Introduction

The human T-lymphotropic viruses also known as human T-cell leukemia-lymphoma viruses (HTLV) are RNA group of viruses those belong to retroviruses family. The family has four species, which include HTLV-1, HTLV-2, HTLV-3, and HTLV-4. These species are known to cause human disease [1].

The viral RNA is packed into the icosahedral capsid, which is contained inside the protein inner envelope. The lipid outer envelope is of host cell origin but contains viral trans membrane and surface proteins. The virion is spherical in shape with a diameter of about 100 nm. The virus genome consists of two identical genomic RNA strands which incorporated into a capsid protein that contain also functional protease integrase, and reverse transcriptase enzymes [5]. The viral genome is dependent on cellular factors for the initial rounds of transcription. The complex retroviral genome codes for the structural proteins Gag (capsid, nucleocapsid, matrix), Protease, polymerase and Envelope from unspliced/singly spliced mRNAs and regulatory and accessory proteins from alternatively spliced mRNA transcripts [5,6].

HTLV-1 is involved in actively spreading epidemics, affecting 15-20 million people worldwide [6]. It was the most clinically significant over all types of virus with at least 500,000 of the individuals infected with HTLV1 eventually develop an often rapidly fatal leukemia, while others will develop a debilitative myelopathy, and yet others will experience uveitis, infectious dermatitis, or another inflammatory disorder. However, HTLV2 is associated with milder neurologic disorders and chronic pulmonary infections. Moreover, there were no specific illnesses have yet been associated with HTLV-3 and HTLV-4 [6].

Up to 2014 the estimated adult seroprevalence of the HTLV-1 was at least 1-2 % but it can also reach 20-40 % among people aged over 50 years in specific clusters. The main highly endemic areas are the southwestern part of Japan, some parts of the Caribbean and its surroundings regions. There are foci in South America, especially in parts of Colombia and French Guyana, some areas of intertropical Africa (such as south Gabon) and in the Middle East (such as the Mashhad region in Iran) and rare isolated clusters in Australia and Melanesia. In Europe, the only country with an endemic HTLV-1 region is Romania. Interestingly and despite different socio-economic and cultural environments, HTLV1 seroprevalence increases gradually with age, especially in women, in all the endemic areas. The general increase with age may be related to a cohort effect, as is well demonstrated in Japan, while the increase seen in older women might also be due to an accumulation of sexual exposures with age [7-12]. While HTLV-2 subtypes are related to highly specific subpopulations and behavior like injection drug use [13].

For many years, studies have established the association of HTLV1 infection with hematologic malignancies namely Adult T Leukemia lymphoma and lymphosarcoma T-cell leukemia. However, current evidence is quite scarce on the prevalence of HTLV-1 infection in other malignancies such as stomach cancer, colon cancer, breast cancer, and lung cancer in Iran [14,15].

In Sudan, there was no data regarding the prevalence of the HTLV-1 among Hematological Malignancy patients. Therefore, this study was the first of its type in our country that seeking to identify the prevalence of the HTLV-1 among Hematological Malignancy patients

Materials and Methods

This study was cross-sectional - It was conducted in the Radio Isotope center of Khartoum state. During the period from April to September 2019 a total of 106 blood samples were obtained from Hematological Malignancy patients including AML, CML, CLL, ALL, Myelodysplastic syndrome and Adult T Leukemia Lymphoma patients. The presence of malignancy was confirmed by using IHC, Molecular and cytogenetic studies. Both males anSerologyical Detection of HTLV-1 antibodies: d females were included.

Specimen's collection and processing

A volume of 6 ml blood was collected from each patient and separated into EDITA anticoagulant containing container (for molecular studies) and plain container (to detect the presence of antibodies). The blood sample in the plain container was centrifuged at 3000 g for 5 min, and then the serum was gently collected into Eppendorf tube and stored at –20°C until the serological analysis.

Specimens analysis

Serologyical Detection of HTLV-1 antibodies: The serum samples were analyzed for the presence of anti - HTLV-1 by a commercially available enzyme-linked immune-sorbent assay (Human T-cell lymphotropic Virus antibody, HTLV-1 Ab ELISA Kit - Creative Diagnostics, Shirley, NY, US). The assay was performed following the instructions of the manufacturer. Positive and negative controls were included in each assay. According to the information included in the kit’s insert, the immunoassay used has a sensitivity and specificity of 100% and 99.95% respectively

Procedure of ELISA test: All reagents and specimens were settled to reach room temperature and the assay was performed following the instruction and procedure that provided in the user manual.

Quality control and calculation of the results: The working reagent, standard, and control were checked for storage, stability, and preparation prior to Starting the work. Each micro plate was regarded separately for the results were calculation and interpretation; the results were calculated by relating each sample absorbance (A) to the cut-off value (C.O.) of the plate.

The equation of (C.O.= NC+0.18) was used to calculate the C.O. value where NC is mean of the three negative controls = (0.02+0.031+0.01)/3 +0.18=0.20

Interpretation of results

1. Positive more than cut-off value.
2. Negative less than cut-off value

Molecular analysis

Samples tested positive for HTLV-1 antibodies were confirmed by using molecular Technique

DNA Extraction Procedure

DNA extraction from blood sample: About 200µl of each Whole blood sample was incubated in lysis buffer (20 µl proteinase K (200mg/ ml) and 5µl of RNAase A). The extraction of DNA was taken place by the DNeasy® Blood Kit (QIAGEN Company) following the manufacturer’s instructions and stored at -80ºC until further analysis. The quality of DNA was checked using Nano-drop test.

Proviral pX-AS gene amplification: Proviral pX-AS gene was amplified in a master mix reaction volume of 25μl containing 5μl of DNA sample and specific forward and reverse primer 1µl for pX-S, 5′-CGGATACCCAGTCTACGTGTT-3′; pX-AS, 5′ CAGTAGGGCGTGACGATGTA-3′) [16]. and completed to 25µl with 13µl Distilled water. The amplification was done by using (TC-3000 conventional PCR Thermal Cycler, USA) through 45 cycles of denaturation at 94°C for 1 min proceeded by initial denaturation at 94°C for 5 min, annealing at 55°C for 1 min and extinction at 72°C for 2 min followed by a final extension at the same temperature for 5 min. Finally, the conventional PCR products were subjected to the gel electrophoresis (2% agarose gels) to visualize the band of the amplified target gene region with ethidium bromide (0.5μg/mL) for 30 min in a UV-gel documentation system.

A method used for data collection

Administrated questionnaire was used to collect patient's data including the gender and age and marital status Data analysis

SPSS version 15 computerized programs analyzed the data, which were collected from questionnaire and laboratory analysis.

Result

A total of 106 patients were enrolled in the study of these, 72 (67.9%) were males and 34 (32.9%) were females. The average age of the study population was 41±12.4 (ranging from 3 to 76) years Table1. The Hematological malignancies were AML 31 (29.2%), CML 37 (34.9%), CLL 9 (8.5%), ALL 17 (16.1%), Myelodysplastic syndrome (MDS) 8 (7.6%) and Adult T Leukemia Lymphoma (ATLL) 4 (3.7%). Most of the patients were newly diagnosed 70 (66.0%) while the rest were received Cancer treatment 36 (34.0%). The history of blood transfusion was reported among 27.3% of patients and the rest were not receiving any cancer treatment Table 1.

The result of serology test reveals that out of 106 serum samples examined 7 (6.6%) were positive for HTLV-1 antibody test including 5 males and 2 females. All positive cases were confirmed by PCR and identified with the pro-viral pX-S gene. Of the HTLV-1 positive cases, three were diagnosed with CML, two were diagnosed with AML in addition to one male patient was diagnosed with ATLL, and another one male patient diagnosed with CLL Table 2. The result also showed that none of the HTLV-1 positive patients has history of Blood transfusion. Furthermore, among HTLV-1 positive cases only one patient was known to be under treatment while the rest were newly diagnosed Table 2.

Discussion

The HTLV-1 infection through the oncogenic properties of it TAX and HBZ Proteins harbor greet risk for hematological malignancies including ATLL [17,18]. In the present study, we demonstrated that the prevalence of HTLV-1 infection among patients with hematologic disorders is 7 (6.6%) out of 106 patients. CML was most frequently identified disorder among the study cases followed by AML and ATLL and CLL, none of the MPD patients was identified with virus. The high prevalence of the virus among CML and AML patients may reflect sampling bias since most of our study populations were diagnosed with one of the two-mentioned disorder. This will need further studies with inclusion of more cases affected with the other disorders to verify our result. Although we include only 4 cases with ATLL the prevalence of HTLV-1 among them was 25% and this may confirm the fact that HTLV-1 have an impact on the development of ATLL disorder. Furthermore, the current study demonstrates that the prevalence of HTLV-1 infection was significantly higher in males compared to females and there was only on patient with history of transfusion history. This may suggest that HTLV-1 was transmitted to those cases by route other than blood transfusion such as sexual contact.

Studies from other countries show variable results of HTLV-1 among hematological malignancy patients. The pre-valance of the HTLV-1 infection was ranging from 18.2% and 23% in south Chile and japan respectively [19,20] to 5.12% and 9.01% in Rio de Janeiro, Brazil and Lagos, Nigeria, Respectively [21,22]. The closest prevalence to our finding is that of Nigeria. This was not surprising because Nigeria located in Africa and near geographically to our country and this may reflect that the geographical distribution of the virus may affect its ability to cause hematological malignancies in certain population. However, our study was differed from that in Nigeria in the sample size and the technique, which were used. We include 106 samples of different hematological malignancies and we confirm the result by detection of pro-viral gene instead they include only 39 samples of lymphoid malignancies and they did not confirm their result.

Conclusion

This was first study in Sudan that high light the prevalence of HTLV-1 among hematological malignancies patients. In conclusion, our results show a similar prevalence of HTLV-1 infection in patients with hematologic disorders in comparison to other studies conducted on the African general population with the most affected group being a patient with CML disorder. We cannot build a clear association of HTLV-1 infection and hematologic disorders due to lacking of the control group. Therefore, larger case control-based studies with inclusion of more sample size are essential to corroborate the evidence.

Ethical Approval

The Ethics Committee of Omdurman Islamic University approved the study.

Consent

Informed consent was obtained from all the study participants.

Conflict of Interests

Authors declare no competing financial interests.

Acknowledgments

The authors acknowledge the efforts of Radioisotope center of Khartoum state for availing their facilities and time for this study.

References

1. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. “Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma”. 1980; 77(12): 7415-7419.
2. Mirvish ED, Pomerantz RG, Geskin LJ. “Infectious agents in cutaneous T-cell lymphoma”. Journal of the American Academy of Dermatology. 2011; 64 (2): 423-431.
3. Roucoux DF, Wang B, Smith D, Nass CC, Smith J, Hutching ST, et al. “A Prospective Study of Sexual Transmission of Human T Lymphotropic Virus (HTLV)-I and HTLV-II”. The Journal of Infectious Diseases. 2005; 191 (9): 1490-1497.
4. Coovadia HM, Rollins NC, Bland RM, Little K, Coutsoudis A, Bennish ML, et al. “Mother-to-child transmission of HIV-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort study”. Lancet. 2007; 369 (9567): 1107-1116.
5. de Thé G, Kazanji M. “An HTLV-I/II vaccine: from animal models to clinical trials?”. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology. 1996; 13(Suppl 1): S191-S198.
6. Desrames A, Cassar O, Gout O, Hermine 0, Taylor GP, Afonso PV, et al. Northern African strains of human T-lymphotropic virus type 1 arose from a recombination event. J Virol. 2014; 88: 9782-9788.
7. Develoux M, Dupont A, Meynard D, Delaporte E. A case of tropical spastic paraparesis associated with HTLV 1 in the Niger Republic. Med Trop (Mars). 1996; 56: 100-101.
8. Diop S, Calattini S, Abah-Dakou J, Thiam D, Diakhate L, Gessain A, et al. Seroprevalence and molecular epidemiology of human T-Cell leukemia virus type 1 (HTLV-1) and HTLV-2 in blood donors from Dakar, Senegal. J Clin Microbial. 2006; 44: 1550-1554.
9. Dumas M, Houinato D, Verdier M, Zohoun T, Josse R, Bonis J, et al. Seroepidemiology of human T-cell lymphotropic virus type I/II in Benin (West Africa). AIDS Res Hum Retroviruses. 1991; 7: 447-451.
10. El-Farrash MA, Badr MF, Hawas SA, El-Nashar NM, Imai J, Komoda H, et al. Sporadic carriers of human T-lymphotropic virus type I in northern Egypt. Microbial Immunol. 1988; 32: 981-984.
11. El-ghazzawi E, Hunsmann G, Schneider J. Low prevalence of antibodies to HI V-1 and HTLV-I in Alexandria, Egypt. AIDS Forsch. 1987; 2: 639.
12. Anyanwu NCJ, Ella EE, Ohwofasa A, Aminu M. Re-emergence of human T-lymphotropic viruses in West Africa. Braz J Infect Dis. 2018; 22: 224-234.
13. Urwijitaroon Y, Barusrux S, Puapairoj C, Romphruk A, Khampeera P. Seroepidemiology of HTLV-1 infection in northeast Thailand: four-year surveillance. Journal of the Medical Association of Thailand = Chotmaihet thangphaet. 1997; 80(Suppl 1): Sl02-S105.
14. Blattner WA, Blayney DW, Robert-Guroff M, Samgadharan MG, Kalyanaraman VS, Sarin PS, et al. Epidemiology of human T-cell leukemia/lymphoma virus. J Infect Dis. 1983; 147(3): 406-416.
15. Yoshida M, Hattori S, Seiki M. Molecular Biology of Human T-cell Leukemia Virus Associated with Adult T-cell Leukemia. Human T-Cell Leukemia Virus. Berlin: Springer-Verlag; 1985: 157-175.
16. Junki K, Ayako M, Keiichiro F, Hisashi T, Takayuki M, Kentaro Y, et al. Quantitative PCR for HTLV-1 provirus in adult T-cell leukemia/lymphoma using paraffin tumor sections. Pathology International. 2016; 66: 618-621.
17. Barrientos A, Lopez M, Sotomayor C, Pilleux L, Calderón S, Navarrete M, et al. Prevalence of human T-Cell lymphotropic virus type 1 and 2 among patients with malignant hematological diseases in South Chile. J Med Virol. 2011; 83(4): 745-748.
18. Miyagi J, Toda T, Uezato H, Ohshima K, Miyakuni T, Takasu N, et al. Detection of Epstein-Barr virus and human T-cell lymphotropic virus type 1 in malignant nodal lymphoma, studied in Okinawa, a subtropical area in Japan. Int J Hematol. 2002; 75(1): 78-84.
19. de Carvalho SMF, de Oliveira MSP, Thuler LCS, Rios M, Coelho RCA, Rubim LC, et al. HTLV-I and HTLV II infections in hematologic disorder patients, cancer patients, and healthy individuals from Rio de. 1997; 15(3): 238-242.
20. Durojaiye I, Akinbami A, Dosunmu A, Ajibola S, Adediran A, Uche E, et al. Seroprevalence of human T lymphotropic virus antibodies among healthy blood donors at a tertiary centre in Lagos, Nigeria. Pan Afr Med J. 2014; 17: 301.