Impact of rectal gonorrhoea and chlamydia on HIV viral load in the rectum: potential significance for onward transmission

Abstract

The aim of this study was to investigate the effect of asymptomatic rectal bacterial sexually transmitted infections (STIs) on rectal HIV viral load (VL). A prospective cohort study of HIV-positive men who have sex with men attending a tertiary centre in London, UK, for their routine HIV care was performed. Forty-two HIV-positive men who have sex with men were recruited between January and August 2014. In participants on antiretroviral therapy (ART), there was no significant difference in rectal VL in those with and without STI (p = 0.4). All rectal HIV VLs were below the limit of detection (<100 copies/µg of total RNA) whether an STI was present or not. In those not on ART, rectal HIV VL was on average 0.6log₁₀ lower post STI treatment. The presence of asymptomatic rectal chlamydia and gonorrhoea was not associated with increased rectal HIV VL in those fully suppressed on ART. In the context of effective ART, the presence of rectal gonorrhoea or chlamydia does not appear to increase rectal HIV VL and the risk of increased viral infectivity.

Keywords

HIV highly active antiretroviral therapy chlamydia gonorrhoea homosexual rectal HIV viral load

Introduction

The incidence of HIV in men who have sex with men (MSM) is stable and high.¹ Anal sex remains a major mode of HIV transmission. With treatment as prevention being highly effective,^2,3 secondary HIV prevention has the advantage of a cohort already attending hospital services. High levels of sexually transmitted infections (STIs) have been identified in HIV–infected MSM.⁴ Although urethral STI are known to increase HIV shedding in the semen,⁵ the impact of rectal bacterial STI on onward transmission of HIV is unknown. In our own cohort of HIV-positive asymptomatic MSM, 17.4% had gonorrhoea (GC) and/or chlamydia (CT), including 9.8% with rectal CT and 4.2% with rectal GC.⁶

We investigated the effect of rectal GC and CT on rectal and plasma HIV viral load (VL) and rectal cytokine levels in infected individuals both on and off antiretroviral therapy (ART).

Methods

From January 2014 to August 2014, 42 HIV-positive MSM attending a tertiary centre in London, UK, for their routine HIV care and undergoing routine asymptomatic STI screening were recruited into this prospective cohort study. A total of 21 participants were on suppressive ART. Further inclusion criteria included no receptive anal sex or use of intra-anal medication 48 hours before acquisition of rectal samples.

At the first visit, demographic and clinical information was collected including CD4 cell counts, plasma VL, herpes simplex virus (HSV)-1 and -2 serostatus, hepatitis C serostatus and ART regimen. Follow-up time was a maximum of three visits over 4 weeks. Sample collection was performed at the first visit. Participants in whom a rectal bacterial STI was detected were offered treatment and invited to return for repeat sampling 2 weeks following treatment.

Ethics approval was obtained from NHS research ethics committee (London-Surrey Borders). All participants provided written informed consent.

Sample collection

Three rectal swabs were taken via proctoscopy for rectal GC/CT testing (Aptima Combo2, Genprobe/Hologic, San Diego, CA), rectal HIV VL quantification, and rectal inflammatory cytokine measurements. For HIV rectal VL detection Dacron-tipped flocked swabs were used (Copan UTM™ viral transport media and flocked swab, Copan Diagnostics, Murrieta, CA). Each swab was held against the mucosa and rotated 360°. Swabs were stored in 1.0 mL of viral transport medium at –70℃ until tested. For inflammatory cytokine detection dry Weck-Cel sponges (Medtronic Xomed, Jacksonville, FL) were used. These were stored in 300 µL of extraction buffer (containing 1 mL protease cocktail, 20 microlitres 10% sodium azide solution + 1.5 g NaCl and 100 mL of PBS solution), at –70℃ until analysis.

Laboratory methods

HIV-1 RNA in rectal secretions was quantified using the Roche Cobas TaqMan 48 analyzer and HIV-1 High Pure Extraction System. The lower limit of detection was 100 HIV RNA copies/mL. However, in order to standardize rectal samples viral load was expressed as HIV RNA copies/µg of total RNA. Total RNA in all rectal swabs was quantified using the Qubit RNA BR assay kit (Q10210, Thermofischer Scientific Inc.). Plasma HIV VL was measured using the Roche COBAS AmpliPrep/COBAS Taqman HIV-1 system. Rectal GC and CT were evaluated using the APTIMA Combo 2 Assay (Hologic/Gen-Probe, Inc., San Diego, CA). Quantitative detection of inflammatory cytokines IL6, IFNγ, and TNF was done using cytokine array (Human cytokine HS X biochip, Randox Laboratories Limited, Crumlin, County Antrim, UK).

Statistical analysis

Differences between HIV VL levels and cytokine concentrations were calculated by independent (two-tailed) t-test. Differences were significant at p value ≤0.05.

Results

Forty-two MSM were recruited. Twenty-one were on ART based on a sample size calculation required to detect a mean difference of 2000 copies/mL in the presence of an STI. Fourteen were on non-nucleoside reverse transcriptase inhibitor-based regimens, six on protease inhibitor-based regimens, and one on a CCR5 inhibitor-based regimen. Six participants had a history of genital herpes simplex virus infection. Rectal STIs were detected in 14 participants. Five had chlamydia only, four had gonorrhoea only, and five had both chlamydia and gonorrhoea.

Rectal HIV viral load

Four rectal samples were inhibitory and not included in the analysis. In the ART group, there was no significant difference in rectal VL in those with and without STI (p = 0.4). All rectal HIV VL were below the limit of detection (<100 copies/µg of total RNA) whether a rectal STI was detected or not (Table 1). All post-treatment rectal HIV VL were also below the limit of detection.

Table 1.

Rectal and plasma HIV viral loads.

Rectal infection status	On ART
	No	Rectal VL^a		Plasma VL^b		No	Rectal VL^a		Plasma VL^b
	No	At recruitment	Post STI Rx	At recruitment	Post STI Rx	No	At recruitment	Post STI Rx	At recruitment	Post STI Rx
STI	5	All < 100	All < 100	All < 20	20,20,20,30,54	7	1.9–4.8 log₁₀	1.9–3.9 log₁₀	3.2–6.1 log₁₀	2.4–4.8 log ₁₀
No STI	13	All < 100	N/A	All < 20	N/A	13	1.9–6–4 log₁₀	N/A	2.87–5.6 log₁₀	N/A

ART: antiretroviral therapy; STI sexually transmitted infection.

Copies/ µg of total RNA.

Copies/mL plasma.

In all, 21 individuals on ART, three rectal samples were inhibitory and not included in the analysis; 21 individuals were ART-naïve, one rectal sample inhibitory and not included in the analysis.

The range of HIV VL in the ART-naïve group is given in Table 1. Of the seven with STIs, only three returned for post-treatment rectal swabs. For this reason statistical significance was not evaluated; however, rectal HIV VL was on average 0.6log₁₀ lower post treatment.

Cytokine levels

Thirteen samples were not suitable for analysis. In the ART group, the presence of STI was not associated with increased inflammatory cytokine levels when compared to those without STI (IL-6 [p = 0.41], IFNg [p = 0.42], TNFa [p = 0.26]). Furthermore, there was no significant change in cytokine level post treatment of STI.

In the ART-naïve group, the presence of STI was not associated with increased cytokine levels when compared to those without STI (IL-6 [p = 0.12], IFNγ [p = 0.16], TNFα [p = 0.09]). In this group, post treatment cytokine levels were only available for two of seven participants and in both participants IL-6, IFNγ, and TNFα levels decreased post treatment.

Discussion

ART markedly reduces the incidence rate of HIV in serodiscordant couples,² thereby suggesting that ART reduces the infectiousness of HIV-infected individuals. However, the role of co-existing STI, particularly non-ulcerative asymptomatic infections in HIV onward transmission is unclear. The majority of the studies on the impact of STIs on HIV shedding have been done in ART-naïve individuals. However, more than 40%⁷ worldwide were on ART in 2014 and this number is likely to increase due to results of the START study⁸ and changes in treatment guidelines. It is therefore important to determine whether co-existing asymptomatic bacterial STIs impact on HIV infectiousness in individuals on effective ART.

This study focused on individuals with asymptomatic STIs as a previous study at our centre demonstrated high rates (17.4%)⁶ and this subset of patients may, by virtue of their lack of symptoms, be less likely to access sexual health screening and treatment. The proposed mechanism for increasing the risk of HIV transmission was through immune activation and we sought to evaluate this by measuring rectal cytokine levels. However, data on their utility in asymptomatic infection is limited and our results did not indicate any correlation in this setting.

As expected, being fully suppressed on ART was associated with an HIV VL below the limit of detection in the rectum irrespective of the ART regimen. Rectal CT/GC did not impact on rectal HIV VL or rectal inflammatory cytokines. Therefore, in the context of effective ART, the presence of rectal GC or CT does not appear to increase rectal HIV VL and the risk of increased viral infectivity. Our findings are supported by observational data from large studies of serodiscordant couples^2,3 that found little impact of CT/GC on onward transmission of HIV.

This was a small study and sample numbers were reduced further due to assay inhibition. However, the sample size was sufficient to detect a difference of 2000 copies/mL or more in the presence of an STI in individuals on ART, despite the four inhibitory specimens. All the participants in this study receive regular outpatient care and STI screening and results may therefore not be applicable to individuals not being monitored. No lymphogranuloma venereum (LGV) infections were detected in our cohort. In addition, although some of our cohort had a history of genital HSV we did not test for concurrent HSV infection during the study, nor did we test for syphilis. We therefore cannot infer that syphilis, LGV, and HSV would not impact rectal HIV VL. Plasma HIV VL may not accurately represent sexual transmission risk, as other factors are involved. However, by measuring rectal HIV VL as well as plasma VL, we hoped to strengthen this association. Although adherence to ART was not measured within the ART group only patients who had fully suppressed plasma VL were studied and this can be considered a proxy for ART adherence.

Our data add to the limited number of published studies on rectal HIV VL levels in MSM. We were able to quantify HIV RNA in rectal secretions and demonstrate an association between plasma and rectal VL. We also examined whether concurrent CT or GC infection affects rectal HIV shedding. Our most salient finding is that in those MSM on ART, GC or CT infection did not alter the likelihood of detecting HIV in rectal secretions, which has important public health consequences as ART becomes more widespread.

In conclusion, our findings demonstrate that when plasma VL is below the limit of detection this correlates with a comparable HIV VL in rectal secretions. In addition, rectal CT and GC did not impact on rectal HIV shedding or rectal cytokines particularly among MSM on ART with an undetectable plasma VL.

Footnotes

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: JW has been a consultant to Diagnostics for the Real World, has received meetings expenses and payment for lectures for Becton-Dickinson Diagnostics, Abbott Molecular and GenProbe/Hologic, and research support from GenProbe/Hologic in the form of diagnostic kits. OD, SC, SO, GC, TD and JF had no conflict.

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 a British HIV Association/ Gilead SpR research award.

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