Importance of extending the use of Polymerase chain reaction in the diagnosis of Venereal syphilis in a blood transfusion center in Burkina Faso, West Africa

Importance of extending the use of polymerase chain reaction in the diagnosis of venereal syphilis in a blood transfusion center in Burkina Faso, West Africa

 

Abibou Simpore^1,2, Cyrille Bisseye^1,3, Bolni Marius Nagalo^1,4,&, Mahamoudou Sanou⁵, Yacoub Nébié², Laure Stella Ghoma-Linguissi¹, Honorine Dahourou², Boukary Sawadogo⁶, Florencia Djigma¹, Siaka Ouattara², Virginio Pietra¹, Jason Nichol⁴, Jacques Simpore¹

 

¹Pietro Annigoni Biomolecular Research Center (CERBA)/ (LABIOGENE), University of Ouagadougou, Burkina Faso, ²National Blood Transfusion Center, Ouagadougou, Burkina Faso, ³Laboratory of Molecular and Cellular Biology (LABMC), University of Sciences and Techniques of Masuku (USTM), Franceville, Gabon, ⁴Endicott College, College of Arts and Sciences, Beverly, USA, ⁵Healthcare Unit Training, (UFR-SDS), University of Ouagadougou, Ouagadougou, Burkina Faso, ⁶Marlboro College, Vermont, USA

 

 

^&Corresponding author
Bolni Marius Nagalo, Pietro Annigoni Biomolecular Research Center (CERBA)/ (LABIOGENE), University of Ouagadougou, Burkina Faso, National Blood Transfusion Center, Ouagadougou, Burkina Faso

 

 

 

 

Despite significant scientific advances in the diagnosis of blood-borne and sexually transmitted diseases, the Human Immunodeficiency Virus (HIV), viral hepatitis and syphilis continue to be major public health issues in Sub Saharan Africa [1, 2]. Although the existence of a wide range of serological testing (such as Treponema pallidum haemagglutination test (TPHA), Fluorescence Treponema antibody (FTA), Rapid Plasma Reagin (RPR), Enzyme-linked immunosorbent assay (ELISA)...) has drastically improved the routine diagnosis of syphilis [3], the majority of these methods is limited because of their low specificity (non-venereal Treponema, cross-reactions, seroconversion ...) and laboratory mistakes [4-6]. Blood transfusion safety begins with the pre-donation interview that eliminates high-risk blood donors, particularly those who have had risky behavior in the last three months prior to donation [7]. Then, to eliminate all the infected blood donations, the laboratory performs a series of blood screenings for infectious diseases markers.

 

In Burkina Faso, the prevalence of syphilis is high and variable: 1.7 %, 5.7 % and 2.1 % respectively for blood donors of Ouagadougou, the prison population and four regional blood transfusion centers [8-10]. The high prevalence and other markers detected in blood donors are corroborated by the work of Nagalo and al. [10]: anti-HIV (1.8 %), HBsAg (13.4 %), and anti -HCV (6.3 %). There is an urgent need to shift to new and effective diagnosis methods that would prevent large numbers of patients from post- transfusion complications. This study is a description of the interest in the use of PCR to diagnose venereal syphilis in a sample of volunteer non-remunerated blood donors in Burkina Faso.

 

 

Samples collection: Two hundred forty-two (242) blood donors were included in this study, from August 2011 to December 2012, with blood donors ranging in age from 17 to 60 and coming from different occupational categories. Reactive or non-reactive RPR plasmas were stored at -20°C for the detection of Treponema pallidum genome.

 

Serological analysis: The presence of antibodies to Treponema pallidum was detected using Rapid Plasma Reagin testing (RPR) (FUTURA SYSTEM, Roma, Italia) and ARCHITECT Syphilis TP assay (Syphilis TP; Abbott Japan, Tokyo, Japan) a two-step sandwich chemiluminescent microparticle immunoassay (CMIA : ARCHITECT-i100SR-ABBOTT).

 

Hepatitis B surface antigen (HBsAg), antibodies to HCV and HIV types 1 and 2 were screened using a fourth generation ELISA (ARCHITECT-i100SR-ABBOTT, Santa Clara, California, United States of America). All the reactive samples for HIV, HBsAg, and HCV were re-tested for confirmation using a second enzyme-linked immunosorbent assay (Bio-Rad, Marnes la Coquette, France). A result is considered positive if both the first and second tests were positive.

 

Treponema pallidum pallidum DNA extraction and Amplification: DNA extraction was performed using the DNA-Sorb-B kit (Sacace Biotechnologies, Como, Italy) following the manufacturer´s instructions. The extracted DNA is used for PCR amplification using the Treponema pallidum 273/668IC PCR kit with electrophoretic Detection (Sacace Biotechnologies, Como, Italy).

 

Statistical Analysis: Data were analyzed using SPSS 17 .0 and Epi Info 3.5.1 softwares. The Chi-square test was used for comparisons. P-values <0.05 were considered statistically significant.

 

 

Sociodemographic characteristics of blood donors: Two hundred forty-two (242) donors were included in this study. The subjects? average age was 26.9 years (range of 17 to 60 years). Donors were mostly from 21-30 year age group (61.6%); 81.0% were males and 71.5% were first-time donors. The majority of donors were recruited in urban areas (79.8%). Repeat donors were mainly from the age group 21-30 (32.2%) (Table 1).

 

Syphilis serology and viral co-infections in blood donors: Of the 242 donors tested, 183 plasmas (75.6%) were reactive to RPR, and only 139 (57.4%) were reactive to CMIA. The difference in the detection of T. pallidum pallidum was significant between RPR and CMIA (p< 0,001). HBsAg, anti-HCV, and anti-HIV antibodies were detected in respectively 24 (9.9%), 28 (11.6%), and 3 (1.2%) blood donors.

 

PCR confirmation of anti-Treponema: Diagnosis results of T. pallidum PCR showed that 170/183 (92.9%) and 125/139 (89.9%) respectively detected by RPR and CMIA were negative by PCR (Table 2). The DNA of T. pallidum subspecies pallidum was found in 16 out of 242 (6.6%) blood donors. Out of 108 samples detected positive using serological methods, 97 (89.8%) were negative by PCR for T. pallidum, and only 11/108 (10.2%) were positive.

 

 

Molecular amplification techniques are struggling to establish themselves in a hostile environment of the African continent, which is marked by economic difficulties, a lack of adequate technical equipment, and a shortage of qualified and competent personnel. However, the high prevalence of blood-borne infections and sexually transmitted diseases has drastically limited the socio-economic development of Sub Saharan African countries. In developed countries, where the prevalence of these infectious diseases is low in comparison to the prevalence in developing countries, blood transfusion centers systematically use the nucleic acid testing (NAT) in the detection of viral pathogens like HIV, HBV, and HCV [11]. By contrast, most blood transfusion centers in Africa continue to use serological techniques in cities and rapid diagnosis testing in rural areas to detect pathogens in blood donation [12]. Beyond the specificity and sensitivity of the PCR, the molecular method is efficient to make a quick and accurate diagnosis. Our study population was made of a large number of non-remunerated young volunteer blood donors and males recruited during social and cultural activities, 196/242 (81.0%). Most of the blood donors (79.8%) were recruited in the city of Ouagadougou and its metropolitan area, and the rest in rural areas (20.2%). Previous studies had indicated that the majority of blood donors in Sub Saharan Africa are young students who live in the big cities [10, 13, 14]. The distribution of prevalence of HbsAg and anti-HCV antibodies in the sampling of our study was respectively 9.9% and 11.6%. The distribution was also higher by 8.7% and 6.3% respectively as found in previous research [15,16]. Twenty-six (26) cases of RPR+/HCV+ were PCR negative, and 19 cases of RPR+/HbsAg+ from the 94.7% (18/19) sample were PCR positive for the screening of T. pallidum DNA, although cases of non venereal treponematosis were not previously included in our screening. The molecular analysis showed the following results: 170/183 (92.9%) and 125/139 (89.9%) respectively positive using serological testing (RPR and CMIA) were negative using PCR. The DNA of T. pallidum subsp pallidum bacteria was found in 16/242 (6.6%) of our sampling. Serological tests (RPR, TPHA, CMIA ...) do not differentiate a venereal syphilis from other treponemal diseases (yaws or bejel) which are not sexually or congenitally transmitted. However the relevance of the extent of the detection of these pathogens in transfusion lies in the fact that there is a risk of transmission through blood. The rates of sensitivity of serological methods used were 81.3% and 87.5% respectively for the RPR and the CMIA in comparison to PCR (Table 3). In addition, these percentages were in keeping with the recommendations by the Center of Diseases Control (CDC) and World Health Organization (WHO) which are 77-100% for the RPR, 82-100% for CMIA [17,18].

 

The study found low levels of specificity regarding serologically testing compared to PCR. The explanation resides in the fact that our diagnosis using PCR is based on detection of the specific subspecies that are responsible for venereal syphilis only. Globally, our results showed the efficiency of PCR in the detection of Treponema pallidum subsp pallidum while different serological testing is unable to differentiate venereal syphilis from other treponematosis [5, 6]. Due to the high prevalence of transfusion infectious diseases, using a more specific technique such as PCR in the diagnosis of venereal syphilis would help reduce the number of infections contracted during a blood transfusion. Moreover, we noticed that these serological tests may give poor estimates of prevalence because of false positives. The diagnosis is often difficult because of the complexity of the interpretation of results (false negatives). A wrong interpretation of results due to the deficiency of the test used would not only affect the patient, but could also lead to complications or development of congenital syphilis, especially in infants or pregnant women who represent the majority of blood recipients in Burkina Faso.

 

 

The results of the study show the urgent need for extending the molecular diagnosis of pathogens in blood transfusion to Treponema pallidum, which will be a more viable alternative to serological testing in the case of poor countries facing a high prevalence of infectious diseases.

 

 

The authors declare no competing interests.

 

 

Abibou Simpore, Cyrille Bisseye, Bolni Marius Nagalo and Mahamoudou Sanou designed the study. Abibou Simpore and Cyrille Bisseye did laboratory work and analyzed data. Abibou Simpore, Laure Stella Ghoma-Linguissi, Bolni Marius Nagalo and Cyrille Bisseye wrote the paper. Boukary Sawadogo corrected the language. Jacques Simpore corrected and approved the final version of the paper. All the authors read and approved the final version of the paper.

 

 

We thank all the participants of this study and The National blood transfusion centre staff. We also thank the Italian Episcopal Conference (CEI) and UEMOA for their financial support.

 

 

Table 1: Show the socio-demographic characteristics of VNRBD

Table 2: Results of the serological screening in the detection of T. pallidum versus PCR

Table 3: Performance of two serological methods using PCR testing as a confirmation test

 

 

1. Lautenschlager S. Sexually transmitted infections: update 2013. Praxis. 2013;102(5):273-8. PubMed | Google Scholar


2. Pallavi P, Ganesh CK, Jayashree K, Manjunath GV. Seroprevalence and trends in transfusion transmitted infections among blood donors in a university hospital blood bank: a 5 year study. Indian Journal of Hematology and blood transfusion. 2011;27(1):1-6. PubMed | Google Scholar


3. Bala M, Singh V, Muralidhar S, Ramesh V. Assessment of reactivity of three treponemal tests in non-treponemal non-reactive cases from sexually transmitted diseases clinic, antenatal clinic, integrated counselling and testing centre, other different outdoor patient departments/indoor patients of a tertiary care centre and peripheral health clinic attendees. Indian journal of medical microbiology. 2013;31(3):275-9. PubMed | Google Scholar


4. De Larranaga G, Trombetta L, Wingeyer SP, Remondino G. False positive reactions in confirmatory tests for syphilis in presence of antiphospholipid antibodies: misdiagnosis with prognostic and social consequences. Dermatology online journal. 2006;12(4):22. PubMed | Google Scholar


5. Morand JJ, Simon F, Garnotel E, Mahe A, Clity E, Morlain B. Overview of endemic treponematoses. Medecine tropicale. 2006;66(1):15-20. PubMed | Google Scholar


6. Farhi D, Dupin N. Serological diagnosis of syphilis. Annales de dermatologie et de venereologie. 2008;135(5):418-25. PubMed | Google Scholar


7. Nebie K, Olinger CM, Kafando E, Dahourou H, Diallo S, Kientega Y, et al. Lack of knowledge among blood donors in Burkina Faso (West Africa); potential obstacle to transfusion security. Transfusion clinique et biologique. 2007;14(5):446-52. PubMed | Google Scholar


8. Ouedraogo AS, Yameogo JT, Poda GE, Kientega Y, Ouedraogo Traore R. Prevalence of anti-CMV antibodies in blood donors in Ouagadougou (Burkina Faso). Médecine et santé tropicales. 2012;22(1):107-9. PubMed | Google Scholar


9. Diendere EA, Tieno H, Bognounou R, Ouedraogo DD, Simpore J, Ouedraogo-Traore R, et al. Prevalence and risk factors associated with infection by human immunodeficiency virus, hepatitis B virus, syphilis and bacillary pulmonary tuberculosis in prisons in Burkina Faso. Médecine tropicale. 2011;71(5):464-7. PubMed | Google Scholar


10. Nagalo BM, Bisseye C, Sanou M, Kienou K, Nebie YK, Kiba A, et al. Seroprevalence and incidence of transfusion-transmitted infectious diseases among blood donors from regional blood transfusion centres in Burkina Faso, West Africa. Tropical medicine & international health. 2012;17(2):247-53. PubMed | Google Scholar


11. Coste J. Screening for viral genomes in blood transfusion. Transfusion clinique et biologique. 2000;7 Suppl 1:11s-17s. PubMed | Google Scholar


12. Allain JP. Moving on from voluntary non-remunerated donors: who is the best blood donor? British journal of haematology. 3 may 2011 ;doi: 10.1111/j.1365-2141.2011.08708.x. PubMed | Google Scholar


13. Kra O, N'Dri N, Ehui E, Ouattara B, Bissagnene E. Prevalence of HBs antigen in blood donors in the Bouake regional centre of blood transfusion in 2001. Bulletin de la Société de pathologie exotique. 2007;100(2):127-9. PubMed | Google Scholar


14. Matee MI, Magesa PM, Lyamuya EF. Seroprevalence of human immunodeficiency virus, hepatitis B and C viruses and syphilis infections among blood donors at the Muhimbili National Hospital in Dar es Salaam, Tanzania. BMC public health. 2006;6:21. PubMed | Google Scholar


15. Nagalo MB, Sanou M, Bisseye C, Kabore MI, Nebie YK, Kienou K, et al. Seroprevalence of human immunodeficiency virus, hepatitis B and C viruses and syphilis among blood donors in Koudougou (Burkina Faso) in 2009. Blood transfusion. 2011;9(4):419-24. PubMed | Google Scholar


16. Bisseye C, Sanou M, Nagalo BM, Kiba A, Compaoré TR, Tao I, et al. Epidemiology of syphilis in regional blood transfusion centres in Burkina Faso, West Africa. The Pan African medical journal. 2013;16:69. PubMed | Google Scholar


17. Larsen SA, Steiner BM, Rudolph AH. Laboratory diagnosis and interpretation of tests for syphilis. Clinical microbiology reviews. 1995;8(1):1-21. PubMed | Google Scholar


18. Peeling RW, Ye H. Diagnostic tools for preventing and managing maternal and congenital syphilis: an overview. Bulletin of the World Health Organization. 2004;82(6):439-46. PubMed | Google Scholar