50 non-pregnant women). Urine samples were collected for microscopic and macroscopic examination, bacterial culture, DNA extraction, followed by amplification with specific primers by a nested polymerase chain reaction for phylogenic analysis.

Results: Out of 100 samples, eight samples were positive for Torque Teno Virus (8%). The infection rate of Torque Teno Virus was more common (75%) in the age group (31-47) years with statistical significance. Three cases (37.5%) of Staphylococcus aureus and 2 cases (25%) of each of Escherichia coli and Proteus mirabilis, and 1(12.5%) Enterococcus fecalis showed co-infection with positive Torque Teno Virus cases. The phylogenetic analysis results for Torque Teno Virus DNA isolated from urine samples revealed that all local isolates (8 isolates) belong to type 1 and 2. According to the closest, the phylogenetic tree results analysis showed the local isolate (No. 6 ) clustered with the reference isolates (Egypt, USA, England, Australia and Saudi Arabia). Isolate (No.1) showed was close to Italian and Brazilian isolates, while isolates number (2 and 5), (3 and 7), and (4 and 8) are very closely related to each other and closed to isolates from Iran.

Conclusion: The genotyping analysis of Torque Teno Virus among women with UTI in Diyala Governorate found that all isolates in the studied group belong to TTV genotypes 1 and 2, local isolate (No. 6) was close to isolates from different regions such as Egypt, USA, England, Australia and Saudi Arabia, local isolate (No. 1) was close to Italian and Brazilian isolates. Moreover, the other six isolates were closed to Iranian isolates.

General Cell Biology & Physiology

General Microbiology

Virology

Developmental Biology

Urinary tract infections

Torque Teno Virus

Molecular detection

Phylogenic analysis

Urinary tract infection (UTI) is an important clinical problem, more than half of women and 1 in 10 men are affected during their lifetime. Many of these infections affect the lower urinary tract, but recurrent pyelonephritis can lead to scarring and chronic kidney disease (1). Urinary tract infection is responsible for 25% of total infections, females are more susceptible to UTI than males, more than 50% of women suffer from UTI during their life (2). Urinary tract infection, especially recurrent UTI, is a common problem, with the prevalence of up to 75% among kidney transplant (KTx) recipients. Older age, female gender and delayed graft function are among the independent risk factors for recurrent UTIs in renal transplant recipients (3).

Factors influencing microbial colonization include environmental characteristics, such as pH, oxygen tension, osmolarity, nutrient availability, adhesion sites, and immune interaction (4). The bacterial etiological agents include Escherichia coli, Klebsiella species, Staphylococcus aureus, coagulase-negative Staphylococci, Proteus mirabilis, Enterococcus species, Pseudomonas aeruginosa, Enterobacter species, non-hemolytic streptococci, Citrobacter species and others (5). Viral pathogens such as human cytomegalovirus, polyomaviruses, John Cunningham virus (JC) or BK virus can replicate in the kidney and cause systemic disease, and symptomatic infectious agents associated with significant morbidity and mortality in immunocompromised host (6).

Torque Teno Viruses (TTV) are small, ubiquitous viruses with a highly diverse, single-stranded, negative-sense DNA genome and a broad host range. They are detected at high rates in healthy and diseased individuals and are considered a significant part of the mammalian virome (7). The genome consists of two main parts, the non-coding and coding regions. The non-coding sequence is conserved, about 1.2 kb in size and regulates replication. The coding region consists of (3–5) open reading frames (ORFs) (8). The horizontal and vertical transmission of Torque Teno Viruses among the human population and interspecies transmission is potentially related to the global widespread of TTV infection (9). Torque Teno Virus has been detected in almost every human tissue type or body fluid reaching near to 100% prevalence. Several studies reported mother to child postnatal transmission of TTV in infancy, but the transplacental transmission of TTV is still unclear (10). Previous studies have demonstrated varying grades of association between TTV DNA levels and immune deficiencies related to age, chronic infections and cancer (11, 12).

Several Iraqi studies were conducted in different cities and reported different percentages such as that of 2% among patients with haemoglobinopathies and haematological malignancies in Basrah Governorate (13), 15% of hemodiyalsis patients had TTV DNA in Kirkuk (14), recently, a high rate (43.33%) in saliva and 40% in tumor biopsy) in oral carcinoma patients than in controls (18.33% in saliva )(15). To the best of our Knowledge, there is no Iraqi study among women with urinary tract infection in Diyala Governorate, thus this study aimed to detect Torque Teno Virus among women with urinary tract infections in Diyala Governorate.

Study Design

A cross-sectional study was performed between the 20th of September to the 20th of January 2021 on 100 women diagnosed with urinary tract infections (50 pregnant and 50 non-pregnant women). The urine samples were chosen according to the criteria of finding pus cells and other indicators that indicate UTI. Their ages ranged from 17–77 years of women admitted to Al-Batool Teaching Hospital for Maternity and Children, Women's Emergency Unit and the Unit of Urological Consultation at Baqubah Teaching Hospital in Diyala Governorate.

Microscopic Examination and Molecular Characterization

Urine samples were collected using disposable gloves and a sterile urine cup to collect 60 − 20 ml of urine. Then, 5ml was taken for direct macroscopic and microscopic examination (16). The samples that contain pus cells were cultivated directly on MacConkey agar and Blood agar (17). All positive specimens were ultracentrifuged at 60000 rpm for 20 minutes, the supernatant removed carefully, and then the sediment collected in sterile 1.5 ml Eppendorf tubes for viral DNA extraction directly (18) by using ZR viral DNA extraction kits (Cat. No. D3015 and D3016, Epigenetics – USA). Then, the samples were labelled and stored at -20 ^oC until the time of analysis.

Primers for Torque Teno Virus gene

Two sets of primers were used to amplify TTV gene fragment, NG059, NG061 (19) are summarized in Tables 1 and 2.

Table 1

The Specific Primer NG059 of the Gene.
Primer *	Sequence	Tm (ᵒC)	GC (%)	Product Size
Forward	5'- ACAGACAGAGGAGAAGGCAACATG- 3'	58.8	50	720 base pair
Reverse	5'-CTGGCATTTTACCATTTCCAAAGTT- 3'	54.7	36	720 base pair

*Integrated DNA technology, Canada.

Table 2

The Specific Primer NG061 of the Gene.
Primer*	Sequence	Tm (ᵒC)	GC (%)	Product Size
Forward	5'-GGCAACATGYTRTGGATAGACTGG-3'	56.1	45.8	271 base pair
Reverse	5'-CTGGCATTTTACCATTTCCAAAGTT-3'	51.8	36.4	271 base pair

*Integrated DNA technology, Canada.

Primers Preparation

The primers were lyophilized and then dissolved in free ddH2O to give a final concentration of 100 pmol/µl as stock solution. The stock was kept at -20^oC to prepare 10 pmol/µl concentration as a working primer, 10 µl of the stock solution was suspended in 90 µl of the free ddH2O water to reach a final volume of 100 µl, that was investigated by IDT (Integrated DNA Technologies Company, Canada).

Nested PCR Principle

A standard PCR has been performed to amplify the N-22 gene (NG059) and (NG061) (20). An amplified pitch (271bp) reverse and forward primer aid and a PCR reaction mix was performed at (25µl) total volumes. As shown in Table (3 and 4).

Table 3

Components of Nested Polymerase Chain Reaction (First Run).
No.	Components	Final Concentration
1	Taq PCR PreMix	12.5µl
2	Forward primer	10 picomols/µl (1µl )
3	Reverse primer	10 picomols/µl (1µl )
4	DNA template	1.5µl
5	Deionized water	9 µl
6	Final volume	25µl

Table 4

Components of Nested Polymerase Chain Reaction (Second Run).
No.	Components	Final Concentration
1	Taq PCR PreMix	12.5µl
2	Forward primer	10 picomols/µl (1µl )
3	Reverse primer	10 picomols/µl (1µl )
4	PCR Product template	1.5µl
5	Deionized water	9 µl
6	Final volume	25µl

Thermocycler was programmed to amplify the genome by MultiGene OptiMax thermal cycler gradient, as shown in Table (5 and 6).

Table 5

The Thermal Cycling Condition for DNA Amplification Specific Primer (NG059) of the Gene (First Run).
No.	Steps	Temperature	Time	Cycles
1	Pre-Denaturation	94 ℃	5 Minutes	1 Cycle
2	Denaturation	94 ℃	1 Second	50 Cycles
3	Annealing	52 ℃	1 Second
4	Extension	72 ℃	1 Second
5	Final extension	72 ℃	7 Minutes	1 Cycle
6	Holding	4℃	-

Table 6

The Thermal Cycling Condition for DNA Amplification Specific Primer (NG061) of the Gene (Second Run).
No	Steps	Temperature	Time	Cycles
1	Pre-Denaturation	94 ℃	5 Minutes	1 Cycle
2	Denaturation	94 ℃	1 Second	50 Cycles
3	Annealing	52 ℃	1 Second
4	Extension	72 ℃	1 Second
5	Final extension	72 ℃	7 Minutes	1 Cycle
6	Holding	4℃	-

Agarose Gel Electrophoresis

Three µl of the processor loading buffer have been mixed with 5 µl of the supposed DNA to be electrophoresis (loading dye). After the mixing process, the process of loading is now to the holes of the gel. Then gel was observed under a UV trans-illuminator to detect DNA band (PCR Products) and compare with ladder (100–1000 bp).

DNA Sequencing and Phylogenetic Analysis

The PCR products sequencing was carried out by sending the PCR DNA products with their specific primers in a freezer bag to Macrogen company in Korea (https://dna.macrogen.com). The sequencing study was designed between the sequence of the standard gene by using the Basic Local Alignment Search Tool (BLAST), which is available at the National Center of Biotechnology Information (NCBI) online at (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The evolutionary analysis was conducted using MEGA6 (Molecular Evolutionary Genetics Analysis software version 6 software).

Statistical Analysis

The Statistical Analysis System- 2012 (21) program was used to detect different study parameters' effect on different factors. Chi-square test was used to significantly compare between percentages (0.05 and 0.01 probability) in this study.

Molecular Detection of TTV

The rate of Torque Teno Virus infection among (50 pregnant and 50 non-pregnant) with urinary tract infection was 8% (8 out of 100) from different regions in Diyala Governorate, while 92% were negative according to the result of nested polymerase chain reaction, as shown in Figure (1).

Distribution of Positive and Negative Torque Teno Virus Infection among Women with Urinary Tract Infection According to Age

The patients in this study were divided into four groups, the highest infection rate 6(75%) were noticed in the age group (31–46) years, followed by one case for each of (17–30 years) and (63–77 years), while no positive case was found among the age group (47–62 years). There was a significant relation with age, as shown in Table (7).

Table 7

Distribution of Torque Teno Virus Infection among Women with Urinary Tract Infection According to Age.
Age	Positive No%	Negative No%
17–30 years	1(12.50%)	39(42.39%)
31–46 years	6(75.00%)	37(40.22%)
47–62 years	0 (0%)	14(15.22%)
63–77 years	1(12.50%)	2(2.17%)
Total	8(100%)	92(100%)
Chi-Square (χ²)	6.25 *	42.348 **
(P ≤ 0.05),* (P ≤ 0.01).

Bacterial Coinfection of TTV Positive Samples

The distribution of positive TTV DNA in urine samples accompanied by positive bacterial growth was detailed in Table (8). three cases (37.5%) for Staphylococcus aureus, followed by 2 cases (25%) for each of Escherichia coli and Proteus mirabilis. While Enterococcus fecalis show a low frequency (12.5%) than others and no positive results were noticed among other species. Statistical analysis did not reveal any significant differences among them.

Table 8

Distribution of Torque Teno Virus Infection According to Culture.
Bacterial isolates	Positive No%	Negative No%
Staphylococcus aureus	3(37.5%)	26(28.26%)
Escherichia coli	2(25%)	20(21.73%)
Proteus mirabilis	2(25%)	1(1.09%)
Enterococcus fecalis	1(12.5%)	11(11.96%)
Klebsiella spp.	0	4(4.35%)
Pseudomonas spp.	0	1 (1.09%)
Streptococcus B-hemolysis	0	7(7.60%)
Candida spp.	0	1 (1.09%)
No growth	0	21(22.83%)
Total	8(100%)	92(100%)
Chi-Square (χ²)	1.000 NS	74.891 **
** (P ≤ 0.01)
NS = Non-significant

Phylogenetic Analysis

The sequences nucleotides analysis of eight local isolates of the TTV from urine produced a significant alignment with strain NA-MU 15 ORF1 gene, partial cds (ID: KY750543.1) length: 269Number of Matches: 1. Pairwise sequences alignment of all eight isolates were performed on the amplicon of ORF1 region (271 bp) compared with reference isolates by using the GenBank library in NCBI site.

The most common approaches to construct the phylogenetic tree are clustering algorithm. that distance-based methods, such as the neighbour-joining method by transforming the sequence data into pairwise distances (dissimilarities), and then using the matrix to estimate the genetic divergence between the eight local isolates and 17 reference isolates using NCBI site and MEGA 6 software. In the current study, the phylogenetic tree results analysis according to the closest, top to bottom, revealed that the local isolate No. 6 clustered with reference isolates (ID: KY750543.1 Egypt, ID: AF397741.1 USA, ID: AJ402241.1, England, ID: AF146809.1 Australia and ID: AY256672.1Saudi Arabia). Isolate No.1 showed a very close identify to ID: AF212332.1Italian and ID: DQ665287.1 Brazilian isolates. Isolates number (3 and 7), (2 and 5) and (4 and 8) were very closely related to each other and close to ID: GQ179967.1Iran isolates. Finally, These results confirmed that they were in P-distance, as shown in (Fig. 2). All local isolates were registered in GenBank under ID (MW513364.1, MW513365.1, MW513366.1, MW513367.1, MW513368.1, MW513369.1, MW513370.1 and MW513371.1).

According to the nested polymerase chain reaction results, the infection rate of TTV was 8% depending on urine samples taken from women with urinary tract infections based on microscopic examination and cultivation who were admitted to Al-Batool Teaching Hospital for Maternity and Children. Women's Emergency Unit and Urological Consultation Unit at Baqubah Teaching Hospital in Diyala Governorate.

Neighbouring countries showed high rates of TTV infection compared to the current study, such as 21% in hemodialysis patients and 26.8% in β- thalassemia patients in Iran (22), 42.9% among hemodialysis patients compared with (19%) for the control group in Saudi Arabia (23). The rate in this study is relatively high compared to other studies, such as the Iranian study, which detected the virus in 4% of healthy individuals and 5.8% in HCV-positive persons (24).

The variation in TTV infection rates in different studies may be due to several factors, such as detection methods. Hence, the current study used a nested PCR technique. While others used quantitative polymerase chain reaction (qPCR). The qPCR method helped detect the most prevalent human TTV genotypes due to no cross-reaction with other viruses (25).

The second cause that may affect TTV infection rates is sample size and type of study population. In the current study, the sample size was (100) urine samples. While the study done by (26) used (607) blood samples and reported (55.4%) co-infected TTV/TTMV in a healthy population, to (96.3%) of TTV / TTMV coinfections of an HBV group in different nationalities by nested PCR in Qatar. Urine samples should be analysed within a short time (less than 1.5–2 hours) after sample collection due to high contamination risk because of multiplying bacteria. Moreover, it is challenging to store due to chemical preservatives resulting in changes in the pH and specific gravity (27). Changes in the environmental pH can induce morphological changes in empty proteinaceous shells occurring in viral capsids after DNA encapsidation (28).

The third cause is using multiple genes in different regions in the genome to detect TTV infection. Using N22 region detected (11%) and re-analyzed TTV prevalence with UTR region found TTV DNA was detected in(95%) of samples in eastern Taiwan indigenes (29). The detection of 5’-UTR primer results showed more prevalence of TTV DNA than N22 primer in patients and healthy control (30). Interestingly, the genome's conserved regions are non-coding areas (UTRs) in TTV and GC rich region, a poly-A sequence downstream and a TATA box and upstream are coding regions (31).

Other factors which may affect the result include immune status, nutrition, therapies, genetic makeup, geographical distribution, seasonal considerations, lifestyle choices (smoking, alcohol consumption, physical activity), and zoonotic transmission (32).

The results of the present study showed that most TTV infections were in the age group (31–46 years) at the rate of 75% (6 cases) while 1case (12.5%) in the age groups (17–30 years) and (63–77 years) with high statistical significance. These results agreed with a study that found TTV prevalence was significantly higher in the very young and elderly by using stool samples from people exhibiting signs of enteritis (954) and from non-diarrheic individuals (76), suggesting that immunological status is essential in infection by using qPCR assay in Canada (33).

A study investigating the association of TTV load and age found a slightly positive correlation with age within a cohort of (379) Italian elderly subjects (34), TTV load was significantly higher in the elderly (50–60 years old) compared to the young group (p < 0.001) during the investigation of TTV DNA load in plasma of (313) healthy persons using real-time PCR in Austria (35).

These results indicate that positive infection with TTV occurred in all age groups due to TTV infecting nearly all people indefinitely without the disease, establishing an acceptable and successful interaction with the host (36).

The current study showed a moderate rate of bacterial growth in relation to positive patient to TTV, who were diagnosed by using many biochemical tests (37), such as Staphylococcus aureus 3(37.5%) sample followed by 2(25%) samples for each of Escherichia coli and Proteus mirabilis. Finally, Enterococcus fecalis 1(12.5%) sample. This result agreed with the result of a study that found that (57.1%) of culture-positive and 100% of culture-negative samples demonstrated TTV DNA's presence, compared to none in the controls in Washington (38). The current study disagreed with a study that concluded that TTV is present in the vagina in many women with typical pregnancy outcomes and that its occurrence is associated with a lack of L. crispatus dominance, an increase in vaginal MMP-8 and a decrease in D-lactic acid in Brazil (39). Potent bactericidal activity was exerted by a minimal number of resident L. gasseri and L. crispatus strains showing the specific ability to produce and release antibiotic-like compounds. It eradicated the microbial pathogens pre-associated with the surface of cervix epithelial cells, providing efficient protection of the cells against the deleterious effects triggered by toxin-producing G. vaginalis and uropathogenic E. coli (40). The vaginal microbiota is a dynamic and often critical factor in this pathogenic interplay because changes in the vaginal microbiota characteristics result in the loss of ordinarily protective Lactobacillus spp., thus increasing the risk of UTI. These alterations may result from the influence of estrogen deficiency, antimicrobial therapy, contraceptives, or other causes (41). The variation in bacterial growth in the study population could be related to the fact that most UTI women have taken antibiotics for treatment.

According to the available information, the present study is considered the first study in Diyala Governorate to deal with a phylogenetic analysis of TTV. The PCR product's sequencing revealed eight local isolates. After nucleotides sequencing and amino acids analysis, a significant alignment with Torque Teno Virus strain NA-MU 15 ORF1 gene, partial with accession number (ID: KY750543.1) length: 269 were produced by using NCBI and MEGA 6 software. The sequences analysis results showed many mutations (transition and transversion) at TTV genome-related by ORF1-N22 region except the local isolate (No. 3) that was 100% identical with the reference isolate Torque Teno Virus strain NA-MU 15 ORF1 gene. All eight isolates nucleotides sequence analysis showed high identity with this reference isolate ranging between 98–100%. Also, high identification ranged between 96–100% with amino acids sequence analysis of all local isolates with this reference isolate.

According to the closest in the phylogenetic tree, the variation between local isolates and reference isolates ranged from 0.01 to 0.03. Distance 0.01 between (isolate No. 6 and reference isolate from Egypt and isolate from the USA), (No.3 and No.7) and (No. 2 and No.5). Distance 0.02 between isolates (No. 6 with reference isolates England, Australia and Saudi Arabia). Also, local isolates (No. 1 and No. 6) and (between local isolates No.4 with No.8). Distance 0.03 (between local isolate No.1 and Italian and Brazilian isolates).

The local isolates may have had multiple recombination and mutation in the Iraqi population this cause the generation of a large number of TTV variants driven by the high mutation rate, which is closer to RNA virus (42). Regarding the phylogenetic tree analysis of eight local isolates, there is a high diversity among them, the phylogenetic tree constructed by the neighbour-joining method according to (43) using MEGA 6 and NCBI software, showed that local isolates clustered with the Egyptian isolate revealed by (44) which reported TTV in a high prevalence among children with thalassemia and non-thalassemic individuals with genotypes 1 and 2. Isolate No. 6 was close to the Egyptian isolate ID: KY750543.1, American isolate ID: AF397741.1, England isolate ID: AF146809.1, Australian isolates ID: AF212332.1 and Saudi Arabian isolate ID: AY256672.1. Local isolate (No. 1) clustered with Italian isolate ID: AJ402241.1and Brazilian isolate ID: DQ665287.1, while local isolates (No.3) showed close relation to isolate (No. 7), isolate (No.2) and isolate (No.5) are very closely related and isolate (No.4) clustered with isolate (No.8) and all these six isolates were close to the Iranian isolate with the accession number ID: GQ179967.1 as shown in Figure (2). This study agreed with the study done by (45) who found that phylogenetic analysis indicated that the most common genotype of TTV among Saudi Arabia is 2. Furthermore, the study found that the data indicates that TTV is widespread genotypes 1, 2 and 3 in Italy in parenterally exposed subjects and that the infection frequently persists (46).

The current study with (17) also disagreed with a Brazilian study which found that the prevalence of TTV in healthy individuals was 69.0% by using ORF N22 primers (47). These results can be explained by the rapprochement between the two countries, sharing water resources, trade exchange and religious tourism, and travelling for study and treatment. Many factors cause high variation in the TTV genome, even in the same geographical area. Amplification of ORF1 (N22 region) gives high diversity among strains when used in phylogenetic analysis (48).

The cause of widely distributed TTV infection among human populations might be explained by multiple transmission modes (9). This could be related to increased travel and communication to a different area for learning, treatment and tourism. Moreover, TTV can transmit by multiple routes such as spreading by exhalation (49) and particles of TTV shed into faeces, demonstrated that they might be highly resistant to the water environment (50).

Genotyping analysis of Torque Teno Virus among women with UTI in the studied group showed that the isolates belong to TTV genotype 1 and 2. Local isolate (No. 6) was close to isolates from different regions such as Egypt, USA, England, Australia and Saudi Arabia. Local isolates (No. 1) was close to Italian and Brazilian isolates. Moreover, the other six isolates were close to the Iranian isolates.

ACKNOWLEDGEMENT

We thank all staff in Al-Batool Teaching Hospital for Maternity and Children and Unit of Urological Consultation at Baqubah Teaching Hospital in Diyala Governorate for their help in this study's sample collection as well as all patients included in this study.

The statement of ethical approval was obtained from the University of Diyala, College of Medicine, Medical Microbiology Branch

COI The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

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Genotyping of Torque Teno Virus among Women with Urinary Tract Infection in Diyala Governorate

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Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS