HSV oropharyngeal shedding among HIV-infected children in Tanzania

Abstract

Herpes simplex virus (HSV) oral shedding has not been studied among HIV-positive children in Africa. We sought to evaluate longitudinal oral HSV reactivation in HIV-positive and -negative children. Twenty HIV-positive antiretroviral-naive and 10 HIV-negative children aged 3–12 years in Tanzania were followed prospectively for 14 days. Oral swabs were collected daily and submitted for HSV DNA PCR analysis. Clinical data were collected via chart review and daily diaries. HSV DNA was detected in 10 (50%) of HIV-positive and 4 (40%) of HIV-negative children. Children who shed HSV had virus detected in a median of 21.4% of samples; shedding was intermittent. Median CD4 count among HIV-infected children was 667 cells/µL in those with positive HSV DNA and 886 cells/µL in those who were negative (p = 0.6). Of the HIV-positive children reporting prior sores, five (83%) had positive HSV swabs, whereas the one HIV-negative child with prior sores did not have a PCR-positive swab. HSV is detected frequently in children with and without HIV. HIV-infected children reporting oral sores have a high rate of HSV detection. Given the proven strong interactions between HIV and HSV, further study of co-infection with these viruses is warranted in children.

Keywords

Herpes simplex virus HSV orolabial reactivation children viral shedding PCR paediatric HIV AIDS oropharynx

Background

HSV-1 seroprevalence in children is generally greater than 80% in developing countries where HIV is endemic.^1,2 Additionally, asymptomatic oral HSV-1 shedding is frequently detected in children with and without HIV,^3,4 and HIV-infected children have frequent HSV-related oral pathology.^5,6

Potent bi-directional clinical interactions between HIV and HSV-2 have been shown in adults, and are supported by strong molecular work.^7–10 In vitro molecular interactions with HIV have been shown for both HSV-1 and HSV-2 which have 80% homology. Although HSV-1 is known to be highly prevalent in children, few clinical studies of HSV-1 in HIV have been performed in this age group.¹¹ We performed a pilot project to determine the frequency of oropharyngeal HSV-1 shedding in a population of children infected with HIV and an HIV-uninfected group with the goal of obtaining preliminary HSV shedding data and determining the feasibility and acceptance of this type of research in children in the developing world in anticipation of future natural history or intervention trials.

Methods

Study design

The study was conducted at the DarDar Pediatric Program (DPP) in Dar es Salaam, Tanzania, a multi-disciplinary paediatric HIV treatment, research and training programme. Children/families meeting entry criteria were recruited at regular clinic visits. Children found to be HIV-negative by routine clinic counseling and testing were offered enrollment as control patients. For this pilot project, we chose to evaluate 20 HIV-1 infected and 10 HIV-1 uninfected children. Children were offered enrollment if they were more than 3 years old and ≤12 years old, not taking or expecting to take acyclovir or its derivatives, and if HIV-1 positive, not eligible for antiretroviral therapy according to the Tanzania National guidelines for HIV care and treatment (antiretroviral naïve). Children were not enrolled if they had an acute illness which by opinion of the attending health care worker made enrollment in the trial unfeasible (including active tuberculosis, malaria or other opportunistic infection requiring treatment). Written informed consent and assent (where appropriate) for study participation was obtained from the caregiver and child (Participants) prior to any study procedures. All consents and written materials were provided in Kiswahili. The human experimentation guidelines of the US Department of Health and Human Services and the individual institutions were followed in the conduct of the clinical research. The institutional review boards of Dartmouth College and Muhimbili University of Health and Allied Sciences approved the protocol.

Study procedures

On enrollment, providers administered a baseline study questionnaire, obtained a brief medical history and concomitant medication information. Participants were then instructed on collection of daily oral HSV swabs, with the collection of the first daily oral swab. A swab was gently rubbed over the lips and then the oropharyngeal mucosal surfaces and placed into a cryovial with 1.5 mL of PCR buffer. Caregivers were then provided with an adequate number of swabs, vials and pre-printed labels to last until their next visit. Participants returned on days 7 and 14 to return swab vials. Participants were also instructed on how to keep a diary of symptoms and signs to review with clinicians at the each visit. Swab vials were stored in as cool a location as possible until they were brought to the clinic, where they were stored at −20°C until shipped to the USA for analysis. Prior research has shown excellent yield for HSV PCR specimen stored at ambient temperatures for an extended time frame.¹²

Laboratory procedures

DNA extraction

DNA was extracted from 400 µL of the patient specimens with the EZ1 Virus Mini Kit and the automated EZ1 Advanced XL instrument (Qiagen). The purified nucleic acid was eluted in 60 µL of elution buffer. High (407,000 copies per mL), low (3000 copies per mL) and negative (sterile viral transport medium) control samples were included with each extraction. Extracts were stored at −20°C until analysed.

RT-PCR

Real-time PCR was carried out using the LightCycler 2.0 system (Roche). Each PCR reaction contained 5 µL of extracted DNA and 15 µL of master mix. The master mix was prepared with the LightCycler Fast Start DNA Master Hybridization Probes, LC HSV1/2 Recovery Template (internal control) and Primer/Hybridization Probes (Roche).¹³ Once the reaction mixture was complete, the capillaries were capped and centrifuged (15 s at 3000 rpm) using the LightCycler specific centrifuge (Roche). All PCRs were performed as follows: an initial cycle of 10 min at 95°C followed by 45 cycles of 10 s at 95°C, 15 s at 55°C and 15 s at 72°C with cycling temperature transitions set at 20°C/s. Amplified DNA was detected with the melting curve analysis consisting of one continuous cycle of 0 s at 95°C, 60 s at 40°C and 0 s at 85°C. High, low, negative, water and HSV1/2 DNA were included as controls with each assay. Data analysis and interpretation were performed using the LightCycler software. Samples with crossing points and melting peaks with Tm’s within 2.5°C of those exhibited by the HSV1/2 control were considered positive. The crossing points of the positive HSV1/2 high and low controls were required to be within ±1 cycle of the average crossing point for that lot in order for the run to be considered acceptable. Negative controls and specimens displayed no crossing points, and only contained melting peaks for the internal control. The lower limit of detection was 300 copies/mL. Two patients had all samples with melting curves in the intermediate range between HSV-1 and HSV-2. As has been previously described, these isolates likely represented HSV-1 isolates with polymorphisms in the probe region.¹⁴ Isolates from these patients and an additional two samples with a melting curve that would be considered to be HSV-2 by this assay were sent to the University of Washington for confirmatory HSV typing. The remaining positive PCR curves were typical of HSV-1.

Statistical methods

Basic univariate comparisons were carried out using chi-squared and ANOVA tests. Where appropriate, nonparametric tests were used to compare medians of variables that were not evenly distributed. Median HSV DNA levels were calculated using all positive HSV DNA values for each subject. Pearson’s correlation coefficient was used to correlate CD4 with HSV viral load.

Results

Compliance with swab samples was good, with 92% (275/280) of HIV-infected and 86% (120/140) of HIV-uninfected children’s swabs available for analysis. Of the 14 possible swabs, HIV-infected children returned a median 14.0 swabs compared to HIV-negative children who had a median 13.5 swabs (p = 0.09, Mann-Whitney U; Table 2). There was no difference in the median number of swabs returned by children who had HSV detected versus those with no HSV detected (14.0 vs. 13.5 swabs), suggesting that HSV detection was not a function of having submitted more swabs. Shedding was detected in 10 (50%) of HIV-positive and 4 (40%) HIV-negative children. In those with any positive samples, HSV DNA was detected on a median of 21.4% of swabs from HIV-positive children and on 28.6% of swabs from HIV-negative children (p = 0.5, Mann-Whitney U; Table 2). Twelve (86%) of 14 children (HIV-positive and -negative) with detectable HSV DNA had positive swabs for more than one day, some with consecutive days and some with non-consecutive days (Table 1).

Table 1.

Daily detection of oral HSV in children with and without HIV.

Shaded = Positive day. X = no sample. HSV2 = sample positive for HSV-2.

Table 2.

Clinical and HSV swab data for 20 HIV-positive and 10 HIV-negative children.

	HIV+ (N = 20)	HIV− (N = 10)
Age (Median)	5.5 (3–11)	7 (3–10)
Number of swabs done per patient (median)	14.0 (13–14)	13.5 (7–14)^a
HSV detected on at least one swab	10 (50%)	4 (40%)
Proportion of HSV+ swabs, in those with any HSV detected (median)	21.4 (7–43)	28.6 (7–71)^a
Median HSV DNA level (log₁₀ copies/mL)	4.4	3.8
Symptoms at time of +HSV PCR	1	1
Prior oral lesions	6 (30%)	1 (10%)
+HSV PCR during study	5	0
Symptoms during study	1	0

p > 0.05 for all comparisons.

Mann-Whitney U.

Median HSV DNA level was 4.4 log₁₀ in HIV-positive and 3.8 log₁₀ in HIV-negative children (p = 0.5, Table 2). In HIV-positive children, median CD4 count (cells/µL) was 667, (range 320–1713) in HSV PCR positive vs. 886 (range 285–2026) in HSV PCR negative children (p = 0.7). CD4 count did not predict HSV DNA level (R²= 0.013). A history of oral sores was reported among six HIV-positive children and one HIV-negative child. Of HIV-positive children reporting prior sores, five (83%) had positive HSV swabs, whereas the one HIV-negative child with prior sores did not have a PCR-positive swab. Overall, two PCR positive episodes were symptomatic, one HIV-positive and one HIV-negative.

Three samples had melting curves between HSV-1 and HSV-2, which were confirmed as HSV-1 by typing. Two swab samples had melting curves consistent with HSV-2. One of the patients had prior HSV-1 reactivation during the study and the other had a single day of HSV-2 detection and no other positive swabs. The sample with possible HSV-2 in the patient who had prior HSV-1 detected on swabs was very low copy number and was not detected in attempts at HSV typing, indicating a possible false positive. One HIV-positive patient, age 8 years old, had a single positive swab that was HSV-2, confirmed by HSV typing.

Discussion

Over a period of two weeks, HSV was detected in 50% of HIV-infected children in Tanzania with high CD4 counts. To our knowledge, this is the first study that has assessed the rate and frequency of HSV reactivation in HIV-infected and HIV-uninfected children.

The rate of asymptomatic HSV shedding from the oropharynx of children with HIV is not well characterized and only one published study has evaluated children with HIV.³ A cross-sectional study in HIV-infected Brazilian and American children, also with unknown HSV serostatus, showed a similarly high rate of HSV detection by PCR (48.3%) from randomly acquired oral swabs. However, that study did not sample subjects longitudinally as we have in our study, where we found shedding on a median of 21% of days in those who shed HSV.

The finding of no difference in the proportion of children with HSV detection between HIV-infected and HIV-uninfected subjects could be explained by a number of factors. First, because this was primarily a pilot descriptive study, our sample size may not have been large enough to have sufficient power to detect small differences. Second, at these high CD4 counts (median ∼780), subjects’ immune systems are relatively intact and there may truly be no difference in HSV shedding rates. In our HIV-infected children, CD4 count did not predict HSV detection or quantitative amount of HSV in our population. Similarly, this could be a function of our small sample size and also due to lack of significant diversity in absolute CD4 counts creating a relatively homogenous population.

HIV-infected children have a high prevalence of oral lesions and pathology, which could be in part due to HSV.⁶ In our HIV-infected children, five of six children reporting a history of prior oral sores had HSV detected at times when they were asymptomatic. Thus, a history of oral sores in HIV-infected children could be an indicator for more active HSV, but this has not been evaluated systematically. In settings where resources are limited and serology for HSV unavailable, the presence of oral sores could be a marker for HSV positivity and reactivation frequency.

Our study had several limitations. First, we did not have HSV serology results on our patients, which likely led to an underestimation of the proportion of patients shedding HSV as some included patients may have been seronegative, estimated to be <20% based on a prior serosurvey in this population.² With this high seroprevalence in the general paediatric population, HSV-1 seroprevalence was not expected to be different between HIV-positive and HIV-negative children, so we do not expect that this would bias our comparison between these groups. Second, our small sample size limits our ability to make comparisons between subgroups regarding factors such as HIV status, CD4 count and HSV viral load. Third, we followed participants for a short period of time and we had limited clinical information, including HIV viral load measurements. Future research should take these factors into account. Fourth, we had no patients with lower CD4 counts and no patients on antiretroviral therapy, limiting the generalizability of our results. If prior HSV research is any indicator, patients with lower CD4 count would likely have increased rate and frequency of HSV detection.

There is a lack of information about viral co-infections in children with HIV. HSV-infection has been shown to be a potent co-infection that likely alters the natural history and infectivity of HIV in adults, data in children are lacking. Our study has garnered preliminary data to help clarify the natural history of HSV infection in a population of children with HIV infection not on antiretroviral therapy. As more children are diagnosed with HIV in the developing world, increasing numbers are being identified that do not meet criteria for ART via local standards. HSV-2 suppression is the first inexpensive therapeutic intervention that has been shown to significantly decrease plasma HIV-1 levels and to delay decline in CD4 count in adults,^15–19 though the long-term effects with regard to safety, toxicity and resistance are still being worked out. There has been no study to determine if there are similar effects with suppression of HSV-1. Given the significant social, emotional and physical costs of being on anti-HIV treatments, delays in ART initiation could have distinct benefits in the paediatric population. Thus, further natural history study with a goal of performing an interventional trial of anti-herpes therapy in children is warranted.

Institutional review

The institutional review boards of Dartmouth College (Dartmouth College Committee for the Protection of Human Subjects, review #21397) and Muhimbili University of Health and Allied Sciences (Senate Research and Publications Committee, Ref. No.MU/RP/ AEC/Vol.XIII/49) approved the protocol.

Footnotes

Acknowledgements

Foremost, we would like to thank the participants and their caregivers for their gracious participation in this study. We would also like to thank Kate Ruoff and Joe Schwartzman in the Dartmouth Lab. Thanks to Meei-li Huang and the lab of Dr Larry Corey for assistance with HSV typing and establishment of our assay.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Foundation for the Treatment of Children with AIDS (FTCA), New York, NY.

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