Performance of Kalon herpes simplex virus 2 assay using dried blood spots among young women in Uganda

Abstract

The study evaluated the performance of the Kalon HSV-2 assay on dried blood spots (DBS) of various dilutions compared with plasma from young women aged 18–24 years in Uganda. We estimated the sensitivity and specificity of three DBS dilutions using plasma as the reference. All three evaluated DBS dilutions yielded low sensitivities and specificities with DBS 1:2 yielding the highest concurrence. Other HSV-2 assays should be examined with regard to their utility for testing DBS.

Introduction

Herpes simplex virus type 2 (HSV-2) causes lifelong infections in exposed individuals and is a useful cumulative marker for unprotected sex. HSV-2 infection has also been shown to be an important co-infection for HIV, facilitating HIV acquisition and transmission, and accelerating disease progression.⁽¹⁾ In Uganda, it is estimated that about half of 15–59 year old adults are infected with HSV-2.⁽²⁾

Numerous studies have outlined the usefulness of dried blood spots (DBS) for the serologic diagnosis of infectious diseases, as well as for large-scale seroprevalence studies.^(3–7) Since DBS do not require immediate refrigeration, occupy little space, and are easily transported, they are an attractive means for biomarker-based studies, in particular in geographic settings with limited laboratory resources.

Recently developed type-specific HSV antibody tests are based on the detection of antibodies to glycoprotein G1 (gG1), a marker for HSV-1 infection, and glycoprotein G2 (gG2), a marker for HSV-2 infection.^{(8, 9)} Validation of DBS for immunoglobulin G (IgG)-based tests has been conducted using HSV-1-positive antibody samples from 22 healthy volunteers.⁽¹⁰⁾ Hogrefe et al. reported that testing data using a single dilution of DBS eluates (1:4) with the HSV type-specific ELISA method were similar to those of sera diluted 1:101 using the standard HerpeSelect ELISA.¹⁰ The efficiency of using IgG eluated from DBS samples was found to be consistent with measurements of IgG concentrations in most corresponding serum samples.

However, results have been inconsistent when using the Kalon ELISA HSV-2 assay measuring antibodies to gG2 with the same testing protocol.⁽¹⁰⁾ When a 1:4 dilution was applied to our samples from young women aged 18–24 years in Kampala (n=277), the estimated HSV-2 prevalence of 2% was significantly lower than that previously reported in the 2004–2005 Uganda HIV/AIDS Sero-Behavioral Survey (UHSBS), which found a prevalence of 21% among women aged 15–19 years and 38% among women aged 20–24 years.⁽²⁾ When an additional 10 DBS specimens from our sample were further analyzed using a dilution of 1:2, the prevalence of HSV-2 increased three-fold.⁽¹¹⁾

In the study reported here, we examined the performance of the Kalon ELISA HSV-2 assay using DBS and plasma samples from the same stored specimen collected during the 2004–2005 UHSBS. The study goal was to examine whether HSV-2 testing results based on DBS at various dilution levels were concordant with plasma-based results obtained from the same participants.

Methods

Ethical considerations

Participants who provided specimens in the 2004–2005 UHSBS consented to the long-term storage and future testing of their delinked blood specimens for which they would not receive results. We obtained additional permission for this study from the Uganda Ministry of Health. The study was also reviewed and approved by the Uganda Virus Research Institute’s Institutional Review Board (GC/127/11/10/15) and the Population Council Institutional Review Board (protocol 433).

Study population

This study was conducted using existing stored DBS and plasma specimens from the 2004–2005 UHSBS. The UHSBS was a nationally representative household-based survey that sampled 19,656 adult respondents during 2004–2005. The main objective of the UHSBS was to obtain national and sub-national estimates of HIV prevalence and selected indicators of HIV-related risk behaviors, program coverage and HIV/AIDS knowledge and attitudes. One of its specific objectives was to determine the magnitude and distribution of HIV and other STIs, such as HSV-2, in Uganda.⁽²⁾ The UHSBS estimated the national adult HIV prevalence at 6.4% and that of HSV-2 at 44%.⁽²⁾

Survey specimens and testing

Survey participants had venous blood samples drawn into 4.5 ml EDTA Vacutainer tubes, from which DBS were produced (using SS903 specimen collection paper) and air-dried overnight in plastic boxes and stored in lots of 20 separated by glassine paper in Ziploc bags containing desiccants. In the field, blood was centrifuged and the plasma was transferred to microvials. Plasma and DBS were transported periodically to a central laboratory in Entebbe for processing and storage at −80°C (plasma) and −20°C (DBS). Plasma and DBS were tested for HSV-2 antibodies using the Kalon HSV type 2 specific IgG assay (Kalon Biological Ltd, Surrey, United Kingdom) within several weeks of collection. Results were classified as positive or negative using cutoffs as specified by the manufacturer.

DBS validation study design

For the purpose of this study, we randomly selected 110 DBS specimens from women aged 18–24 years whose plasma-based equivalents tested positive and 110 DBS specimens whose plasma-based equivalents tested negative using the Kalon HSV type 2 assay.

From each of these 220 DBS, a 6-mm-diameter (28-mm²) disk, containing approximately 50–75μl of blood per spot, was punched out from the filter paper and soaked overnight at 4 °C in 150μl of phosphate-buffered saline (PH 7.4). After the overnight elution step, the eluates were diluted at three different levels, 1:4, 1:3, and 1:2. Each specimen was tested in triplicate with the Kalon HSV-2 ELISA assay as directed by the manufacturer’s instructions. Laboratory testing and analysis was conducted at the Molecular Research Laboratory (MOLAB) in Kampala as part of the Makerere University-University of California, San Francisco (MU-UCSF) Research Collaboration on 2 May 2011.

Data analysis

Frequencies of reactivity/non-reactivity, sensitivity and specificity with 95% confidence intervals were generated using SAS® software, version 9.3 (SAS Institute Inc., Cary, North Carolina, United States). DBS-based estimates of sensitivity and specificity were obtained using the plasma-based results as the reference. We determined the dilution level for DBS-based HSV-2 testing that yielded the highest (relative) sensitivity and specificity.

Results

The final sample size included 210 individuals due to missing test results for 10 DBS. Of the 210 plasma specimens, 104 (49.5%) were reactive and 106 (50.5%) were non-reactive for HSV-2 antibodies (Table 1). DBS 1:2 dilution yielded 116 (55.2%) reactive and 94 (44.8%) non-reactive results. DBS 1:3 produced 124 (59.0%) reactive and 86 (41.0%) non-reactive results, whereas DBS 1:4 yielded 110 (52.4%) reactive and 100 (47.6%) non-reactive results.

Table 1.

HSV-2 seropositivity for plasma-based and DBS dilution-based assays using the Kalon ELISA HSV-2 test among young women in Uganda, 2004–2005.^†

	DBS 1:2
	Non-reactive‡	Reactive§	Total
Plasma
Negative¶	78	28	106 (50.5%)
Positive††	16	88	104 (49.5%)
Total	94 (44.8%)	116 (55.2%)	210 (100%)
	DBS 1:3
	Non-reactive‡	Reactive§	Total
Plasma
Negative¶	68	38	106 (50.5%)
Positive††	18	86	104 (49.5%)
Total	86 (41.0%)	124 (59.0%)	210 (100%)
	DBS 1:4
	Non-reactive‡	Reactive§	Total
Plasma
Negative¶	72	34	106 (50.5%)
Positive††	28	76	104 (49.5%)
Total	100 (47.6%)	110 (52.4%)	210 (100%)

Abbreviations: DBS, dried blood spots; HSV-2, herpes simplex virus 2

^†Women were aged 18–24 years, N=210.

^‡Non-reactive, DBS negative for HSV-2 antibodies

^§Reactive, DBS positive for HSV-2 antibodies

^¶Negative, plasma negative for HSV-2 antibodies

^††Positive, plasma positive for HSV-2 antibodies

The 1:2 ratio of buffer and eluate yielded the highest sensitivity (84.6%) and specificity (73.6%) (Table 2). The 1:3 dilution had the next highest sensitivity (82.7%), but had the lowest specificity (64.2%). The 1:4 dilution showed the lowest sensitivity (73.1%) with a specificity of 67.9%. Overall, 127 (60.5%) of the 210 specimens had concordant results between plasma and all three DBS dilutions (not shown in tables). Of the 127 concordant results, 69 (54.3%) were concordant positive and 58 (45.7%) were concordant negative. A total of 83 (39.5%) cases had a discordant result between plasma and at least one of the DBS dilutions. Of the 83 discordant results, 35 (42.2%) were discordant positive and 48 (57.8%) were discordant negative.

Table 2.

Relative sensitivity, specificity, and 95% CIs of DBS dilutions tested with the Kalon ELISA HSV-2 test compared with plasma among young women in Uganda, 2004–2005^†

Dilution	Sensitivity % (95% CI)	Specificity % (95% CI)	Overall concordance (%)
DBS 1:2	84.6 (76.2–90.9)	73.6 (64.1–81.7)	79.0
DBS 1:3	82.7 (74.0–89.4)	64.2 (54.3–73.2)	73.3
DBS 1:4	73.1 (63.5–81.3)	67.9 (58.2–76.7)	70.5

Abbreviations: DBS, dried blood spots; HSV-2, herpes simplex virus 2

^†Women were aged 18―24 years, N=210.

Discussion

The Kalon HSV-2 has been shown to be sensitive and specific for HSV-2 diagnosis using plasma.^{(2, 12)} In our study, a dilution of 1:2 showed the highest sensitivity and specificity compared to the 1:3 and 1:4 dilution levels. This estimated sensitivity and specificity is low relative to the plasma-based reference results. Specifically, our findings differ from the estimated higher sensitivity and specificity found in a South African population which used the HerpeSelect ELISA serological assay.⁽¹³⁾ However, earlier studies evaluating the plasma-based HerpeSelect ELISA test in sub-Saharan populations suggested a lower specificity than the plasma-based Kalon HSV-2 ELISA assay.⁽¹⁴⁾

In addition, patterns of reactivity varied by dilution level. Although not the optimal dilution based on sensitivity or specificity, DBS 1:3 had the highest proportional reactivity to HSV-2 antibodies (59.0%). All three dilutions yielded higher frequencies of reactivity than plasma (49.5%), specifically DBS 1:2 (55.2%), DBS 1:3 (59.0%), and DBS 1:4 (52.4%). We found low concordance between plasma-based and DBS-based results, which contrasts with the high concordance reported by Hogrefe et al. using the HerpeSelect assay.⁽¹⁰⁾

Limitations

There are several limitations in our study. The sampling frame was limited to young women aged 18–24 years in Uganda; thus, our results may not be generalizable to males or older adults. Additionally, the sample size was relatively small. Further, we did not compare the quantity of IgG in the DBS punch specimens to that in plasma specimens. Finally, this study only used the Kalon ELISA to evaluate DBS for HSV-2 diagnosis. Other HSV-2 assays, such as the HerpeSelect HSV-2 ELISA and Biokit HSV-2 Rapid Assay are used in African countries and need to be further evaluated to compare their utility with DBS.

Conclusion

In summary, our study examined the performance of the Kalon HSV-2 assay using DBS in a population of young women in Uganda. DBS testing with the Kalon HSV-2 assay revealed relatively low sensitivity and specificity compared to plasma-based results on the same individuals. While DBS would be an appealing and relatively simple means for biomarker-based studies, particularly in resource-constrained settings, the accuracy of this testing format would need to be substantially improved before its use could be recommended for epidemiological studies.