Evaluation of the Rapid Dual HIV and Syphilis Tests in Women Attending Antenatal Clinic at the University Teaching Hospital, Lusaka, Zambia. [Original Research]
Pierre Yassa, MD, PhD1, Rajiv Hira, MBBS, MPH2,3, Charity Sibinda, BSc1, PT Kantenga, MSc4, Ryan Kim, MSc5, Sadik Seeda, BSc5, Valery Mulenga, BSc5, Abdon Mukalay, MD, MPH, PhD6, Davies Glynis,MPH5, Sam Chisela,MD, MMED4,Tamba Tamba, MD7, Grace Tembo, MSc7
1.University of Zambia, Department of Dermatology & Venereology
2. MGM University of Health Sciences, Navi Mumbai, India.
3. Emory University-SPH, Atlanta, USA.
4. University Teaching Hospital Lusaka Zambia
5. Alere, United States of America
6. University of Lubumbashi, School of Medicine, DR of Congo
7. Ministry of Community Development, Mother and Child Health, Zambia.

[emedpub – International Infectious Diseases Vol 1:13] [Date of Publication: 01.03.2015]
ISSN 2231-6019

January 3, 2015 at 12:52 AM

ABSTRACT:

Objective

The purpose of this study was to evaluate the sensitivity and specificity of SD Bioline Dual Rapid HIV and Syphilis Tests using Uni-gold HIV and Treponema pallidum hemaglutination assay as reference tests in women attending antenatal clinic at the University Teaching Hospital, Lusaka, Zambia.

Methods

To evaluate the sensitivity and specificity of SD Bioline Dual Rapid HIV and Syphilis tests, a  prospective cross-sectional study was conducted at the University Teaching Hospital in Lusaka using previously collected, stored blood samples of antenatal women submitted to cytology laboratory.

Results

The study found that compared with the reference tests, the sensitivity and specificity of the SD BIOLINE HIV and Syphilis Dual test were 100%.

Conclusion

With high laboratory performance of the SD BIOLINE HIV and Syphilis Dual test established in Lusaka, the test is recommended for regular screening in antenatal clinics.

INTRODUCTION:

HIV and syphilis are major public health concerns affecting women and their infants in the world. Over a million women and families are facing the trauma of repeated pregnancy loss, stillbirths or newborn infected with or suffering from HIV and syphilis (1). Globally, 2.5 million children under the age of fifteen are living with HIV and 90% of these are in Africa. New infections of HIV are increasing in the younger age group and about 500,000 infected children under fifteen years die each year due to this pandemic. Approximately, 90% of the infections in children are contracted through vertical transmission from an infected pregnant mother to the unborn baby (2). With appropriate testing and treatment strategies, the risk of mother to child transmission can be reduced to 1% (2). In 2008, approximately 1.4 million pregnant women were estimated to have had active syphilis worldwide and 80% of these had attended antenatal clinics. Globally, 520 adverse outcomes were discovered to be caused by maternal syphilis such as stillbirths and early deaths. Most of the adverse outcomes occurred in women who had attended antenatal but did not get treated for syphilis and hence this highlights the need to accelerate quality of antenatal care aswell as the antenatal coverage (3).

In Zambia, syphilis continues to be a serious public health concern as it leads to a high morbidity and mortality among new-borns and children (4,5,6). According to Kapina et al (7), antenatal prevalence rate was estimated at 17.4% and 7.1%, for HIV and syphilis, respectively in 2007. Further, a very high probability of syphilis positive patients to acquire HIV was established for the first time in medical literature in 1990 (8). This was because the spirochete causes up-regulation of gene expression, such as the CCR5 co-receptor used in HIV entry. HIV-induced meningeal inflammation can also facilitate the penetration of the spirochete in the central nervous system and hence the development of symptomatic neurosyphilis (9) Co-infection of syphilis in HIV positive patients can lead to an accelerated reduction in CD4+ T-cells and this leads to an increase in HIV viral load in semen and plasma leading to the risk of concurrent opportunistic infections and re-infections (10). Syphilis can also be vertically transmitted from an infected pregnant woman to the unborn baby through the placenta (11).

There are many ways of preventing vertical transmission of HIV and syphilis from mother to child. This includes; treating the mother for syphilis by the use of penicillin which is the drug of choice (12) and for HIV, the mother can be put on HAART by following the Options A, Option B and Option B+ (13,14). However, for development of an effective antenatal program, screening for both HIV and syphilis using a single laboratory test is considered more desirable. A number of dual tests from different countries as cited by Claire (15) were evaluated and the SD BIOLINE HIV and syphilis dual rapid test kit from Korea has been reported to have a higher sensitivity and specificity compared to other dual tests. The SD BIOLINE HIV and Syphilis dual rapid test kit is a solid phase immunochromatograghic assay for the qualitative detection of antibodies (Ab) to all isotopes specific to HIV-1/2 and Treponema pallidum simultaneously in human serum, plasma and whole blood (16).

In Zambia, presently there are two non treponemal tests in use: qualitative, the Rapid Plasma Reagin (RPR) and the quantitative Rapid Plasma Reagin tests. The qualitative RPR is the standard screening test adopted in Zambia for screening syphilis and is prone to false positives in certain conditions such as malaria, chronic auto-immune skin conditions, etc.  The errors in the screening tests were also observed by other authors (16). Failing to detect syphilis using qualitative RPR at the rate of 36% was considered too high as observed by the Yassa et al  in 2013 (17) in a study done in Zambia. Misdiagnosis of syphilis and HIV by the use of weak screening tests can lead to failure of the control program and an increase in morbidity and mortality.

To date, there are few data on the dual rapid diagnostic test performance in the field although they have been evaluated in laboratory based studies and have shown encouraging performance in terms of specificity and sensitivity compared to reference standard assays (15,16). Hence, this study was undertaken with the purpose to validate the sensitivity and specificity of SD BIOLINE HIV and syphilis dual rapid test among women attending antenatal clinic at the University Teaching Hospital in Lusaka.

MATERIAL AND METHODS:

Study Design: This was a prospective cross-sectional study in which the sensitivity and specificity of the rapid, dual SD BIOLINE rapid HIV/Syphilis Korean test kit was evaluated using previously collected blood samples from antenatal women submitted to the cytology laboratory.

Study Site: The study was conducted at University Teaching Hospital, Lusaka at the STI laboratory.

Study Population: The tested population were women who attended antenatal care from December 2013 to March 2014.

Inclusion Criteria: Samples from pregnant women were included in the study.

Exclusion Criteria: Samples from non pregnant women were excluded from the study.

Sample Size: The recent prevalence of HIV and Syphilis in women attending antenatal at UTH was not known. In order to estimate the prevalence within 5%(0.05) and considering 95% confidence level, a minimum sample size of 200 will be needed in order to obtain a statistical significance of<0.05 as calculated below, assuming that HIVandSyphilis infections in this study area were 50%.

Where n=sample size

p=proportion of population with HIV and/ or Syphilis infections in pregnancy (50%~0.5)

q=1-p

SE=     level (95%).

n=200

Laboratory Procedures

Sample Collection: Plasma from previously collected blood samples obtained from women attending antenatal care at UTH and stored at the cytology laboratory were used.

Reference test kits

  1. 1. Treponema  Pallidum Hemaglutination (TPHA) Protocol

All the samples obtained from women attending antenatal care were tested for syphilis using TPHA according to the manufacturer’s instructions of the kit.

  1. HIV UNIGOLD Protocol

All the samples obtained from women attending antenatal care were tested for HIV using UNI-GOLD according to the manufacturer’s instructions.

Evaluation test kit: SD Bioline dual HIV/Syphilis RDT Protocol

The kit used in this study was developed by Standard Diagnostics, Inc. Gyeonggi-do, South Korea.The SD Biolinedual HIV/Syphilis rapid test is a solid phase immune-chromatograghic assay for the qualitative detection of antibodies to all isotypes (IgG, IgM, IgA) specific to HIV-1/2 and Treponemapallidum simultaneously in human serum, plasma or whole blood.

The SD Biolinedual HIV/Syphilis rapid test contains a membrane strip which is pre coated with recombinant HIV-1 capture antigen (gp41), recombinant HIV-2 capture antigen (gp36) and recombinant HIV sub O antigen on test band 1 region and recombinant Treponema pallidumantigens (17kDa) on test band 2 region, respectively.

The recombinant HIV-1/2 antigen colloid gold conjugate , recombinant  Treponema pallidum antigen colloid gold conjugate, the specimen sample and sample diluents move along the membrane chromatograghically to the test region and form a visible line as the antigen-antibody-antigen gold particle complex with high degree of sensitivity and specificity

This test device has a letter of HIV, Syphilis and c as test line HIV (HIV1/2), TEST LINE Syp (Syphilis) and control line on the surface of the device. Both the test line and control line in result window are not visible before applying the specimen.

The control line is used for procedural control and should always appear if the test procedure is performed properly and also if the test reagents of control line are working(quality check).

Test Procedures: The test device was removed from the pouch, placed on a flat surface and 20ul of sample was added to the sample well, followed by 3 drops of assay diluents. A purple colour line appeared across the result window and results were read within 15-20minutes. A negative test result was indicated by a single purple line (c or control) within the results window and the presence of a second purple line signifies the test was positive for either HIV (HIV1/2) or Syphilis. The presence of three purple lines indicated that the test was positive for both HIV and Syphilis.

And the absence of the control line shows that the test was unequivocal (quality check failure) and required repeating.

Data Collection Tool: In this study all the information was collected using data collection forms that included the ID of the patient, age and results for HIV/ Syphilis dual, Uni-gold HIV, and TPHA tests. The variables that were considered in the study included both dependent and independent variables.

Data Analysis: Data that were generated in this study were coded, entered, validated, and analysed using statistical package for social science (SPSS).Cross tabulation between index and standard test for both SD Bioline HIV/Syphilis, TPHA, and HIV Uni-gold were performed.  For the sensitivity and specificity two by two tables were used.

The sensitivity and specificity was analysed using the two by two tables which assessed the accuracy of the dual rapid HIV and syphilis test in comparison to the reference standard assays (TPHA and Uni-Gold).

Ethical Considerations: Waiver was obtained from the Research & Ethics Committee of UTH. The blood samples were obtained from cytology laboratory upon authorization by the section head and the Chief Biomedical Scientist and there was no direct contact with the patients. The information obtained from the study was strictly used for research purposes and high confidentiality was maintained throughout the study.There was no name, physical address or contact details of the patient obtained so as to maintain privacy.The samples were coded using sample IDs and not patient name.

RESULTS:

Using SD Bioline dual tests, 71/200 (35.5%) tested positive for HIV-1/2 and were also confirmed positive using the Uni-gold HIV reference test. The study results also show that 19/200 (9.5%) blood samples tested positive for syphilis using SD Biolinedual tests. The same 19 samples also tested positive using TPHA test(Tables 1,2).

Table 1. SD BiolineDual HIV test results (n=200)

Uni – gold HIV test Total
Negative Positive
SD Bioline HIV test Negative 129 0 129
Positive 0 71 (35.5%) 71
Total 129 71 200

The SD BIOLINE dual HIV sensitivity and specificity were calculated as follows;

Specificity=× 100%=100%

Sensitivity =×100%=100%

Discriminant ability=100+100/2=100%

The SD Bioline in this study have detected 181 true negative from the 200 blood samples tested and all the  19  blood samples that tested positive by SD Bioline syphilis were confirmed by TPHA ( Table2)

Table 2. SD Bioline syphilis dual sensitivity and specificity were calculated using values from the crosstabulation (n=200)

TPHA Total
Negative Positive
SD Bioline Syphilis Negative 181 0 181
Positive 0 19 (9.5%) 19
Total 181 19 200

Sensitivity=× 100%=100%

Specificity =×100%=100%

Discriminant ability=100+100/2=100% ( Figure 2)

Positive predictive value=19/(19+0)=100%

Negative predictive value=181/(0+181)=100%

DISCUSSION:

This study showed that the sensitivity and specificity of the dual HIV/syphilis test using serum was acceptably high at the University Teaching Hospital in Lusaka. With sensitivity and specificity of 100%, it meant that the SD Bioline test was able to detect both the true positives and the true negatives exactly as the reference test.

These results were similar to those found by Claire et al in 2014 (15) in their literature review of Dual HIV / Syphilis tests conducted in six counties. In that review, evidence showed that Ghana, Togo and Mexico obtained similar results with different reference tests other than Uni-gold test that was used in the Zambian study.

In 2013, a study was carried out in Kenya and HIV specificity was 100% and sensitivity was 99.7% using Uni-gold as standard and this was similar to the findings of this study, probably because of similar methods used (15).

Similar evaluation studies in Kenya and Laos using TPHA as a reference tests were carried out  in 2014(15), and the specificity and sensitivity of both studies  were at 100% for dual tests.  The reference test used in Zambia was similar to the one used in Kenya, that was the TPHA (Randox Lab UK). However, in Laos the TPHA German was used with the same outcome of 100% sensitivity and specificity (15).

In view of the limited community access to laboratory, these point-of-care tests will prove to be very important for clinical settings.  The implementation of this affordable and user friendly test i.e. rapid, dual SD Bioline HIV/syphilis test at the national level in Zambia will improve screening, diagnosis, and time to treatment (15). This duo test integrated in antenatal HIV and syphilis screening may reduce the adverse outcomes of syphilis in pregnant women and help to decrease the vertical transmission of HIV from mother to child (18- 22).

Limitation of the study: The use of algorithm for characterizing serum specimens for HIV and T. pallidum testing in Zambia was different from that used in other studies as reported by Claire  (15).

CONCLUSION:

With high laboratory performance of the SD Bioline HIV and Syphilis Dual rapid test established in Lusaka, the test is recommended for regular screening in antenatal clinics.


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