Prevalence of Human T-Lymphotropic Virus Type 1 among Blood Donors from Mashhad, Iran

The city of Mashhad is the capital of Khorasan, the northeasternprovince of Iran, which has been recognized as an area wherehuman T-lymphotropic virus type 1 (HTLV-1) infection is endemic.All serum samples from blood donors are routinely screened forHTLV-1 by using enzyme-linked immunosorbent assay (ELISA). Inthe present study, 28,926 donors (81.86% male and 18.14% female)with a mean age of 32 years (range, 18 to 65 years) were screenedin a 6 months period (July to December 1999). Of these donorsin the primary screening, 228 (0.78%) tested positive by ELISA.The positive samples were confirmed by Western blot (WB) analysis.The WB results indicated that, of 228 positive ELISA specimens,91.2% (208 specimens) were HTLV-1, 4.82% (11 specimens) wereHTLV, 3.5% (8 specimens) were indeterminate, and 0.44% (1 specimen)was not confirmed. HTLV refers to samples in which the completeviral antigen banding patterns on WB strips were not present.In order to further evaluate the detection methodologies used,the HTLV-1-seropositive samples, the indeterminant samples,and/or HTLV samples were examined and confirmed by PCR. TheHTLV samples were determined to be HTLV-1, the remaining sampleswere indeterminant, and the negative sample could not be confirmedfor HTLV-1 by PCR. The prevalence of HTLV-1 infection in ourstudy was 0.77% among blood bank donors, which reconfirms thecity of Mashhad as an area where the virus is endemic comparedto other regions in the world. The incidence was correlatedwith increasing age, and it was higher in females than in males.

INTRODUCTION

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Introduction

Human T-lymphotropic virus type 1 (HTLV-1) was first identifiedin humans in 1980 (11) and 1982 (5). It is the etiologic agentof two distinct human diseases, adult T-cell leukemia or lymphoma(1) and a chronic, progressive demyelinating disorder knownas HTLV-1-associated myelopathy/tropical spastic paraparesis(2). HTLV-1 is distributed worldwide, but it is endemic onlyin certain parts of the world such as southwestern Japan, theCaribbean basin, Africa, part of South America, southern Italy,Taiwan, and the United States (6). Routes of infection includetransfusion, sharing of needles or syringes with infected individuals,sexual contact, and breast-feeding; transplacental transmissionis also suspected (7, 9). Cellular blood products are the mainsource of transfusion-associated HTLV transmission, whereasfresh frozen plasma, cryoprecipitate, or coagulation factorconcentrates appear not to cause infection (4, 10).

To avoid HTLV-1 transmission by transfusion, screening of blooddonation for HTLV-1/2 infection has been mandatory in severalcountries: in 1986 in Japan; in 1989 in the United States; in1990 in Canada, in 1989 in French Caribbean and in 1991 in theentire French territory; in 1993 in The Netherlands; in 1994in Sweden, Denmark, and Iran; and more recently in Portugaland Greece. Such screening is still under debate in other countries.The present study was carried out to validate the efficacy ofserological screening of blood for HTLV-1 contamination by usinghybridization and PCR methods.

MATERIALS AND METHODS

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Materials and Methods

Subjects. A total of 28,926 blood donors were tested for HTLV-1/2 duringJuly to December 1999. The donors were 83% male (23,680) and17% female (5,246), with a mean age of 32 years (ages rangedbetween 18 and 65 years). All donors fulfilled the criteriafor blood donation, which included a clinical examination andan interview to record the history of previous infectious diseases,surgery, blood transfusion, heart diseases, anemia, and informationon foreign travel. Redonation rate during the 6-month studyperiod was 1.5% (439 individuals). All HTLV-1-positive subjectswere informed of the test result and were prohibited from redonation.

Serological assays. Serum samples were screened for HTLV-1/2 by using enzyme-linkedimmunosorbent assay (ELISA; Vironostika HTLV I/II, Organon Teknica).All repeatedly positive samples were confirmed by Western blotting(WB; HTLV blot 2.4 kit; Gene Labs Diagnostics, Ltd.). Our indexof HTLV-1 seropositivity was reactivity to GAG (P19 with orwithout P24) and two ENV (GD21 and rgp46-I) (Table 1).

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TABLE 1. Interpretation of WB pattern

Synthesis and purification of oligonucleotides. Oligonucleotides were synthesized on a Biosearch 8600 automatedDNA synthesizer and purified by high-performance liquid chromatographyand/or by polyacrylamide gel electrophoresis.

DNA purification and PCR. The peripheral blood mononuclear cell DNA was extracted by anonenzymatic method and then analyzed for HTLV-1 sequence. PCRamplification was performed with two primer sets, tax (forward,GGA TAC CCA GTC TAC GTG TTT G; reverse, CGG AAC ATT GGT GAGGAA GGC) and long terminal repeat (LTR; forward, CCA GAC TAAGGC TCT GAC GTC TC; reverse, CCT GAG CTC TAA ACT TAC CTA GACG; GenBank accession no. LO3562), resulting in 522- and 394-bpproducts, respectively. The PCR mixture contained a 1-µgsample DNA, 10 pmol of each primer, 200 µM concentrationsof each deoxynucleoside triphosphate, 50 mM KCl, 10 mM Tris(pH 8.3), 1.5 mM MgCl₂, and 1 U of Thermus aquaticus (Taq) enzyme(CinnaGen, Inc., Tehran, Iran). The reaction mixture was incubatedfor 5 min at 94°C and then subjected to 30 cycles consistingof 1 min at 94°C, 1 min at 53°C, and 30 s at 68°C.The final annealing step was performed for 5 min at 68°Cin a DNA thermal cycler (Perkin-Elmer 480). For positive control,MT-2 cell line DNA was used. The reaction mixtures were storedat 4°C until they were analyzed by agarose gel (1.5%) electrophoresis.To confirm the PCR fidelity, two blood samples were amplifiedand sequenced by using an automated sequencer (Applied Biosystems377).

Statistical methods. Descriptive results were presented as crowd frequencies, ratios,and 95% confidence intervals. The correlation between differentfactors was evaluated by chi-square test.

RESULTS

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Results

A total of 28,926 blood samples were analyzed for HTLV-1 contamination.In the primary screening, 228 (0.77%) samples tested positiveby ELISA. All samples were assayed in duplicate, and the positivesamples were confirmed by WB analysis. The WB results indicatedthat, of these 228 positive ELISA specimens, 91.2% (208 specimens)were HTLV-1, 4.82% (11 specimens) were HTLV, 3.5% (8 specimens)were indeterminate, and 0.44% (1 specimen) was not confirmed.HTLV refers to samples in which the complete viral antigen bandingpatterns on WB strips were not present.

The prevalence of the infection was 0.77% (after correctionfor the redonation rate of 1.5% [439 of 28,926]) among bloodbank donors. In order to address the issues of false negatives,150 samples (previously tested negative by ELISA) were randomlyselected and analyzed by using WB. These analyses also testednegative for HTLV contamination of selected samples. Similarly,to validate the methodology used and to determine the possibilityof false positives, 20 samples of HTLV-1 ELISA and WB positiveswere randomly selected and examined by PCR. All of the sampleswere confirmed by PCR.

In order to confirm and determine the HTLV strains, the HTLV-1-seropositivesamples, indeterminant samples, and HTLV samples were examinedby PCR. The 11 HTLV-positive samples were determined to be HTLV-1.The PCR products corresponding to the tax and LTR regions (Fig.1) of the HTLV-1 genome were sequenced and resulted in a completehomology with the cosmopolitan strain of HTLV-1. The eight indeterminantsand the one negative sample tested negative for HTLV-1 by PCR.

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FIG. 1. Amplification of tax and LTR regions in blood donors. All 522- and 394-bp fragments were amplified from the tax and LTR regions of HTLV-1 genome, respectively. PCR products were electrophoresed in a 1.5% agarose gel, stained in ethidium bromide and photographed. Lane 1, negative control; lanes 2 and 3, blood donors; lane 4, MT-2 cell line DNA; lane 5, molecular weight marker; lanes 6 to 10, blood donors.

A significant correlation exists between increasing age andincidence of infection (P value of correlation, 0.0001 for menand 0.0002 for women). It is also concluded that seroprevalencerate in females is higher than males (Fig. 2).

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FIG. 2. HTLV-1 seroprevalence by age group (in years) and gender among Mashhad blood donors. Lightly shaded bars, male; darkly shaded bars, female.

DISCUSSION

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Discussion

There is no defined treatment for patients infected with HTLV-1,but the accurate knowledge of seroprevalence rates in differentpopulation groups may be helpful in establishing prophylacticmeasures to reduce rates of viral transmission from infectedindividuals. The overall 0.77% HTLV-1 seroprevalence rate foundin Mashhad blood donors is greater than that seen in similarstudies in the United States (0.004%), France (0.004%), andBrazil (0.42%). Higher seroprevalence in blood donors has beenfound in Jamaica (2.1%) (12). Such comparisons must be madecautiously because screening tests, specificity in marker levels,and medical selection of blood donors can vary from one studyto another, but we have attempted to accomplish standard screeningtests. The present study confirms, by using both serologicaland PCR detection methodologies, that Mashhad is a region whereHTLV-1 infection is endemic. In a previous study, seropositivitywas reported to be 3% among the patients reporting to the clinicwith HTLV-1-associated disease symptoms in Iran (13). ELISAkits have high sensitivity and low specificity; thus, it maynot be a reliable screening tool. Therefore, positive ELISAresults should be confirmed by WB or PCR. The WB seropositivityparameters used (HTLV blot 2.4 kit) a recombinant spiked WBassay, which is more stringent than those previously used withwhole-virus lysate WB. The epidemiology of HTLV-1/2 has beenlargely defined through the use of antibody testing. PCR hasalso been a useful tool for facilitating epidemiological studiesfor distinguishing virus type and for quantifying viral presence(3). Age and sex relationships have been identified as contributingfactors to HTLV-1 seroprevalence in all areas where this virusis highly endemic (8). Female predominance could be relatedto a preferential sexual transmission from husband to wife.Our study also revealed a strong age-dependent rise in seroprevalencerate. This pattern is also well documented in previous studies,which could be explained by a cohort effect (14) and by cumulativeeffect of infections occurring over the lifetime of individuals,such as by heterosexual transmission. The age-dependent risein HTLV-1 prevalence could also be explained by a birth cohorteffect (12).

ACKNOWLEDGMENTS

We thank Antoine Gessain, Fata Kashanchi, and Absar Alum foruseful insights. We are also appreciative of the great effortsmade on behalf of this project by the personnel of the bloodbank center in Mashhad.

FOOTNOTES

* Corresponding author. Mailing address: Department of Human Genetics, Bu-Ali Research Institute, Bu-Ali Sq., Ferdowsi Sq., Mashhad University of Medical Sciences, Mashhad 91966, Iran. Phone: (98-511) 762-7601. Fax: (98-511) 761-8088. E-mail: m-abbaszadegan{at}mums.ac.ir.

REFERENCES

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