CHAPTER 1. INTRODUCTIONBackground of studyThe World Health Organization (WHO) recommendation of safe blood transfusion is provision of compatible blood which is cross matched and had been screened at least for five transfusion transmitted infections (TTIs); human immunodeficiency virus (HIV), hepatitis C (HCV),hepatitis B (HBV), syphilis and malaria parasite (WHO, 2011).Blood products, such as blood components for both transfusions and plasma derivatives, are essential therapeutics in modern medicine. Red blood cell transfusions are vital in saving lives during emergencies and in other cases where interventions are necessary (Heiden, 2010) For example, blood coagulation factor concentrates dramatically improve the life expectancy and quality of life of hemophilia patients.
Until recently, blood products were considered to be purely physiological materials that were not expected to be harmful (Su et al., 2003). However, HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) were found to be associated with blood transfusions in Chinese adults (Shepard et al., 2005; Shang at al., 2007; Shan et al., 2007).
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Transfusion therapy has been the mainstay of several medicosurgical therapeutics since 1930 (Zafar, 2000). There are 3 types of blood donors: voluntary/unpaid, family/replacement, and paid.2 A voluntary blood donor intentionally donates blood without pursuing any remuneration, whereas are placement donor is requested to do so by the patient or his associates (WHO, 2014). According to World Health Organization (WHO) Global Database on Blood Safety (GDBS) 2008, total around 91.8 million blood donations are collected annually. But, approximately 48% of these emanate from high-income countries, astringent to 15% of earth’s population. Ten nations vouch for 65% of blood collections worldwide ,and India is the third highest bidder in this respect following United States and China (Agravat et al.
, 2004). With almost 9.8 million units of yearly collections and 84% voluntary donors, India is expected to bang on the WHO target of 100% voluntary donations by 2020, much before due date ( WHO, 2014).Blood transfusion aggravates the risk of transfusion transmissible infections (TTIs) like hepatitis B (HBV), hepatitis C (HCV), Human Immunode?ciency Virus (HIV), syphilis, and less commonly to malaria, toxoplasmosis, brucellosis, other viral infections (Mollison and Engelfriet, 2005).
Early reports of the transfusion-related transmission of syphilis led to the World Health Organization (WHO) recommendations for syphilis testing of blood donors (Takpo et al., 2007 ). These recommendations have been questioned, since many syphilis antibodies among blood donors are the result of previous infections or even unspeci?c reactions. Furthermore, Treponema pallidum does not withstand cold storage (Tagny, 2011). The WHO recommends several syphilis screening tests: the enzyme immunoassay (EIA)and T. pallidum haemagglutination assay (TPHA) as speci?c tests, or the Venereal Disease Reference Labor to y (VDRL) and rapid plasma reagin (RPR) as non specific test (WHO, 2010).Objectives: The primary objective of this study is to compare laboratory practices for screening blood donors for syphilis and hepatitis B virus at blood transfusion facilities in some private hospital in Port Harcourt, Nigeria with the recommendations of the World Health Organization and the National Blood Transfusion Service, Nigeria (NBTS).
This survey is to compare the current syphilis and Hepatitis B virus screening practices in Nigeria with the recommendations of the WHO and NBSG regarding the use of assays for screening blood donors and their performance. Also the prevalence of syphilis antibodies and Hepatitis B virus in blood donors will be estimated. Additionally, the survey determined whether written SOPs or guidelines were in place for syphilis screening and whether donors with positive syphilis tests were referred for clinical follow-up.CHAPTER 2. LITERATURE REVIEW.2.1 HEPATITIS B VIRUS2.
1.1 HEPATITIS B VIRUS INFECTIONHepatitis B virus (HBV) is a noncytopathic, hepatotropic virus of the Hepadnaviridae family that causes variable degrees of liver disease in humans. Infection with HBV can be either acute or chronic; while adult infections have a relatively low rate of chronicity (around 5%), neonatal infections usually have a high persistence rate (McMahon, 2010).Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV) that affects the liver (WHO,2014). It can cause both acute and chronic infections (WHO,2014).
Many people have no symptoms during the initial infection. Some develop a rapid onset of sickness with vomiting, yellowish skin, tiredness, dark urine and abdominal pain. Often these symptoms last a few weeks and rarely does the initial infection result in death (WHO,2014). It may take 30 to 180 days for symptoms to begin (WHO,2014). In those who get infected around the time of birth 90% develop chronic hepatitis B while less than 10% of those infected after the age of five do (CDC,2011). These complications result in the death of 15 to 25% of those with chronic disease (WHO, 2014). 2.
1.2 HBV GENOMEThe genome organisation of HBV. The genes overlap.The genome of HBV is made of circular DNA, but it is unusual because the DNA is not fully double-stranded. One end of the full length strand is linked to the viral DNA polymerase.
The genome is 3020–3320 nucleotides long (for the full-length strand) and 1700–2800 nucleotides long (for the short length-strand) (Kay and Zoulim, 2007).The negative-sense (non-coding) is complementary to the viral mRNA. The viral DNA is found in the nucleus soon after infection of the cell. The partially double-stranded DNA is rendered fully double-stranded by completion of the (+) sense strand and removal of a protein molecule from the (?) sense strand and a short sequence of RNA from the (+) sense strand. Non-coding bases are removed from the ends of the (?) sense strand and the ends are rejoined.
There are four known genes encoded by the genome, called C, X, P, and S. The core protein is coded for by gene C (HBcAg), and its start codon is preceded by an upstream in-frame AUG start codon from which the pre-core protein is produced. HBeAg is produced by proteolytic processing of the pre-core protein. In some rare strains of the virus known as Hepatitis B virus precore mutants, no HBeAg is present (Buti et al.,2005). The DNA polymerase is encoded by gene P. Gene S is the gene that codes for the surface antigen (HBsAg).
The HBsAg gene is one long open reading frame but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-S1, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large (the order from surface to the inside: pre-S1, pre-S2, and S ), middle (pre-S2, S), and small (S) Glebe and Urban (2007) are produced Beck and Nassal (2007) The function of the protein coded for by gene X is not fully understood but it is associated with the development of liver cancer. It stimulates genes that promote cell growth and inactivates growth regulating molecules (Li et al.
,2010).2.1.3 Structural Protein22.214.171.124 Hepatitis B surface antigenEnvelope polypeptides are encoded by the combination of the pre S and S gene regions.The major protein of hepatitis B surface antigen (HBsAg) particles is the smallest geneproduct (SHBs).
The middle protein (MHBs) contains the pre-S2/S component.The large surface protein (LHBs) contains pre-S1, pre-S2 and HBsAg, and isincorporated in intact virus particles (Reifenberg et al., 2006). In viraemic carriers, MHBsand SHBs products predominate in the liver, whereas in non-viraemic carriers, LHBsproducts predominate. LHBs show direct toxic or immunomodulatory effect andinteraction with cytokines (Ayada et al., 2006) which may result in massivehepatocellular necrosis, regeneration and the eventual development of HCC. 2.1.
3.2 Core ProteinsThe hepatitis B core antigen (HBcAg) (25 kD) is assembled into the capsid, which isessential for viral packaging. Its synthesis is restricted to liver tissues, and is an importanttarget for immune recognition in chronic infection. Soluble Hepatitis B e antigen(HBeAg) (21kD) peptide is released into the circulation, and is a reliable marker foractively replicating virus, and hence, for high infectivity (Wu et al., 2007).
Seroconversion from HBeAg to anti-HBe is commonly associated with the clearance ofwild type (wt) HBV and the resolution of acute liver disease.2.1.
4 Nonstructural Proteins126.96.36.199 HBV Polymerase EnzymeThe HBV polymerase is a 56 to 70kD polypeptide. HBV polymerase has the followingdomains; the amino-terminal region; terminal protein (tp) which acts as a prime promoterfor synthesis of the minus strand cDNA, spacer domain, the RNA-dependent DNApolymerase (reverse transcriptase), and the RNase H domain at the carboxy-terminus. Mutation of polymerase affecting its activity will affect the amount of virus produced, as well as the number of templates available to encode viral proteins (Bajunaid,2013).
2 X-geneThe X gene protein (154 amino acids (aa.)) encoded by the X gene (nt. 1372-1834),exhibits numerous activities affecting intracellular signal transmission, gene transcription,cell proliferation, DNA repair, and apoptosis (Francois et al., 2001). HBx trans-activatesand upregulates viral and cellular genes as the transcriptional expression of humantelomerase l; reverse transcriptase (hTERT) (Qu et al.
, 2005), through activation oftranscription factors, modulation of cell signalling pathways, RNA stabilization, andalteration of nucleocytoplasmic translocation and inhibition of serine protease activity(aa. 67-69 and 135-138 (Blackberg and Kidd-Ljunggren; 2003).2.
1.5 GENOTYPES AND SEROTYPESWith about 240 million chronic carriers worldwide and more than 686 000 deaths per year (WHO, 2014), Hepatitis B virus (HBV) infection remains a serious public health problem, particularly in endemic areas including Southeast Asia and Sub-Saharan Africa (WHO, 2015). To date, eight genotypes named A – H are recognized (Norder et al., 2004) and two additional genotypes, I and J, have been proposed (Tran et al., 2008, Tatematsu et al.
, 2009). HBV genotypes may have distinct geographical distributions. In Africa, especially genotypes A, D and E are prevalent, with genotype E being dominant throughout West Africa (Hübschen et al.
, 2008, Cooksley, 2010, Kramvis, 2014, Pourkarim et al., 2014, Kramvis, 2016, Hübschen et al., 2011). Several studies have implicated HBV genotypes in disparate disease progression, clinical outcome, therapeutic response and the degree of protection provided by vaccination (Cooksley, 2010, Kramvis, 2014, Pourkarim et al., 2014).
The human HBV is a member of the family Hepadnaviridae and has been classified into 10 genotypes (A-J), which can be further sub-divided into over 40 sub-genotypes (Kay and Zoulim, 2007; Kurbanov et al., 2010; Locarnini et al., 2013). The geographical distribution of genotypes is shown in HYPERLINK “https://www.sciencedirect.
com/science/article/pii/S0168827815000495″ l “f0005” Fig. 1 (Locarnini et al., 2013).Geographical distribution of the HBV genotypes and sub-genotypes. Genotype I and J are not shown as they have not been ratified by the ICTV; genotype I is found in Southern China and Vietnam whilst genotype J was identified from a Japanese World War II person who lived in Borneo (Locarnini et al., 2013).2.
1.6 HBV REPLICATIONDuring infection, HBV penetrates into the cells after surface binding, then the virus is transported into the nucleus without processing, where replication starts by unwinding circular DNA which is converted into a covalently closed circular DNA (cccDNA) that acts as a template for transcription of HBV pregenomic, and messenger RNAs (Beck and Nassal, 2007). Transcription starts from the core promoter to yield the 3.5 kb pregenomic RNA, which is packaged with polymerase into immature core particles, and then serves as a template for reverse transcription and negative strand DNA synthesis. The incomplete positive strand DNA is then synthesized.
The mature core particles are packed into HBsAg and pre-S proteins in the endoplasmic reticulum then are exported from the cell. 2.1.
7 IMMUNE RESPONSE TO HBVThe release of HBV DNA from the protective nucleocapsids (NCs) (NC disassembly or uncoating), a prerequisite for CCC DNA formation (Cui et al., 2013), may potentially expose the viral DNA to host DNA sensing mechanisms. Indeed, foreign or mis-localized cellular DNA represents one of the major pathogen-associated molecular patterns (PAMPs) that are recognized by their corresponding cellular receptors or sensors, the pattern recognition receptors (PRRs) (Kawai and Akira, 2010; Paludan and Bowie, 2013).
Thus, many DNA viruses are detected by DNA sensors in the host cell cytoplasm (cyclic GMP-AMP synthase or cGAS, and others) (Gao et al.,2013; Sun et al., 2013;Cai et al., 2014), endosomes (Toll like receptor 9 or TLR9 in select immune cells) ((Kawai and Akira, 2010; Paludan and Bowie, 2013), and even in the nucleus (IFN?-inducible protein 16 or IFI16) (Monroe et al.,2014;Kerur et al.
, 2011). DNA sensors are important 86 innate immune factors in triggering early antiviral defenses such as type I IFN production and in regulating the adaptive immune response to clear viral infections. A major signal transducer in cytosolic DNA sensing is the ER-associated protein, stimulator of interferon genes (STING), which acts downstream of DNA sensors like cGAS, although STING-independent pathways of DNA sensing have also been reported (Paludan and Bowie,2013;Cai et al.,2014; Burdette and Vance 2013). Signaling through STING and downstream effectors such as interferon regulatory factor 3 (IRF3) leads to type I IFN production, which has strong antiviral activities against a variety of viruses through the induction of a large number of interferon stimulated genes (ISGs) (Tanaka and Chen, 2012). If and how HBV is detected by the innate immune system has remained an important yet unresolved issue.
In contrast to many other viruses, HBV is generally thought to be a “stealth” virus because it does not induce a type I interferon (IFN) response during natural or experimental infections (Wieland et al., 2014). This is thought to be related to the unique replication cycle of the virus. In particular, the viral DNA is synthesized only after pgRNA packaging into the protective NCs, sequestered away from cytosolic DNA sensors. However, some recent reports have suggested that HBV may be able to trigger the innate immune response under certain conditions in infected hepatocytes or non-parenchymal liver cells such as Kupffer cells, which are not productively infected (Chang et al., 2012; Shlomai et al.
, 2014). In addition to cytotoxic effector lymphocytes that directly kill infected cells, soluble immune effectors (such as type I and type III IFN, TNF?, IL-6) elicit strong, non cytolytic antiviral effects targeting multiple stages of the HBV replication cycle, including transcriptional and post-transcriptional suppression of viral RNA expression, blocking of nucleocapsid (NC) assembly and destabilization of pre-formed NCs (Weiland et al., 2014; Chang et al., 2012). Intriguingly, as demonstrated in an HBV transgenic mouse model, IFN can also stimulate, rather than suppress, HBV gene expression and replication when viral replication levels are low (Tian et al., 2011), suggesting that HBV may have evolved to co-opt the host antiviral response to enhance its own replication.
We recently developed an immortalized mouse hepatocyte cell line, AML12HBV10, which supports high levels of HBV replication in a tetracycline (tet)- regulated manner and is highly responsive to HBV suppressive effects of certain antiviral cytokines like IFN (Xu et al., 2010). Furthermore, we have found that AML12HBV10 cells could support efficient HBV CCC DNA formation, which was likely facilitated by the rapid and efficient uncoating of the viral NCs to expose the genomic DNA for CCC DNA conversion in these cells (Cui et al.
, 2015). Here, we reported that the increased exposure of RC DNA in AML12HBV10 cells led to the triggering of an innate immune response that was dependent on viral DNA and host DNA sensing and signaling mechanisms and was able to modulate viral gene expression and replication.2.1.8 INFECTIOUS DOSE OF HBVRecent studies in HBV infected chimpanzees using a wide dose range of a single monoclonal HBV inoculum demonstrated that also the size of the viral inoculum contributes to the outcome of HBV infection (Asabe et al.
, 2009). As shown in , animals inoculated with 1010, 107 and 104 genome equivalents (GE) of HBV cleared the virus within 8–30 weeks after its first detection, in a virus dose-related fashion similar to what we have previously observed in several other animals that had been inoculated with 108 GE HBV (Thimme et al., 2003). In contrast, both of the animals that were inoculated with 101 GE became chronically infected, one of which (like many chronically infected humans) ultimately cleared the virus in the context of an acute disease flare 42 weeks after first detection, while the other remained heavily infected for at least 55 weeks at which point the study was terminated. This suggests that a virus dose window exists between 104 and 101 GE within which the host-virus dynamics favor persistent infections, while higher doses favor viral clearance. Importantly, viral clearance was heralded by early CD4+ T cell priming either before or at the onset of detectable viral spread, and it coincided with a sharply synchronized influx of HBV-specific CD8+ T cells into the liver and a corresponding increase in intrahepatic CD8 mRNA, serum ALT activity and histological evidence of acute viral hepatitis. Interestingly, the first detectable peripheral CD4 T cell response occurred during or before the phase of detectable viral expansion in the animals that cleared the infection in this study (Asabe et al.
, 2009). In contrast, the CD4 response was delayed until after the onset of viral expansion in the animals that developed persistent infection at which point the virus had infected 100% of the hepatocytes (Asabe et al., 2009) and there was an uncoordinated influx of HBV-specific CD8+ T cells into the liver and a correspondingly asynchronous increase in intrahepatic CD8 mRNA and serum ALT activity (Asabe et al., 2009).2.
1.9 SIGNS AND SYMPTOMS188.8.131.52 Extrahepatic manifestations.Symptoms outside of the liver are present in 1–10% of HBV-infected people and include serum-sickness–like syndrome, acute necrotizing vasculitis ( HYPERLINK “https://en.wikipedia.
org/wiki/Polyarteritis_nodosa” o “Polyarteritis nodosa” polyarteritis nodosa), membranous glomerulonephritis, and papular acrodermatitis of childhood ( HYPERLINK “https://en.wikipedia.org/wiki/Gianotti%E2%80%93Crosti_syndrome” o “Gianotti–Crosti syndrome” Gianotti–Crosti syndrome) (Trepo and Guillevin , 2001). The clinical features are fever, skin rash, and polyarteritis. The symptoms often subside shortly after the onset of jaundice but can persist throughout the duration of acute hepatitis B (Liang, 2009).
Membranous glomerulonephritis is the most common form (Liang, 2009) Other immune-mediated hematological disorders, such as essential mixed cryoglobulinemia and aplastic anemia have been described as part of the extrahepatic manifestations of HBV infection, but their association is not as well-defined; therefore, they probably should not be considered etiologically linked to HBV (Liang, 2009).184.108.40.206 Intrahepatic manifestationsHepatitis B virus can cause a variety of liver diseases including acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma.Acute infections: After an incubation period of six weeks to six months, this is inversely proportional to the infective dose of the virus. The spectrum of the acute infection varies from mild to severe attacks. Clinically, acute infections manifest by fever, anorexia, nausea, malaise, vomiting, jaundice, dark urine, clay coloured stools, and abdominal pain.
1 to 2 % of acute disease results in fulminant hepatitis, with a case fatality ratio of 63 to 93 % (Bracho et al., 2006). Viraemia may reach up to 1010 virions per ml. HBV replicates in extrahepatic tissues, and particularly in peripheral blood mononuclear cells (PBMCs), which may serve as a reservoir for the maintenance of infection (Mazet-Wagner et al., 2006).
Acute exacerbations of infection may develop in chronically infected patients (Kao, 2002).Chronic Hepatitis B: Chronic infection with hepatitis B virus either may be asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of several years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (HCC; liver cancer). Across Europe, hepatitis B and C cause approximately 50% of hepatocellular carcinomas (El-Serag and Rudolph, 2007, El-Serag, 2011) Chronic carriers are encouraged to avoid consuming alcohol as it increases their risk for cirrhosis and liver cancer. Hepatitis B virus has been linked to the development of membranous glomerulonephritis (MGN) (Gan et al., 2005).
Hepatocellular Carcinoma: Hepatocellular carcinoma (HCC) is one of the 10 most common cancers in man. The annual incidence is 250 000 worldwide. Risk factors are; HBV and HCV infections especially those acquired early in life or after prolonged course, cirrhosis, male sex, aflatoxin and smoking, peak incidence in 30-50 year age group. The relative risk of developing HCC is over 200-fold for HBsAg carriers over matched controls. Whilst most HCC arise in cirrhotic liver, this is not always the case (Liu et al., 2006).2.
1.10 TRANSMISSIONPossible forms of transmission include sexual contact, (Fairley and Read, 2012) blood transfusions and transfusion with other human blood products (Buddeberg et al., 2008), re-use of contaminated needles and syringes (Hughes, 2000), and vertical transmission from mother to child (MTCT) during childbirth. Breastfeeding after proper immunoprophylaxis does not appear to contribute to mother-to-child-transmission (MTCT) of HBV (Shi et al., 2011). 2.
1.11 DIAGNOSIS OF HBV2.1.
11.1 Serological Tests Serological tests are the main stay of diagnosing and differentiating the various viruses causing hepatitis, and for blood bank screening, as they are quick, cheap, and detect HBsAg carriers. Acute HBV is characterized by the presence of HBsAg in serum and the development of IgM core antibodies (anti-HBc IgM), which may be the only marker in active hepatitis, and it correlates with inflammatory activity. In the convalescent stage, HBsAg and HBeAg are cleared with the development of anti-HBs, anti-HBe and antiHBc antibodies. Anti-HBs is also elicited by vaccine.
In chronic HBV infections, HBsAg generally persists for life. Total anti-HBc tests for both IgM and IgG antibodies to HBV core protein, they indicate current or past infection by HBV respectively. IgM anti-HBc disappears six months after the acute infection.
The IgG anti-HBc appears shortly after HBsAg in acute disease and persists for life. Different methods exist for detection of HBsAg as immunodiffusion, reverse passive haemagglutination assays, and the more sensitive enzyme linked immunoassays ELISA and radioimmunoassays with detection limit of = ; 0.1 ng /ml of HBsAg.
Ordinary serological tests may not detect mutant HBsAg and would therefore give rise to false negative results. New immunoassays are designed for the detection of hepatitis B surface escape mutants, and are specifically useful in the monitoring of liver transplant recipients on HBIG prophylaxis (Ijaz et al., 2001).2.1.
11.2 Molecular Biology Techniques Different molecular techniques have been used to detect HBV DNA. HBV DNA is detectable in serum by slot or dot blot hybridization assays (Shao et al., 2007) with detection limit of 1.
5 pg per ml (4.0 x105 genomes/ml). PCR detects 103 pg /ml (approximately 100 to 1000 genomes). However, the high sensitivity of PCR is limited by the increased risk of false positive results. Clinical significance of HBV PCR is the same as detection of HBsAg and indicates current HBV infection.
HBV DNA monitoring and quantitative PCR are essential in determining the response and follow up of chronic HBV infection to treatment. Nucleic acid sequence analysis is used to identify genetic variants of the virus, and to epidemiologically type nosocomial transmission of HBV (Gunson et al., 2006). 2.
1.12 EPIDEMIOLOGYApproximately 5 % of the world`s population reaching 350 millions, have chronic HBV infection, which is the leading cause of chronic hepatitis, cirrhosis and HCC worldwide. It is estimated that 500, 000- to 1000, 000 persons die annually from HBV related liver disease (Hou et al.
, 2005). Most infections occur at birth or during early childhood. Infections usually cluster in households of chronically infected patients.Geographical Distribution Areas of high endemicity where prevalence is ; 8% are China, Indian subcontinent and Africa. Intermediate endemicity areas show prevalence of 2 to 7 % in North Africa, India and Russia. Low endemicity ; 2 % seen in Western Europe and North America. In areas of high endemicity, the lifetime risk of HBV infection is ; 60 % (Bajunaid, 2013).
The main risk factors for HBV progression to HCC include HBeAg positivity and HBV DNA levels. Seminal studies from Taiwan established these associations (Chen et al.,2010). The incidence of HCC was 1169/100,000 person-years for HBsAg and HBeAg-positive persons, 324/100,000 person-years for HBsAg positive, HBeAg-negative and 39/100,000 person-years for those who were HBsAg negative. The Risk Evaluation of Viral Load Elevation and Associated Liver Disease (REVEAL-HBV) study established HBV DNA levels as the main determinant of progression to HCC.
However, even HBsAg positive carriers with low levels of HBV DNA and normal ALT had an almost 5-fold greater risk for HCC than HBsAg negative controls (Chen et al., 2010; El-Serag,2012).In 2004, an estimated 350 million individuals were infected worldwide. National and regional prevalences range from over 10% in Asia to under 0.5% in the United States and Northern Europe. The primary method of transmission reflects the prevalence of chronic HBV infection in a given area. In low prevalence areas such as the continental United States and Western Europe, injection drug abuse and unprotected sex are the primary methods, although other factors may also be important (Redd et al.
, 2007). In moderate prevalence areas, which include Eastern Europe, Russia, and Japan, where 2–7% of the population is chronically infected, the disease is predominantly spread among children. In high-prevalence areas such as China and South East Asia, transmission during childbirth is most common, although in other areas of high endemicity such as Africa, transmission during childhood is a significant factor (Alter, 2003) The prevalence of chronic HBV infection in areas of high endemicity is at least 8% with 10–15% prevalence in Africa/Far East (Komas et al.
, 2013). As of 2010, China has 120 million infected people, followed by India and Indonesia with 40 million and 12 million, respectively. According to World Health Organization (WHO), an estimated 600,000 people die every year related to the infection.In the United States about 19,000 new cases occurred in 2011 down nearly 90% from 1990 (Schillie et al., 2013),2.2 SYPHILIS2.2.
1 SYPHILIS INFECTIONTransfusion-transmitted syphilis, which is caused by Treponema pallidum subspecies pallidum, is one of the oldest recognized infectious risks of blood transfusion (Gardella et al.,2002).Syphilis is a sexually transmitted infection caused by the bacterium HYPERLINK “https://en.
wikipedia.org/wiki/Treponema_pallidum” o “Treponema pallidum”Treponema pallidum subspecies pallidum (CDC, 2015 a). The signs and symptoms of syphilis vary depending in which of the four stages it presents (primary, secondary, latent, and tertiary) (CDC,2015b). The primary stage classically presents with a single chancre (a firm, painless, non-itchy skin ulceration) but there may be multiple sores (CDC,2015b). In secondary syphilis a diffuse rash occurs, which frequently involves the palms of the hands and soles of the feet (CDC,2015b). There may also be sores in the mouth or vagina (CDC,2015b). In latent syphilis, which can last for years, there are few or no symptoms (CDC,2015b). In tertiary syphilis there are HYPERLINK “https://en.
wikipedia.org/wiki/Gumma_(pathology)” o “Gumma (pathology)” gummas (soft non-cancerous growths), neurological, or heart symptoms (Kent and Romanelli, 2008). Syphilis has been known as “the great imitator” as it may cause symptoms similar to many other diseases (CDC,2015b; Kent and Romanelli, 2008).Syphilis is most commonly spread through sexual activity (CDC,2015b). It may also be transmitted from mother to baby during pregnancy or at birth, resulting in congenital syphilis (CDC,2015b; Woods, 2009).
Other human diseases caused by related Treponema pallidum subspecies include yaws (subspecies pertenue), HYPERLINK “https://en.wikipedia.org/wiki/Pinta_(disease)” o “Pinta (disease)” pinta (subspecies carateum), and HYPERLINK “https://en.wikipedia.
org/wiki/Nonvenereal_endemic_syphilis” o “Nonvenereal endemic syphilis”bejel (subspecies endemicum) (Kent and Romanelli, 2008). Diagnosis is usually made by using blood tests; the bacteria can also be detected using dark field microscopy (CDC,2015b). The Center for Disease Control recommends all pregnant women be tested (CDC,2015b).2.2.
2 SIGNS AND SYPMTOMSSyphilis can present in one of four different stages: primary, secondary, latent, and tertiary, (Kent and Romanelli, 2008) and may also occur congenitally (Stamm,2010). It was referred to as “the great imitator” by Sir William Osler due to its varied presentations (White, 2000).Primary stagePrimary syphilis is typically acquired by direct sexual contact with the infectious lesions of another person (CDC, 2006). Approximately 3 to 90 days after the initial exposure (average 21 days) a skin lesion, called a HYPERLINK “https://en.wikipedia.org/wiki/Chancre” o “Chancre”chancre, appears at the point of contact. This is classically (40% of the time) a single, firm, painless, non-itchy skin ulceration with a clean base and sharp borders 0.3–3.
0 cm in size (Kent and Romanelli, 2008) The lesion may take on almost any form. In the classic form, it evolves from a HYPERLINK “https://en.wikipedia.org/wiki/Macule” o “Macule” macule to a papule and finally to an erosion or ulcer (Eccleston et al.
, 2008). Occasionally, multiple lesions may be present (~40%) (Kent and Romanelli, 2008) with multiple lesions more common when coinfected with HIV. Lesions may be painful or tender (30%), and they may occur in places other than the genitals (2–7%). The most common location in women is the cervix (44%), the penis in heterosexual men (99%), and anally and rectally relatively commonly in men who have sex with men (34%) (Eccleston et al., 2008).
Lymph node enlargement frequently (80%) occurs around the area of infection, (Kent and Romanelli, 2008) occurring seven to 10 days after chancre formation (Eccleston et al., 2008). The lesion may persist for three to six weeks without treatment (Kent and Romanelli, 2008). Secondary stageSecondary syphilis occurs approximately four to ten weeks after the primary infection (Kent and Romanelli, 2008).
While secondary disease is known for the many different ways it can manifest, symptoms most commonly involve the skin, mucous membranes, and lymph nodes (Mullooly and Higgins, 2010). There may be a symmetrical, reddish-pink, non-itchy rash on the trunk and extremities, including the palms and soles (Dylewski and Duong,2007). The rash may become HYPERLINK “https://en.
wikipedia.org/wiki/Maculopapular” o “Maculopapular” maculopapular or HYPERLINK “https://en.wikipedia.org/wiki/Abscess” o “Abscess” pustular. It may form flat, broad, whitish, wart-like lesions known as HYPERLINK “https://en.wikipedia.org/wiki/Condyloma_latum” o “Condyloma latum” condyloma latum on mucous membranes.
All of these lesions harbor bacteria and are infectious. Other symptoms may include fever, sore throat, malaise, weight loss, hair loss, and headache.5 Rare manifestations include liver inflammation, kidney disease, joint inflammation, HYPERLINK “https://en.wikipedia.
org/wiki/Periostitis” o “Periostitis” periostitis, inflammation of the optic nerve, HYPERLINK “https://en.wikipedia.org/wiki/Uveitis” o “Uveitis” uveitis, and interstitial keratitis (Bhatti, 2007). The acute symptoms usually resolve after three to six weeks; (Bhatti, 2007). about 25% of people may present with a recurrence of secondary symptoms. Many people who present with secondary syphilis (40–85% of women, 20–65% of men) do not report previously having had the classic chancre of primary syphilis (Mullooly and Higgins, 2010).
Latent stageLatent syphilis is defined as having serologic proof of infection without symptoms of disease (White, 2000). It is further described as either early (less than 1 year after secondary syphilis) or late (more than 1 year after secondary syphilis) in the United States (Bhatti, 2007). The United Kingdom uses a cut-off of two years for early and late latent syphilis (Eccleston et al., 2008). Early latent syphilis may have a relapse of symptoms. Late latent syphilis is asymptomatic, and not as contagious as early latent syphilis (Bhatti, 2007).
Tertiary stageTertiary syphilis may occur approximately 3 to 15 years after the initial infection, and may be divided into three different forms: gummatous syphilis (15%), late HYPERLINK “https://en.wikipedia.org/wiki/Neurosyphilis” o “Neurosyphilis” neurosyphilis (6.5%), and cardiovascular syphilis (10%) (Bhatti, 2007). Without treatment, a third of infected people develop tertiary disease (Bhatti, 2007).
People with tertiary syphilis are not infectious (Kent and Romanelli, 2008). Gummatous syphilis or late benign syphilis usually occurs 1 to 46 years after the initial infection, with an average of 15 years. This stage is characterized by the formation of chronic HYPERLINK “https://en.
wikipedia.org/wiki/Gumma_(pathology)” o “Gumma (pathology)” gummas, which are soft, tumor-like balls of inflammation which may vary considerably in size. They typically affect the skin, bone, and liver, but can occur anywhere (Kent and Romanelli, 2008). Neurosyphilis refers to an infection involving the central nervous system. It may occur early, being either asymptomatic or in the form of syphilitic meningitis, or late as meningovascular syphilis, general paresis, or HYPERLINK “https://en.wikipedia.org/wiki/Tabes_dorsalis” o “Tabes dorsalis” tabes dorsalis, which is associated with poor balance and lightning pains in the lower extremities.
Late neurosyphilis typically occurs 4 to 25 years after the initial infection. Meningovascular syphilis typically presents with apathy and seizure, and general paresis with dementia and HYPERLINK “https://en.wikipedia.org/wiki/Tabes_dorsalis” o “Tabes dorsalis” tabes dorsalis (Kent and Romanelli, 2008). Also, there may be Argyll Robertson pupils, which are bilateral small pupils that constrict when the person focuses on near objects but do not constrict when exposed to bright light.
Cardiovascular syphilis usually occurs 10–30 years after the initial infection. The most common complication is syphilitic aortitis, which may result in aneurysm formation (Kent and Romanelli, 2008). Congenital syphilisCongenital syphilis is that which is transmitted during pregnancy or during birth. Two-thirds of syphilitic infants are born without symptoms. Common symptoms that develop over the first couple of years of life include enlargement of the liver and spleen (70%), rash (70%), fever (40%), neurosyphilis (20%), and lung inflammation (20%).
If untreated, late congenital syphilis may occur in 40%, including saddle nose deformation, HYPERLINK “https://en.wikipedia.org/wiki/Higoumenakis_sign” o “Higoumenakis sign” Higoumenakis sign, saber shin, or HYPERLINK “https://en.wikipedia.org/wiki/Clutton%27s_joints” o “Clutton’s joints” Clutton’s joints among others (Woods, 2009).Infection during pregnancy is also associated with miscarriage (Cunningham et al.,2013).
2.2.3 AETIOLOGYTreponema pallidum subspecies pallidum is a spiral-shaped, Gram-negative, highly mobile bacterium (Eccleston et al., 2008). Three other human diseases are caused by related Treponema pallidum subspecies, including yaws (subspecies pertenue), pinta (subspecies carateum) and HYPERLINK “https://en.wikipedia.
org/wiki/Nonvenereal_endemic_syphilis” o “Nonvenereal endemic syphilis” bejel (subspecies endemicum). (Kent and Romanelli, 2008). Unlike subtype pallidum, they do not cause neurological disease (Woods, 2009). Humans are the only known natural reservoir for subspecies pallidum (Stamm, 2010) It is unable to survive more than a few days without a host. This is due to its small genome (1.14 HYPERLINK “https://en.
wikipedia.org/wiki/Base_pair” o “Base pair” Mbp) failing to encode the metabolic pathways necessary to make most of its macronutrients. It has a slow doubling time of greater than 30 hours (Eccleston et al., 2008). 2.2.4 TRAMSMISSIONSyphilis is transmitted primarily by sexual contact or during pregnancy from a mother to her fetus; the spirochete is able to pass through intact mucous membranes or compromised skin (Stamm, 2010).
It is thus transmissible by kissing near a lesion, as well as oral, vaginal, and anal sex (Kent and Romanelli, 2008). Approximately 30% to 60% of those exposed to primary or secondary syphilis will get the disease (Bhatti, 2007). Its infectivity is exemplified by the fact that an individual inoculated with only 57 organisms has a 50% chance of being infected (Eccleston et al., 2008). Most (60%) of new cases in the United States occur in men who have sex with men. Syphilis can be transmitted by blood products, but the risk is low due to blood testing in many countries.
The risk of transmission from sharing needles appears limited (Kent and Romanelli, 2008). It is not generally possible to contract syphilis through toilet seats, daily activities, hot tubs, or sharing eating utensils or clothing (CDC, 2010).2.
2.5 DIAGNOSISEarly reports of the transfusion-related transmission of syphilis led to the World Health Organization (WHO) recommendations for syphilis testing of blood donors (Takpo et al., 2007).
These recommendations have been questioned, since many syphilis antibodies among blood donors are the result of previous infections or even unspecific reactions. Furthermore, Treponema pallidum does not withstand cold storage (Tagny, 2011). However, as not all blood components can be assumed to be kept cold for a sufficient amount of time, if at all, and as syphilis may also serve as a potential surrogate marker for high risk behaviour in relation to HIV infection, syphilis screening continues to be a requirement in many countries. The WHO recommends several syphilis screening tests: the enzyme immunoassay (EIA) and T. pallidum haemagglutination assay (TPHA) as specific tests, or the Venereal Disease Reference Laboratory (VDRL) and rapid plasma reagin (RPR) as non-specific screening tests (WHO,2010).Following a documented case of transfusion-transmitted syphilis in Ghana in 2011, (Owusu et al.,2011). The techniques used for syphilis screening are different from one country to another: the VDRL or RPR alone for some, and the VDRL and TPHA for others (Takpo et al.
,2007). Tests and algorithms should be selected so that they correspond with the prevalence of the disease and match the technical expertise of the personnel and the availability of reagents and equipment (Tagny, 2009). The selection criteria for a screening strategy must include simple techniques, reliability, sustainability, and cost-effectiveness. Although they are not recommended for blood banks in Africa, rapid test techniques may be preferred because of their affordability, user-friendliness, the availability of test materials, and good sensitivity and specificity; furthermore they do not require sophisticated laboratory materials (Tagny, 2009).Direct testingDark ground microscopy of serous fluid from a chancre may be used to make an immediate diagnosis.
Hospitals do not always have equipment or experienced staff members, and testing must be done within 10 minutes of acquiring the sample. Sensitivity has been reported to be nearly 80%; therefore the test can only be used to confirm a diagnosis, but not to rule one out. Two other tests can be carried out on a sample from the chancre: direct fluorescent antibody testing and nucleic acid amplification tests. Direct fluorescent testing uses antibodies tagged with HYPERLINK “https://en.wikipedia.org/wiki/Fluorescein” o “Fluorescein” fluorescein, which attach to specific syphilis proteins, while nucleic acid amplification uses techniques, such as the polymerase chain reaction, to detect the presence of specific syphilis genes. These tests are not as time-sensitive, as they do not require living bacteria to make the diagnosis (Eccleston et al.
, 2008).2.2.6 PREVENTIVE MEASURESVaccineAs of 2018, there is no vaccine effective for prevention (Stamm, 2010). Several vaccines based on treponemal proteins reduce lesion development in an animal model and research continues (Cameron and Lukehart, 2014).SexCondom use reduces the likelihood of transmission during sex, but does not completely eliminate the risk.
(Cameron and Lukehart, 2014). The Centers for Disease Control and Prevention (CDC) states, “Correct and consistent use of latex condoms can reduce the risk of syphilis only when the infected area or site of potential exposure is protected. However, a syphilis sore outside of the area covered by a latex condom can still allow transmission, so caution should be exercised even when using a condom.” (CDC, 2010). Abstinence from intimate physical contact with an infected person is effective at reducing the transmission of syphilis. The CDC states, “The surest way to avoid transmission of sexually transmitted diseases, including syphilis, is to abstain from sexual contact or to be in a long-term mutually monogamous relationship with a partner who has been tested and is known to be uninfected.” (CDC, 2010). Congenital diseaseCongenital syphilis in the newborn can be prevented by screening mothers during early pregnancy and treating those who are infected (Schmid, 2004).
If they are positive, it is recommend their partners also be treated. (Hawkes et al., 2011). Congenital syphilis is still common in the developing world, as many women do not receive antenatal care at all, and the antenatal care others receive does not include screening. It still occasionally occurs in the developed world, as those most likely to acquire syphilis (through drug use, etc.
) are least likely to receive care during pregnancy (Schmid, 2004). Several measures to increase access to testing appear effective at reducing rates of congenital syphilis in low- to middle-income countries (Hawkes et al., 2011). Point-of-care testing to detect syphilis appeared to be good although more research is needed to assess its effectiveness and into improving outcomes in mothers and babies (Shahrook et al.,2014). 2.2.7 EPIDEMIOLOGYSyphilis is still a public health problem in the world.
The World Health Organization estimated that approximately 12 million new cases are reported each year in the world with more than 90 percent from developing countries (Centers for Disease Control CDC, 2007; World Health Organization WHO, 2001). Moreover, syphilis has acquired a higher potential of morbidity and mortality with the increasing prevalence of HIV infection. If syphilis is rare in developed countries, it is much more common in developing countries where prevalence can reach 25% amongst blood donors (Tagny & al., 2009, 2010).The infection is transmitted from person to person through contact with a syphilis ulcer (during vaginal, anal, or oral sex). An infected mother can infect her fetus via the placenta. Furthermore, intravenous drug addicts or other infected person can transmit syphilis through infected blood products i.e.
through blood transfusion or use of infected needles for example (Workowski & Berman, 2006).2.3 BLOOD TRANSFUSIONBlood transfusion is a life saving intervention that is essential in the management and care of patients. In 2005, all member states of WHO signed a document that commits them to the provision of safe and adequate blood and blood products to patients (WHO, 2010). This concern stems from the fact that there is a wide spectrum of blood borne infections which can be transmitted through the blood of apparently healthy and asymptomatic blood donors. These transfusion transmissible infectious agents include hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1/2), human T-cell lymphotropic viruses (HTLV-I/II), Cytomegalovirus (CMV), Parvovirus B19, West Nile Virus (WNV), Dengue virus, trypanosomiasis, Syphilis and malaria (Allain et al.,2009). This scenario is worsened by the method of replacement of blood either by family members or paid donors as against WHO recommended voluntary donors (WHO, 2010).
Regular voluntary, unpaid blood donors are the safest group of donors as the prevalence of blood borne infections is lowest among these donors (WHO, 2010).2.3.1 CLASSIFICATION OF BLOOD DONORS IN NIGERIA. There are 3 main classifications of blood donors in Nigeria; the voluntary (non remunerated) donors, family (replacement) donors (FDs) and commercial (paid) donors (Ahmed et al., 2007). The voluntary donors are the altruistic individuals who donate blood with the sole aim of saving a life, without regard to any form of inducement. They are usually mobilized through the mass media or blood donation drives to schools, churches and mosques.
An earlier study had hypothesized that the dearth of voluntary donors in Nigeria and Sub-Saharan Africa is probably associated with the fact that the mentality of altruism (regarding blood donation) is not yet generally accepted in the typical African culture, compared to what is obtainable in the most developed countries (Osaro and Charles, 2011). The family (replacement) donors include those that donate for a hospitalized family member, friend, or associate and is largely nonremunerated, depending entirely on the good will of friends and family members. Osaro et al. had concluded that the continued importance of family donors in Sub-Saharan Africa could be linked to the fact that it may actually cost less to procure and is also well adapted to the extended family support system of many Nigerian and African communities (Osaro and Charles, 2011).
However, FDs may be under pressure to donate blood when their relatives are admitted to hospital and in need of a blood transfusion, even when they know that they are potentially at risk of sexually transmitted diseases as a result of high-risk behaviours. They may be more likely to conceal a relevant medical history and the risky sexual behaviours that predispose them to infections and thus pose a threat to the safety of the blood supply. Despite this, family donations remain dominant in the African continent because family and community ties are often considerably stronger than in other types of society; making the gift of blood is a natural contribution to relieve sufferers in hospitals (Ne’bie’ et al., 2007). Additionally, potential donors may be less willing to donate to someone not known to them. The WHO states that blood from VNRDs who give blood out of altruism is the safest source of blood (Takpo et al.
,2007). Persistent blood shortages coupled with increased poverty in Nigeria (and most African countries) created another population of donors who give blood strictly for financial gratification; these constitute the commercial (paid) donors. These have continued to increase in number and prominence in Nigeria, fuelled by the very huge deficit in blood supply and utilization. Ahmed et al. succinctly captured this phenomenon in a report among blood donors seen at the University of Maiduguri Teaching Hospital, Northeast Nigeria over a 12 years period (Ahmed et al., 2007). They observed a progressive decrease in the percentage of voluntary blood donation, over the study period, from 31% to 5%, against an increase from 20% to 63% in the frequency of commercial blood donation (Ahmed et al., 2007).
The above study equally emphasized the wide gap between blood supply and demand in Nigeria by showing that the mean annual increment in the number of blood donations (4%) was well below the mean annual increment in in-patient numbers (11%) (Ahmed et al., 2007). 2.3.2 WORLD HEALTH ORGANISATION (WHO) AND NATIONAL BLOOD TRANSFUSION SERVICE (NBTS) STANDARD OF BLOOD TRANSFUSION IN NIGERIA.The WHO recommends that each country should decide on the TTIs to be screened for as part of the blood screening programme and develop a screening strategy appropriate to its specific situation, influenced by the incidence and prevalence of infection, the capacity and infrastructure of the blood service, and the costs of screening (WHO, 2009).
WHO recommends that universally, blood for transfusion be screened for HIV, HBV, HCV and Syphilis. In selected countries depending on epidemiological evidence, screening should be done for the following also; malaria, Chagas disease, Human T-cell lymphotropic viruses 1 & 2 and Human Cytomegalovirus (WHO, 2010). The World Health Organization (WHO) had projected that Sub-Saharan Africa will attain sustainable blood transfusion safety by the year 2012, through the implementation of sets of policies geared toward provision of safe, affordable, and readily available blood units in hospitals to serve the needs of patients. (Tagny et al.,2008). Up till now, however, this goal is far from achieved in Nigeria and a number of other African countries, with attendant negative impact on health indices.
Effective healthcare delivery globally is known to be supported by a robust supply of safe blood units which could indeed be lifesaving in a number of clinical scenarios. Correspondingly, from the road traffic accident victim with acute hemorrhage at the emergency room to the obstetric patient with antepartum/postpartum hemorrhage or the under-five child presenting with anemic heart failure, prompt administration of appropriate units of blood could well make the difference between life and avoidable demise. Earlier, extensive inquests into the causes of the high maternal mortality in Nigeria and Sub-Saharan Africa had highlighted the huge contribution of the very ineffective blood transfusion services (Umeora et al., 2005; Bates et al., 2005).In Nigeria, the national blood transfusion guideline stipulates that donor blood should be screened for specified Transfusion-Transmissible Infections including Human Immunodeficiency Virus (HIV), HBV, HCV and Syphilis (NBTS, 2006). The National Blood Policy Nigeria established a National blood transfusion policy through a published set of guidelines in December 2006. The publication was a fall-out of the baseline survey on blood transfusion practices which was earlier conducted in the country in August 2005. Salient conclusions from the survey included; (NBTS, 2006).? Only half a million units of blood were collected from both private and public sources in 2004? At the time of the survey, blood need in Nigeria was estimated to be 1.5 million units? In the public sector, the donor population was made up of 25% commercial donors and 75% of replacement donors. Voluntary unpaid donors were negligible? In the private sector, the donor population was made up of 75% commercial donors and 25% of replacement donors. Voluntary unpaid donors were insignificant.The National blood policy is essentially made up of sets of action plans which are all geared toward the provision of safe, available, and affordable blood donor units, where and when they might be needed in the country. It is structured into blood transfusion services under the following strata;the national blood transfusion service (NBTS), the zonal blood service centers, state and local government areas blood service centers, the armed forces blood service, and private and other nongovernmental health establishments ( NBTS, Nigeria,2006). The essence of the above stratification was to ensure universal coverage of the country, right to local government councils.2.3.3 RISK FACTORS OF BLOOD TRANSFUSIONFactors contributing to transfusion-related transmissions in sub-Saharan Africa include: high rates of transfusion in some groups of patients (particularly women and children); a high prevalence of human immunodeficiency virus (HIV) in the general and blood donor populations; inadequate screening facilities; and lack of infrastructure and capacity to ensure sustainable operations (Holmberg, 2006; Bournouf and Radesevich,2000). Since a person can transmit an infection during its asymptomatic phase, transfusions can contribute to an ever-widening pool of infection in the population. The economic costs of the failure to control the transmission of infection include increased requirement for medical care, higher levels of dependency and the loss of productive labour force, placing heavy burdens on already overstretched health and social services and on the national economy (WHO, 2002; Kitchen and Barbara, 2001). It should, therefore, be mandatory that blood is screened for transfusion-transmissible infectious disease markers such as antibodies to HIV, hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis, and hepatitis B surface antigenaemia (Choudhury and Phadke, 2001;Nwabuisi et al., 2002).2.3.4 PREVALENCE OF HEPATITIS VIRUS AND SYPHILIS AMONG BLOOD DONORS.Hepatitis B and other Hepatitis virus among blood donorsIn Federal Medical Centre, Umuahia, screening is carried out for HIV, HBV, HCV and syphilis. Human immune deficiency virus, HBV and HCV are of particular concern because of their prolonged infectivity, carrier state and the fact that they also cause various debilitating disorders which may eventually be fatal (Wallace, 2008). Though these viruses can also be transmitted through other means, infectivity estimates for the transfusion of infected blood products are much higher (92%) than for other modes of transmission owing to the much larger viral dose per exposure than for other routes (Baggaley et al., 2006). It is also important to discourage and as much as possible eliminate commercial and replacement blood donation by relations of the person as such persons have been shown to be likely to test positive for blood transmitted infections (Eldryd et al., 2004). Although blood transfusion contributes relatively little to the overall HIV and other pathogen transmission, prevention of infection through blood transfusion is a priority for ethical reasons. Apart from this, whatever quantity of the pathogen that is present in blood for transfusion is most likely to be transmitted to the recipient as the blood will act as a direct vehicle. The median overall risks of becoming infected with HIV, HBV, and HCV from a blood transfusion in sub-Saharan Africa were 1, 4.3, and 2.5 infections per 1000 units, respectively (Jayaraman et al., 2010). While in the developed countries the estimate is 1/2 600 000 for HIV, 1/6 500 000 for HCV, 1/1 700 000 for HBV (Traineau et al., 2009). According to the World Health Organization (WHO), each year about 340 million new infections are due to sexually transmitted diseases such as chlamydia, gonorrhea, syphilis and Trichomonas (WHO, 2001). Syphilis remains a major public health problem in sub-Saharan Africa, including Burkina Faso. It is diagnosed routinely in all blood donors using non-treponemal and treponemal tests such as Rapid Plasma Reagin test (RPR) and T. pallidum haemagglutination Test (TPHA) (Wiwanitkit, 2002). Syphilis among blood donorsBlood donors with high-risk sexual behaviour and other risk factors may be infected by syphilis and compromise the safety of blood used for transfusion. The medical selection of the blood donors consists of information of the donor, the finding of the risk factors in the behaviours and the medical history using a questionnaire, the physical examination in order to find clinical signs of the infection. Donor deferral follows identification of any risk. Medical selection is crucial because it could permit to defer more than half of infected donors, especially the ones in the early period of infection here laboratory tests are not efficient (de Almeida Neto & al, 2007; Tagny, 2009). In some European countries, the prevalence of T. pallidum infection in the general population and thus in blood donors has been increasing since last two decades. An increase in syphilis infections has been associated to the high incidence of HIV. Moreover, an infected blood donor with syphilis is more than 5 times more likely to be HIV-positive. However, the prevalence of syphilis is still very low in developed countries and the very rare cases of recipient contamination raised the question of whether syphilis screening was still necessary for blood donors. In developing countries, the prevalence of positive serologic tests for syphilis can reach 25%. The prevalence is however very variable from one area to another and from a country to another. In such settings, the poor quality of laboratory screening due to the lack of equipment, training personnel, reagents and standard procedures highlights the need of the systematic and better screening for syphilis to help ensure a safer blood supply. Very little systematic information is available on the profile of positive blood donors including differences between donors with recent versus past infection. The exclusion of donors with past and treated infection is still a matter of discussion. Abusive exclusion reduces the blood supply and could be problematic in developing countries. However, past history of syphilis may be high-risk sexual behaviour associated to transmitted transfusion infection such as syphilis itself and HIV. The transfusion risk of syphilis is closely related to risk factors in the blood donor, in particular the sexual behaviours, the disease being primarily transmitted by sexual route. The rates of infection are highest amongst homosexual (gay) men – or men who have sex with men (VallMayans & al, 2006). Recent syphilis infections have been shown to be associated with younger age, male-male sex, two or more sex partners, past syphilis treatment, past syphilis history, HIV seropositivity. Risk factors usually associated with transfusion transmitted syphillis also include more than one sexual partner, prostitution, bisexuality ( men having sex with both men and women), intravenous drug use, and skin scarification (tattoing,blood rituals). In developing countries, most blood donors infected are first-time donors. The prevalence of syphilis is one of the highest amongst the TTI screened in developing countries. The problem of this disease, first of all, is its high prevalence in blood donors in various areas of Africa. The recent prevalence were 3.7 % in Congo (Batina & al 2007), 7.9 % in Ghana (Adjei & al., 2003; Ampofo & al., 2002) and 9.1 % in Cameroon (Mbanya & al., 2003; Tagny & al., 2009). It is just as high in females as in males, in the different age groups and in voluntary donor as well as family donors. The family blood donation and remunerated blood donation, mostly found in developing countries is statiscally associated with higher prevalence of the disease (Batina & al., 2007; Tagny & al., 2010). The donors who have been positive for syphilis during the previous donation are less likely to donate again, whereas donors who were negative for the presence of syphilis in the past would be more likely to donate again. In countries, which use a medical questionnaire for selection of blood donor, there are usually questions related to infection with syphilis. These questions concentrate particularly on sexual behavior (a number of sexual partners, use of condoms, past history of sexually transmitted diseases) and sometimes on specific symptoms observed during clinical examination. However, medical selection remains ineffective for several reasons: – Difficulty of understanding the questions due to the level of education (ignorance of the transmissible infections by blood transfusion) (Nébié & al., 2007; Agbovi & al., 2006), linguistic and cultural (taboos) barriers; – Discrete expression of the disease in its primary phase. The syphilitic rosella is not clearly visible on dark skin. – Suppression of clinical signs and symptoms by the various antibiotics following self – medication (ampicilline, penicillin). Thus, the biological screening of this disease remains essential to defer blood donors at risk. Identified safe donors must be retained in the pool of repeated donors and frequently informed and educated to avoid risky behaviours (Claude, 2011).2.3.5 SYPHILIS AND SCREENINGOF BLOOD DONATION.At the beginning of the 20th century newer tests were developed. Present-day, several labs tests, treponemic or not treponemic exist, among which rapid tests, immunological tests, and genomic (Young & al., 2000). Neither there is a specific type of method absolutely indicated, nor is there any confirmatory algorithm for testing based on the different assays available. In fact, the laboratory assessment of syphilis is generally based on the detection of antibodies against T. pallidum antigens in blood by the use of either specific or nonspecific reagents. The detection of genomic particle are more specific but not affordable for most of laboratories (Marfin & al., 2001; Orton & al. 2002). The detection of specific Treponema antigens is possible using methods as passive agglutination, as T. pallidum hemagglutination (TPHA) assay or the T. pallidum particle agglutination (TPPA) assay, indirect immunofluorescence as the fluorescent treponemal antibody absorbed (FTA-ABS) assay or enzyme immunoassay (EIA) for the detection of specific IgG and IgM or total Ig. Non-treponemal methods are based on non-treponemal lipid antigens (cardiolipin), using frequently the flocculation technique. Of these, the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR) tests are the most commonly used. These tests are cheap, fast and more sensitive (Montoya & al. 2006; WHO, 2006). They are able to identify the contaminated blood donors few days before the treponemal test and thus useful for acute infection. However, VDRL and RPR cannot be automated and are time-consuming if used for large scale testing. Moreover, they produce more false positive results. These tests are routinely used to screen blood donors. False positives on the rapid tests can be seen in viral infections such as hepatitis, tuberculosis, malaria, or varicella. Thus, non-treponemal tests should be followed up when possible by a treponemal test. The treponemal tests are based on monoclonal antibodies and immunofluorescence; they are more specific and more expensive. The tests based on enzyme-linked immunoassays are the more specific and are usually used to confirm the results of simpler screening tests for syphilis. According to the guidelines published by the U.S. Centers for Disease Control and Prevention, the diagnosis of syphilis should be based on the results of at least two tests: one treponemal and the other non treponemal (CDC, 2006; CDC, 2004 ). According to WHO, blood banks may choose Venereal Disease Research Laboratory (VDRL), rapid plasma reagin (RPR), or enzyme immunoassay (EIA). VDRL and RPR are sensitive for recent syphilis infection, but not for past infection. Screening should be performed using a highly sensitive and specific test for treponemal antibodies: either TPHA or enzyme immunoassay. In populations where there is a high incidence of syphilis, screening should be performed using a non-treponemal assay: VDRL or RPR. EIA can detect past or recent infection, but may result in rejecting non-infectious blood with distant past infection (Cole & al., 2007). However, one should remember that the reliability of the screening and the diagnosis include the performances as well as the quality assessment notably the use of standard operating procedures, norms, training of the personnel and management of quality. The screening for syphilis is frequently carried out on the African blood donor, and national policies often include the disease in the list of ITT to be screened at the time of blood donation. More than 90 % of blood collected in Africa in the year 2004 was screened for syphilis (Tapko & al., 2005). The techniques used for screening are different from one country to another: VDRL or RPR alone for some, VDRL + TPHA for others (Tagny, 2009). Developing countries are characterized by a difficult epidemiologic, sociological and economic environment which limits the implementation of a high quality of blood safety. Thus, this context requires that tests and algorithms should be selected so that they correspond with the high prevalence of the disease, limited technical know-how of the personnel and limited availability of reagents and equipments. The selection criteria of screening strategy must include simple techniques, reliability, sustainability and cost effectiveness. Regular supply of electricity, freezer and ELISA kits is mostly found in big cities and barely available in small towns. Several blood banks use rapid test technique as it does not required sophisticated lab materials (Tagny & al., 2009). Screening strategies must also take into account the training of technicians, guarantee their capacity to carry out the test and provide reliable results (Claude, 2011).CHAPTER 3: MATERIALS AND METHODS3.1. Study design and settingThe data of blood donor recorded from January 2017 to April 2018 and Bio-data and positivity of the diseases will be collected at the blood bank of the Meridian Hospitals. Situated at 21 Igbokwe str D/Line. Port Harcourt. Rivers State. Nigeria. This hospital also has as its objective the management of the numerous accidents along the Aba road which is major highway and other surrounding roads due to the reckless driving of taxi drives, management which often requires blood transfusions.3.2. Study populationDonors were either volunteers, or relatives or friends of patients who came to replace blood used or expected to be used by patients. Voluntary donors either belonged to an association of blood donors or came individually on their own account to donate blood. 3.3 Sample collectionBlood samples will be aseptically collected from each subject by venipuncture in 5-ml red-top vacutainers (Becton Dickinson, NJ, USA) and allowed to clot naturally at room temperature. Serum specimens will be separated by centrifugation at 3000 g for 5 min and will be used for the analyses.3.4 Hepatitis B surface antigenemiaHBV was detected using a one-step immunoassay-based DIASpot HBsAg test kit (DIASpot Diagnostics, USA) for qualitative detection of hepatitis B surface antigen (HBsAg) in serum. This test has a relative sensitivity and speci?city of 99% and 97.0%, respectively. Followed with and HBsAg ELISA rapid kit.3.5. Syphilis serologySyphilis will be diagnosed using the Venereal Disease Research Laboratory (VDRL) test (Omega Diagnostic, UK) and the Treponema pallidum hemagglutination assay (TPHA) test (Omega Diagnostic, UK). Active syphilis will be diagnosed if an individual’s blood tested positive with both tests. All samples positive for one test and negative for the other will be excluded from the analysis.