Quantitative RPR Test as a Guide for the Diagnosis and Treatment of Syphilis in Zambia.[Original Research]
Yassa P,MD PhD*, Bwalya CB,BBS**, Hira RS, MBBS***, Kwenda, PhD*, Kunda M, MD*, Sarenje K,BSc*, Kaswa J,MD****, Skwe H, BSc**, Sheba Ambali C, DipSMM**, Mwika Kabeya M, MD, MPH*, Tembo Mumba G, MOH*, Lumamba R, DipIT*, Makwaza G,DipHR*
*University Teaching Hospital, Lusaka, Zambia.
**University of Zambia.
***Emory University-SPH, Atlanta, USA.
*** *CNPP, Kinshasa, DR Congo.
[emedpub – International Infectious Diseases: Vol 1:12] [Date of Publication: 08.18.2013]
August 18, 2013 at 3:54 AM
Correspondence: Pr Yassa Pierre, University Teaching Hospital, STI Clinic, Lusaka. Perets31@gmail.com
Syphilis is prevalent in Zambia. A study was designed to determine correlation between the rapid plasma regain (RPR) test and the Treponema Pallidum Hemaglutination Assay (TPHA).
Methods: This was a prospective, cross-sectional study. One hundred seventy seven randomly selected plasma samples that were reactive using qualitative rapid plasma regain (RPR) test were compared with Treponema Pallidum Hemaglutination Assay (TPHA). The samples were collected the Lusaka urban clinics namely, Chawama, Mutendere, Kanyama, Kalingalinga and Chilenje clinics.
Results: The results were tabulated using the SPSS program. Among clinical samples, an increasing trend of syphilis prevalence was observed with increase age groups. Of 177 RPR+ samples, 108 (61%) were confirmed TPHA+. The RPR+ samples with titres of 1:2 and 1:4 all tested negative on the TPHA and those with 1:16,1:32 and 1:64 all tested positive using TPHA. However, RPR+ samples with the titre of 1:8 showed mixed frequency of positivity with TPHA. Co-infection with HIV was established in 118/177 (66.7%) of the samples.
The quantitative RPR test can be used as a confirmatory test for diagnosis and treatment of syphilis in the absence of TPHA test in resource-limited settings.
Infection with syphilis continues to be a serious public health concern in Zambia due to its associated morbidity and mortality among newborns and children (1,2) and its facilitation in the transmission of HIV infection (3,4). In recent years, many STI clinics in Zambia have reported a high prevalence of syphilis cases. In a study conducted in Zambia in 2007, prevalence of syphilis was established at 6.5% for antenatal women and that for 7.4% for men attending outpatient clinics. Highest seropravalence for syphilis was documented in the Coppperbelt, Lusaka and the Eastern provinces of Zambia. It was also found that most clinics in Zambia did qualitative Rapid Plasma Reagin (RPR) test for syphilis and based the diagnosis and treatment on the qualitative RPR results. Since qualitative RPR test detects the non-treponemal antibodies, concerns about false-positive results in the absence of active clinical signs, led to the design of this comparative study.
This was a prospective cross-sectional study to determine correlation between the quantitative RPR and TPHA tests.
The study was conducted at the University Teaching Hospital, STI clinic, Lusaka. UTH is a tertiary referral and teaching hospital with a bed capacity of 1665 and 250 baby cots.
The target population was all syphilis suspected patients seeking health services at UTH and the Lusaka urban clinics namely, Chawama, Mutendere, Kanyama, Kalingalinga and Chilenje clinics. The five clinics were randomly selected to ensure total representation of the Lusaka district Urban Clinics.
All qualitative RPR+ samples were included in the study.
The RPR negative samples were not considered in the study.
The sample size was 170 qualitative RPR+ samples. It was calculated using the formula
n = z²p (Q-p)
Venous blood was collected from the suspected patients seeking health services at UTH-STI clinic and Lusaka Urban Clinics. The samples were stored at 20ºC prior to testing.
Protocol for RPR qualitative Screening procedure
The qualitative test determines whether the substance being tested for is present or absent, the RPR Card Test is an 8-minute macroscopic non-treponemal flocculation test which was used for the detection of non-treponema antibodies. The micro particulate carbon RPR antigen enhances the visual discrimination between reactive and nonreactive results. The reagin-type antibody binds with the antigen that is composed of a complex of cardiolipin, lecithin and cholesterol particles with activated charcoal; the result of this antigen-antibody reaction is macroscopic flocculation. This was carried out to investigate the presence of non treponemal antibodies using the fresh serum separated from blood cells by a centrifuge. The samples and reagents were allowed to reach room temperature and also to ensure that all reagents were fully resuspended before use. One drop of serum was dispensed onto a circle of white reaction card of RPR test kit. Gently the antigen dispensing bottle prior to use was shaken. Holding in a vertical position one drop was dispensed on the same circle containing serum and results observed after 8 minutes.
Protocol for RPR quantitative procedure
Quantitative testing is a measure of the amount of a substance present in the positive sample either to guide treatment or to quantify the infection. The samples which tested positive for qualitative RPR were retested using a quantitative RPR method, the tests were carried out as described below according to the manufacturer`s instructions.
- For each sample, 25ul of diluent was dispensed into each well in one column of the plate. For titration of controls dispensing commenced from row 3.
- 25ul was transferred from row 2 of the original screening plate to row 1 of the quantitative plate.
- This was then mixed and 25ul discarded.
- 25ul was again transferred from row 2 of the original screening plate to row 2 of the quantitative plate.
- Preparations of the 25ul doubling dilution from row 2 to row 8 (for controls doubling commenced from row 3).
- 75ul of the well mixed controls were added to row 1.
- 75ul of the well mixed test cells were added to rows 2 to 8.
- This was mixed by gentle tapping (the final dilutions in row 1 and row 2 became 1/80.
This was then covered and left to stand for 45 to 60 minutes at room temperature and thereafter the results interpreted (OMEGA DIAGNOSTIC LTD, Omega Scotland, UK).
Protocol for Treponema Pallidum Hemaglutination Assay (TPHA)
In order to compare the two tests all the qualitative and quantitative RPR+ samples were retested with TPHA to confirm their positivity and easy calculation of the P value. The protocol was done according to the manufacturer`s instructions. (OMEGA DIAGNOSTIC LTD, Omega Scotland, UK).
Procedure for testing for HIV
The qualitative RPR+ samples were tested for HIV as per the procedure. The qualitative immunoassay Determine HIV-1/2 was used to test for HIV and using Uni-Gold™ Recombigen ®HIV as a confirmatory test. Sample were applied on a test-card and results were interpreted after 15 minutes.
There was no direct contact of investigators with the patient since RPR positive samples were tested in the laboratory using multiple tests. No names were attached to the samples; instead the samples were given numbers for better analysis of the results and confidentiality. Furthermore, waiver permission was granted by relevant ethics authorities.
A total of 177 qualitative RPR reactive samples were obtained from the urban clinics and UTH-STI clinic and were reported as qualitatively reactive regardless of the degree of reactivity of the results. The reactive samples were retested qualitatively to confirm their reactivity and quantitative RPR test results done were reported in titres while the TPHA test results were reported as positive or negative. A qualitative reactive result was indicated by the presence of aggregates throughout the test circle, ranging from slight to marked and intense.
Data was analysed using SPSS software. There was an increasing trend in syphilis prevalence with increasing age (table 1). The following tables and figure were the results obtained.
Table 1: Frequency and Percentages of age groups of patients (n=177)
|Variable||Measure||15 – 25||26 – 30||31 – 35||Above 36|
Figure 1: Frequency of gender distribution
Table 2: Quantitative RPR
Table 3: Correlation between Quantitative RPR &TPHA tests
|Quantitative RPR Test (Titers)||Total|
Table 4: HIV test results (n=177).
Table 5: RPR titres 1:8 versus HIV test (n=48).
In this study it was found that the least affected age group presenting with qualitative RPR reactivity was between 15 to 25 years. This could have been attributed to the fact that it was the age group of youth, most of who were not married and possibly selective with their sexual activities. While the most affected age groups with the highest percentages were found to be 31-35 years and above 36 years, the reason why these age groups are affected could be that most of the people are financially stable and they can afford to engage in multi-sexual practices. Also, this age group had a high prevalence of HIV; hence, having opportunistic infections leading to the production of non-treponemal antibodies that could cause qualitative RPR positivity (5,6). The study also showed that more men had qualitative RPR reactivity than that seen in women. This could be attributed to the fact that men were more sexually active and the symptoms of syphilis show up early in men, generally within two weeks.
The study showed that 39% of the qualitative RPR reactive samples were negative on the TPHA test which is specific for treponema pallidum, indicating that these were not syphilis infections but likely to be false positives. False positives occur because qualitative RPR test detects non-treponemal antibodies which are not only produced by syphilis infection but also produced by other infections such as, intercurrent viral and bacterial infections (Infectious mononucleosis, Epstein-Barr viral infections, viral hepatitis, herpes simplex infections, chancroid, and lymphogranuloma venereum, tuberculosis, malaria, measles)(7). Also, False positive results may occur as long term (chronic) biological false positivity in Hansen’s disease and collagen diseases, such as systemic lupus erythematosus and rheumatoid arthritis, as well as in narcotics addiction (especially methamphetamines) and in some forms of neoplasm which means that people with these condition continue to have qualitative RPR reactivity for life (7) and if the test is not confirmed they can continue to be treated repeatedly for syphilis for lifetime. Therefore, if 39% received treatment for syphilis then they were misdiagnosed and were exposed to potential side effects of syphilis drugs besides missing the actual infections they were suffering from.
The area of interest in this study was on the comparison of quantitative RPR versus TPHA as the confirmatory test and to investigate at which titre or dilution of the quantitative RPR does the TPHA give a positive result. It was found that those samples which had titres or dilutions of 1:2 and 1:4 on quantitative RPR tested negative on the TPHA whilst those which had titres of 1:16, 1:32 and 1:64 tested positive on the TPHA. It was also found that the titres of 1:8 were more complex for interpretation because of 48 samples with dilution of 1:8, 12 tested positive on TPHA while 36 tested negative on TPHA (table 5). Therefore, the study showed that titres of 1:8 in HIV negative individuals or >= 1:16 in HIV positive/negative individuals was actually confirmed by a positive result using TPHA while titres of <=1:8 in HIV positive individuals gave a negative result using TPHA. This means that the titres of <=1:8 in HIV positive individuals does not mean syphilis infection because many conditions can lead to production of non-treponemal antibodies which could show a reactive result on quantitative RPR test. To support the statement above the p-value of the test was calculated using SPSS which was found to be =0.000 indicating a strong significance.
A study done in 2005 (8) showed that the phospholipid antibodies detected by non-treponemal tests are not only produced in syphilis and other treponemal disease but also in response to a variety of conditions unrelated to syphilis. The incidence of false-positivity is generally 1% to 4% (8). The rate of false-positives during pregnancy is no greater than that seen in the general population, but is higher among intravenous drug users. Generally, up to 90% of false-positive reactions have a titer of <1:8 and can persist for >6 months. False-positive reactions can also occur with treponemal tests but this is less common than with non-treponemal tests. Both types of tests can also yield false-negative results due to the prozone phenomenon. Such false-negatives occur in 1% to 2% of patients, especially in pregnant women and HIV-infected patients. Serum from such patients should be tested at a 1:16 dilution and treatment given accordingly (8). The quantitative RPR test if fully utilized can be the best diagnostic test for syphilis because it can be used for both guiding treatment and monitoring infection progression after treatment. Also the 2008 European Guidelines on the Management of Syphilis showed that quantitative RPR can be used as a diagnostic test better than TPHA because it can be used to guide treatment.
Described in another study were the point-of-care of immunochromatographic test for the simultaneous detection of both non-treponemal and treponemal antibodies in the sera of patients with syphilis that acts as both a screening and a confirmatory test. These results indicate that the dual test could be used for the serological diagnosis of syphilis in primary health care clinics or resource-poor settings and therefore improve rates of treatment where patients may fail to return for their laboratory results. When sera have quantitative RPR titers of ≥1:8, the dual Point of Care test has a sensitivity of 99.7% (9).
The prozone phenomenon in syphilis testing refers to a false negative response resulting from overwhelming antibody titers which interfere with the proper formation of the antigen-antibody lattice network necessary to visualize a positive flocculation test. This prozone effect in syphilis testing can be expected in cases of disproportionately high antibody titers, such as secondary syphilis, or with human immunodeficiency virus (HIV) coinfection.
Treponemal antigen tests, such as the TPHA are inappropriate for use as screening tests. Treponemal tests may remain reactive for years with or without treatment. Therefore, treponemal tests should not be used to evaluate response to therapy, relapse or re-infection in previously treated patients. Also, treponemal tests do not differentiate venereal syphilis from endemic syphilis (yaws and pinta). Treponemal tests are used mainly as confirmatory tests to verify reactivity in non-treponemal tests. However, in populations of low disease prevalence, treponemal tests can be used for screening, utilizing a rapid test or TPHA. Then, all positive patients would either be treated presumptively because the serious consequences of untreated infection far outweigh the effect of overtreatment, or have a follow-up quantitative RPR to determine if they have active infection before treatment. Treponemal tests are also used as diagnostic tests in patients with non-reactive non-treponemal tests but with signs and symptoms of late syphilis. Treponemal tests are technically more difficult to perform and more expensive than non-treponemal tests. As with non-treponemal tests, false-positive reactions can occur with treponemal tests, nevertheless in the absence of immunosuppression, a non-reactive treponemal test is indicative of no past or present infection (8).
In this study the prevalence of 66.7% HIV sero-positivity was significantly high as expected. HIV test was done so as to investigate the likelihood of having co-infection samples from individuals who might have had some opportunistic infections due to HIV leading to the production of non–treponemal antibodies. The high prevalence of HIV in qualitative RPR positives also indicated that the positivity of the qualitative RPR could have been caused by the production of non-treponemal antibodies in immune compromised individuals with HIV infection. The immune system plays an important role in protecting against syphilis. Impairment of both cell-mediated and humoral immunity by HIV may limit the host’s defenses against many micro-organisms, thereby altering the clinical manifestations or natural course of the infections leading to false positivity of the qualitative serological tests for syphilis. The likelihood of syphilis infected patients contracting HIV is very high, the molecular pathogenic mechanisms explains the role of the spirochete in facilitating HIV transmission which may include up-regulation of gene expression, such as that of the CCR5 co-receptor used in HIV entry. HIV-induced meningeal inflammation may also facilitate the penetration of spirochetes into the central nervous system (CNS) and thus contribute to the development of symptomatic neurosyphilis (10). The complication of having both syphilis and HIV co-infections cannot be overemphasized, like many acute infections in the HIV-infected patients, syphilis may further decrease CD4+ T-cell counts (CD4 cell counts) and increase HIV RNA in plasma and semen which in turn, may contribute to risk of re-infection and increase occurrence of opportunistic infections; hence leading to the production of non-treponemal antibodies (11).
The study recommends that all the hospitals and Clinics in resource-limited settings should reconsider adopting the use of quantitative RPR test after reactive qualitative RPR test so as to quantify the disease to avoid unnecessary treatment of patients for syphilis and to rule out the false positive results in suspected patients. This will enable reduce the cost of managing the syphilis infections. Another study should be taken up to find the correlation of CD4+ count in HIV sero-positive patients with the titers in quantitative RPR test for the proper diagnosis of syphilis in HIV-infected patients. The Clinicians and the laboratory personnel must be educated on the importance of the use of quantitative RPR test as a guide in the diagnosis and treatment of syphilis. Safer-sex messages should include reducing the number of sexual partners; knowing the health status and HIV infection status of partners; avoiding unsafe sexual practices, not just for HIV, but for all STIs and using barrier protection methods such as condoms.
This study has some limitations such as, though non-reactive patterns are slightly granular or “rough” prozone effects maybe experienced and sometimes the end point might be greater than 1:256 which might be difficult to interpret. Also, it cannot differentiate the treponemal infections such as Pinta, yaws, bejel and other treponemal diseases because they all produce positive reactions. Contaminated and hemolyzed serum should not be used because of the possibility of nonspecific reactions in the first screening test. Reaction times longer than specified may cause false positive results due to a drying effect. Temperature of the reagents and samples should be kept between 20 and 30C.
Qualitative RPR cannot be used to guide treatment because not all qualitative RPR reactive or positive results mean syphilitic infection. The two tests, namely, TPHA and quantitative RPR test are equivalent at the dilution of >=1:16 in any individuals regardless of the HIV status. In the presence or absence of the TPHA, quantitative RPR test can be used to obtain the same reliable results that could have been obtained if the TPHA was used at the above mentioned titers. Thus, quantitative RPR can be used not only for monitoring the progress of treatment in syphilis patients but it can also be used to guide the diagnosis of syphilis as a confirmatory test just like its TPHA counterpart.
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