Syphilis, an ancient disease, is still a public health problem worldwide. The World Health Organization (WHO) estimated that there are 12 million new cases of syphilis each year, with more than 90% occurring in developing nations (Cesar de Almeida et al., 2009). Prevalence of syphilis among blood donors varies between countries. In Malaysia, prevalence of syphilis among blood donors in 2007 was 0.1% (BTS, 2007).
Blood transfusion service in Malaysia was started in 1950 at Kuala Lumpur Hospital. The first transfusion transmitted infection screening introduced was syphilis screening in 1950s. The earliest report available on prevalence of syphilis among blood donors in Malaysia was in 1974, which was as high as 3.2% (Malaysia, 1974).
Syphilis is a sexually transmitted infection caused by bacterium Treponema pallidum. It is spread through sexual contact with an infected person in the first year of infection, where the probability of transmission exceeds 60% (Alberta, 2007). Syphilis is also spread during pregnancy or at delivery as well as through blood transfusion, especially in fresh blood transfusion. Therefore, in many countries it is required that blood banks screen every donation for syphilis.
Routine screening of blood donors for syphilis has been justified for two reasons. First, preventing transfusion transmission of syphilis and secondly, serving as a surrogate marker for infections caused by other pathogens such as human immunodeficiency virus (HIV) (Shimian et al., 2009).
In the past, blood transfusion was a potential route of infection, especially if fresh blood was transfused. After donated blood is stored at 40C, any infection risk is essentially reduced as the organism is very sensitive totemperature and destroyed at low temperature.T. pallidum survives at the most 5 days in blood stored at 40C (Kaur and Basu, 2005). Improved donor selection, serologic screening of donors for the presence of syphilis, and the shift from transfusion of fresh blood components to refrigerated blood components have greatly decreased rates of transfusion transmitted syphilis.
Syphilis infection is also commonly used as a marker of donor suitability. Although syphilis is not a specific marker for HIV infection, it indicates donor who are at risk of sexually transmitted diseases because of their sexual behaviour. Since this may increase their risk of exposure to HIV infection, donors with such patterns of risk behaviour should be deferred.
Because the causative agent T. pallidum cannot be grown using conventional techniques, serology is the primary method for laboratory diagnosis of syphilis. Syphilis serological test are divided into two types i.e. non-treponemal and treponemal test. Traditional screening for syphilis infection involves using a non-treponemal test, followed by a confirmatory treponemal test.
The non-treponemal tests uses cardiolipin antigen to detect cardiolipin antibodies. The Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) test are the most commonly used non-treponemal tests. These tests are sensitive, but there are susceptible to nonspecific reactions.
Treponemal tests uses T. pallidum or its components as the antigen to detect treponemal antibodies. Treponemal antibodies may be detected by a variety of techniques based on agglutination, enzyme immunoassay (EIA), immunofluorescence, immonublotting and immunochromatography. These include T. pallidum haemagglutination (TPHA), T. pallidum particle agglutination (TPPA), Particle gel immunoassay (PaGIA), EIA, Fluorescent treponemal antibody absorption (FTA-Abs) test and immunoblotting. These tests are specific and reactive results are likely to represent true syphilis infection.
Current practice in National Blood Centre (PDN), Kuala Lumpur for syphilis screening consists of RPR as a standard initial test and TPPA as a standard confirmatory test. All blood donations will be tested with RPR and blood which are reactive for RPR will be confirmed with TPPA. This current practice has limitations in terms of high rates of false positives and unidentified positive cases.
RPR may not identify individuals very early in primary syphilis or in late latent stages (Chuck et al., 2008). RPR tests have detectable reactivity approximately two weeks after exposure (Orton, 2001). RPR tests are not very specific, with high frequency of false positive results. False negative results have also been reported with high titre sera due to a prozone phenomenon. Confirmatory testing using TPPA is less sensitive than the RPR test in the early stage of syphilis and false positive results have also been reported (Aktas et al., 2007). TPPA tests have detectable reactivity approximately four weeks after exposure (Orton, 2001). Data from 2008 PDN annual report showed that reactive rate for RPR was 0.113% and true positive rate (both RPR and TPPA reactive) was only 0.076%.
In contrast to syphilis screening, HIV and hepatitis screening protocol in PDN have greater diagnostic accuracy. This protocol consists of primary screening test, confirmatory test and few supplementary tests. Furthermore, nucleic acid amplification technology (NAT) for HIV and hepatitis has been introduced in November 2007 to detect the presence of viral nucleic acid, DNA or RNA. Currently all blood donation samples will be subjected to NAT testing for HIV and hepatitis during primary screening. This technique enables detection of low levels of virus in the samples and thus reduces the window period of infection.
The limitations of current syphilis screening protocol necessitate a reappraisal of screening and confirmatory tests for syphilis in blood donors. Therefore, this study will be conducted to evaluate the performances of RPR and TPPA which currently used for syphilis screening in PDN and also EIA which is increasingly being used for syphilis screening in blood bank setting. Thus, TPPA and EIA tests are perform in parallel with RPR test which is use as routine primary screening for blood donor in
PDN. Three serological assays will be evaluated i.e. Immutrep Carbon Antigen (Omega Diagnostics, United Kingdom), Serodia-TP.PA ( Fujirebio Inc, Japan) and Syphilis Total Antibody (TA) EIA II (Newmarket Laboratories Ltd, United Kingdom). EIA is regarding to gold standard test for this study. The discrepant result will be determined with EIA results, if EIA is positive, that sample is determine as true positive.
Study justification
Syphilis is one of the most prevalent sexually transmitted infections worldwide. Although the incidence of syphilis declined after the introduction of penicillin 60 years ago, but it is still remain a public health concern especially in developing countries. The disease is still endemic and has resurged in some urban area. The declining trend of syphilis infection was reversed due to increased in high risk behavior practices especially heterosexual. The potential for concomitant infection with HIV to influence both treponemal antibody response and clinical course of syphilis is an additional factor to consider in the selection and use of serological tests for syphilis.
The above changes in the epidemiology of syphilis necessitate a reappraisal of screening and diagnostic methods. In spite of the recent increases, screening for syphilis especially in blood bank setting involves very large numbers of specimens being examined to detect very small number of positive findings. The screening test should be very sensitive to detect very low titer of antibodies. The confirmatory test should be very sensitive as well as very specific to exclude false positive detected by screening test.
The current protocol for syphilis screening in PDN has some limitation with high rate of false positive and possible false negative cases. False positive cases may result in the wastage of donations and unnecessary deferral of donors. In false negative cases, there is potential for spread of the disease. Therefore, there is a need for evaluation of syphilis serological tests to improve methods of syphilis screening in blood donors. This can lead to a safer blood supply, reduced wastage of donation and better management of blood donors who have positive syphilis serological test.
Benefit of the study
Result of this study would show which test would be more superior and appropriate for screening and confirming syphilis infection in blood bank setting to ensure safe blood supply. Improvement in this screening strategy also can reduce wastage of donation and unnecessary donor deferral, and better management of the blood donors who have positive syphilis serological test. Besides that, infective donation would not go undetected and cause post transfusion syphilis.
LITERATURE REVIEW
Syphilis is caused by bacterium T. pallidum. It may be found in blood and other body fluids and can be transmitted through blood transfusion. Syphilis is a chronic disease. Once in the bloodstream, the bacteria spread throughout the body, damaging many organs over times, especially the central nervous system.
While T. pallidum may be found in the bloodstream, levels are variable, even in acute primary syphilis, and the bacteraemia is often short-lived. In addition, the treponemes are relatively fragile and heat-sensitive; storage below +20°C for more than 72 hours results in irreparable damage to the organism such that it is no longer infectious. Thus, although clearly potentially infectious, the risk of transmission through the transfusion of blood and blood components stored below +20°C is very low (WHO, 2009).
Blood components stored at higher temperatures (above +20°C), such as platelet concentrates, or those not stored at lower temperatures for any length of time, such as blood collected and used within 48 hours, present a significantly higher risk of transmitting syphilis. Thus, although the risk of transmission of syphilis from unscreened donations is variable, the screening test is nonetheless considered essential as most blood transfusion services provide some blood components that are either stored above +20°C or are not stored below +20°C for sufficient time to kill any organisms present (WHO, 2009) .
Transfusion-transmitted syphilis was first reported in 1915, but by 1941, 138 cases had been reported (Orton, 2001). With the use of refrigerated blood storage, transfusion-transmitted syphilis has reduced. Since 1965, only two cases of transfusion-transmitted syphilis have been reported in English literature (Cable, 1996). The last reported case of transfusion transmitted syphilis in the United States was in 1966 (Brian, 2009).
Syphilis is reported as rarely transmitted by transfusion. Therefore, clinicians are unlikely to recognize a case when it occurs. Transfusion-transmitted syphilis may, in fact, be more common than reported. Transfusion cases may be excluded erroneously because it is difficult to exclude sexually transmitted syphilis. Transfusion cases have no chancre, which is the diagnostic hallmark of syphilis. Moreover, many transfusion recipients are treated with antibiotics coincidentally and T. pallidum is sensitive to most antibiotics. This may prevent or modify the clinical picture and it is often unrecognizable clinically (Cable, 1996).
The natural history of syphilis is very variable and may lead to various clinical presentations. The incubation period is 9-90 days with an average of 21days (Orton, 2001). In early stages, syphilis is a highly infectious disease with major health and life consequences, as it progresses from one stage to another. Clinical manifestation of syphilis can be divided into several stages: primary, secondary, latent and tertiary.
Primary syphilis is characterized by chancre at the point of contact and regional lymphadenopathy. Signs of secondary syphilis usually occur approximately 6 months after untreated primary stage. Presentation may include rash, fever, malaise, lymphadenopathy, mucosal lesion, genital wart and meningitis. This is followed by a latent period where the individual is asymptomatic. Approximately 15% to 40% of patients with untreated syphilis progress to the tertiary stage where serious physiological and neurological consequences result in increase health care costs (Alberta, 2007).
The immune response to syphilis infection involves production of non-specific (cardiolipin) antibodies as well as specific treponemal antibodies. The first demonstrable response to infection is the production of specific treponemal immunoglobulin M (IgM), which may be detected at the end of the second week of infection, and immunoglobulin G (IgG) appears later at about 4 weeks (Egglestone and A.J.L., 2000). Non-specific antibodies and anti-treponemal IgM both decline after successful treatment but anti-treponemal IgG generally persist and often remains detectable after many years after the disease has been cleared (Cole et al., 2007).
Various test methods for the diagnosis of syphilis have been developed. The most widely used methods are serological tests. Serological test for syphilis was first developed by August von Wasserman in 1906 and this allowed for testing of blood donors beginning in the 1940s (Orton, 2001). Serological tests are generally divided into two category i.e. non-treponemal tests and treponemal tests.
The non-treponemal tests use an antigen which contains standardized amounts of cardiolipin, cholesterol and lecithin. It detect cardiolipin antibodies formed by the host in response to lipoidal material released from damaged host cells as well as to lipoprotein-like material released from the treponemes (Aktas et al., 2005). Cardiolipin antibodies rise significantly in active infection due to cellular damage. Therefore, these tests identify those individuals who may have been recently infected. These cardiolipin antibodies also present in other diseases and non diseased states and can result in positive result in patient without syphilis. There is also possibility of classifying infective material as normal (Brian, 2009).
The majority of non-treponemal tests are flocculation test. With a reactive serum, antigen antibody complexes are formed which remain suspended as flocculates. Non-treponemal tests such as RPR are widely available, rapid and inexpensive. There are, however, a number of limitations associated with non-treponemal tests (Nesteroff, 2004). Firstly, they lack of sensitivity in late stage infection: 30% of patients with late latent or late active syphilis will show a non reactive result. Secondly, 1-2% of patients with secondary syphilis exhibits a prozone reaction (excess antibodies in diluted serum inhibits flocculation with the antigen, giving rise to weakly reactive, atypical or occasionally false reactive results). Finally, antibodies detected by non-treponemal tests are not only produced as a consequence of treponemal infection, but also in response to other conditions where tissue damage occurs referred to as biological false positive reactions (Nesteroff, 2004).
Treponemal test detect specific antibodies and thus identify donations from anyone who has ever been infected with syphilis, whether recently or long in the past, and whether treated or not. These tests are importance in diagnosis of syphilis. It is used primarily to verify reactivity in the non-treponemal tests. It may also be used to confirm a clinical impression of syphilis in which the non-treponemal test is non reactive, but there is evidence of syphilis. When treponemal tests are used for screening purposes, about 1% of general population will have false positive reaction. However, a reactive treponemal test result on a sample that is also reactive in a non-treponemal test is highly specific.
TPPA is a particle agglutination test, which detects presence of treponemal antibodies by the principle of agglutination particle enabling macroscopic reading of a result. Thus, it is more specific and sensitive, simple and cheap routine test for syphilis. However, it is less sensitive than RPR in the early stage of syphilis.
EIA tests can detect treponemal IgG and/or IgM. It is very specific, sensitive and reliable method for detection of treponemal antibodies. The advantages of EIA are suitability for automation, objective reading of results and the ability to link EIA plate readers to laboratory computer systems, reducing potential for transcription errors. EIA has better sensitivity and specificity than TPPA (Brian, 2009). However, it is more expensive compared to TPPA.
The FTA-Abs is generally regarded as gold standard confirmatory test, but it has a number of limitations and can lead to false positive and false negative. It is subjective test and difficult to standardise. It relies on subjective reading and is dependent on technician's experience. Approximately 1-2% of normal population will show false positive FTA-Abs result (Nesteroff, 2004). False positive results also have been reported for patients with other spirochete infections or immunological disorder (e.g. Systemic lupus erythematosis, rheumatoid factors, antinuclear and anti-DNA antibodies or cross reacting antibodies such as those from Borrelia burgdorferi) (Hagedorn et al., 2002). In very early syphilis, the FTA-Abs may be negative in up to 20% of patients (Clyne and Jerrard, 2000).
Because of the limitations of FTA-Abs test, possibility of using EIA as an alternative has been evaluated in various studies. EIA assays are more sensitive and yield more objective results than FTA-Abs assay (Ebel et al., 2000). Studies have shown that EIA is a reliable method for detecting antibodies to T. pallidum. Several studies have been done to compare the accuracy of EIA and FTA-Abs in detecting syphilis infection. The sensitivity and specificity of EIA was 100% and 99.9%. One study shown that the specificity of EIA is higher than FTA-Abs and conclude that EIA may be an alternative to FTA-Abs (Castro et al., 2003). Another study also have shown that EIA is slightly more sensitive than FTA-Abs (Aktas et al., 2007). A report suggested that a recombinant antigen-based EIA is the most sensitive treponemal test with high specificity (Chew, 2008).
Accurately diagnosing the disease is critical to reduce the burden and spread of the disease. In cases of false positives, individuals may undergo treatment unnecessarily. Contacting sexual partners may result in unnecessary stigma for the individual, their family and those persons contacted for testing. False positives result in considerable unnecessary time commitment for healthcare professional responsible for contacting partners and administering the tests. In blood bank setting, false positives result also may result in the wastage of donations and unnecessary deferral of donors.
Cases of false negatives, where patient symptoms have gone unnoticed, undiagnosed and untreated have the greatest potential for spread of the disease. Syphilis can be successfully treated but can also be difficult to diagnose. Improving the accuracy of testing and diagnosis of the disease can have major positive impacts on outcomes for individuals, population health and the health care system.
An important principle of syphilis serology is the detection of an antibody response by a screening test, followed by confirmation of a reactive result by further testing. Any specimen found reactive with screening test should be confirmed by confirmatory testing before a diagnosis is made because of the low prevalence of syphilis. Screening tests should be readily available, economical and suitable for large numbers of specimens. They should have high sensitivity, to identify as many patients with the disease as possible, and a high negative predictive value. The confirmatory tests should ideally have high sensitivity and specificity, to identify any false positives detected by screening, and a high positive predictive value.
There are 3 screening alternatives i.e. screening with a non-treponemal test, screening with treponemal test, or screening with combination of both tests. The decision on which tests a laboratory uses depends on several factors including cost, ease of use and convenient, compatibility with other tests, rapid result, performancecharacteristics and whether the aims is to detect all stages of syphilis or only infectious syphilis.
To minimize the risk of syphilis infection through the route of transfusion, WHO recommended (WHO, 2009);
Previous guidelines for serological diagnosis for syphilis, produced by the WHO recommended the use of non-treponemal test such as RPR or VDRL and treponemal test TPHA/TPPA for screening purposes. These guidelines are still in place in many countries but with the development of sensitive and specific treponemal antigen based EIA, they have been extended in some countries, including the United Kingdom (West et al., 2002).
The current practice using RPR/VDRL and TPPA/TPHA has three major limitations potentially leading to higher rates of incorrect diagnosis and unnecessary costs (Chuck et al., 2008):
EIA which is a treponemal antigen targeted assay, has the following benefits (Chuck et al., 2008):
There are several studies evaluate the performance of different assays in detecting syphilis in terms of sensitivity, specificity and positive and negative predictive value. Ohnimaa et al. (2007) reported that sensitivity for RPR was 79.9%-100%, TPPA was 85%-100% and EIA was 96.7%-100% and specificity was 85%-100%, 98%-100% and 98.3%-99.9% respectively (Alberta, 2007). Comparative evaluation of 15 serological assays for syphilis detection demonstrated that the sensitivity of Newmarket Laboratories Syphilis EIA II and Serodia TPPA was 95.2%-100% and the specificity was 98.5%-100% for both (Cole et al., 2007). These authors concluded that Newmarket Laboratories Syphilis EIA II and Fujirebio Serodia TPPA were the most suitable for detecting positive specimens from all disease stages of syphilis. Another report demonstrated that sensitivity of RPR, TPPA and EIA was 86%-100%, 85%-100% and 82%-100% respectively and specificity was 93%-98%, 98%-100% and 97%-100% respectively (Herring et al., 2006)
RPR test is not very specific, with a high frequency of false positive results (Aktas et al., 2007). Up to 50% of positive RPR results may be false positives especially in low- risk populations (Castro et al., 2003). False negative results have also been reported with high titer antibody due to prozone phenomenon (Aktas et al., 2007). One study demonstrated that sensitivity and specificity for the RPR test was 96.4% and 97.5% respectively, and positive and negative predictive values for the RPR test of 72% and 99.8%, respectively (Silletti, 1995).
EIA is an appropriate alternative to the use of combination RPR/VDRL and TPHA/TPPA test for screening for syphilis. EIA has practical advantages as a screening test and it is recommended that laboratories with large workloads use this approach (Egglestone and A.J.L., 2000). As a screening test, EIA is replacing VDRL/TPHA in UK. EIA use in syphilis serology is supported by 100% sensitivity and at least 99.8% specificity (Zrein et al., 1995). It is suitable assay for use in blood bank screening that would replace non-treponemal VDRL/RPR and treponemal TPHA/TPPA assays. Study on evaluation of EIA techniques demonstrated that reactive rates by the RPR test were 65.5% and EIA were 73%. This study also demonstrated that the sensitivity of EIA was 100% and specificity was 93%-100% (Castro et al., 2003).
Non-treponemal tests as a screening test does not detect most adequately treated cases, lack of sensitivity in late stage infection and can yield false negative results. Because of these limitations, many laboratories have been using combination of a non-treponemal and treponemal test for screening. However, with the increased sensitivity in primary syphilis of the TPPA and EIA, these assays now meet the criteria for an appropriate screening test by themselves. Today the use of two different screening tests is probably unnecessary and greatly increased the cost. As the consequence, the number of laboratories screening with single treponemal test is increasing (Nesteroff, 2004)
OBJECTIVES
General Objective
Specific Objectives
Hypothesis
1. There are differences in the performances of RPR, TPPA and EIA assays in detecting syphilis infection in blood donors.
Conceptual Framework
METHODOLOGY
Study design:
Cross sectional.
Study Location
This study will be conducted at National Blood Centre (PDN), Kuala Lumpur. PDN is located at Jalan Tun Razak, Kuala lumpur. PDN is responsible in planning and establish the blood transfusion services and all activities of blood donation in whole country. As a National Blood Centre, PDN is a reference centre for transfusion services in whole country. PDN collected, received, screened and processed a large number of blood donation i.e. more than 100,000 blood donation a year. Beside this, PDN also provide training to medical staff and laboratory technologies to their staff as well as staff from all hospital in Malaysia.
Study sample
Target population:
Blood sample from blood donors at PDN and PDN mobile sessions.
Source population
Blood sample from blood donors at PDN and PDN mobile sessions that will be subjected for syphilis screening.
Sampling frame
Blood sample from blood donors at PDN and PDN mobile sessions from June 2010 to December 2010.
Inclusion criteria
1. Blood sample from blood donors at PDN and PDN mobile session for syphilis screening from June 2010 to December 2010.
Exclusion Criteria
1. Blood sample from reactive donor counselling clinic (clinic for counselling and management of donor who have positive screening test for transfusion transmitted diseases).
Sample Size
Sample size was estimated using Power and Sample Size Calculation version 3.0.10 available at http://www.mc.vanderbilt.edu/prevmed/psintro.htm. A total of 1631 samples needed for the power of study of 80% with the significant level of 5%. Additional 326 samples are needed to achieve the power of 100%. Therefore, total samples needed are 1957.
From sample size calculation, a minimum of 1957 samples are required. In this study, a total of 2000 blood samples will be taken.
Serological tests
Three serological assays will be evaluated in this study. There are:
All the samples will be subjected to all three serological assays. The tests will be performed according to the manufacturer's directions. RPR and TPPA will be done manually. EIA will be done fully automated using Evolis machine. EIA is regarding to gold standard test for this study. The discrepant result will be determined with EIA results, if EIA is positive, that sample is determine as true positive. Characteristics of all assays are described in Table 1.
Table 1: Characteristics of assays evaluated in the study
Immutrep Carbon Antigen assay procedure
Serodia-TP.PA assay procedure
Syphilis Total Antibody (TA) EIA IIassay procedure
This assay will be carried out fully automated using Evolis machine.
Definition
Sensitivity
The proportion of true positive samples that are correctly identified by the assay as positive.
Specificity
The proportion of true negative samples that are correctly identified by the assay as negative.
Positive predictive value
The proportion of true positive samples when the assay is positive.
Negative predictive value
The proportion of true negative samples when the assay is negative.
Agreement
Degree of agreement between two assays.
Data processing and analysis
Table 2 : Statistical formula for analysis between RPR and EIA
Table 3 : Statistical formula for analysis between between TPPA and EIA
a = true positive b = false positive
c = false negative d = true negative
Calculation formula for sensitivity, specificity, positive predictive value and negative predictive value
Sensitivity = a
a+c
Specificity = d
b+d
Positive predictive value = a
a+b
Negative predictive value = d
c+d
Observed agreement= a+d
Total
Statistical analysis
Statistical analysis will be performed using Statistical Package and Social Sciences (SPSS) version 15.0. Univariate and multivariate analysis will be used.
HASIL KAJIAN
Table 4: Comparison of reactivity of RPR, TPPA and EIA assay.
Table 5 : Comparison of the RPR, TPPA and EIA test performance and characteristic
TPPA and EIA are more sensitive and specific than RPR. EIA is the most sensitive and specific in detecting syphilis infection and suitable for screening test in blood bank setting with large workload.
DISCUSSION
CONCLUSION AND RECOMMENDATION
