The impact of syndromic treatment of sexually transmitted diseases on genital shedding of HIV-1

Objectives: To examine the impact of sexually transmitted diseases (STD) syndromic treatment on genital shedding of HIV and the impact among women in whom STD treatment was not successful.

Design: Seventy-one HIV-infected women were included; 60 had symptomatic STD [72% with genital discharge syndrome (GDS) and 28% with genital ulcer syndrome (GUS)] and 11 controls did not have symptomatic STD. Cervical HIV load in 94% women was measured at baseline and after STD treatment.

Results: Cervical HIV load at entry was significantly higher in women with symptomatic STD than in controls [median, 3.15; interquartile range (IQR), 1.90-3.34 versus median, 1.90; IQR, 1.90-2.19 log₁₀ RNA copies/swab, respectively; P = 0.024]. Women with STD were also more likely to have detectable cervical HIV RNA (68% versus 27%; P = 0.016). Cervical HIV load was significantly higher in women with GUS than in those with GDS (median 3.46; IQR, 2.84-4.18 versus median, 2.83; IQR, 1.90-3.31 log₁₀ copies/swab; P = 0.019). There was no significant reduction in genital HIV shedding after syndromic treatment of GDS or GUS. However, significant decreases were limited to only those with clinical improvement (median, 2.91; IQR, 1.90-3.45 versus median, 2.25; IQR, 1.90-3.08 log₁₀ RNA copies/swab, respectively; P = 0.006). GUS was significantly associated with treatment failure, independent of plasma HIV RNA load and CD4 T-cell count (odds ratio, 4.79; 95% confidence interval, 1.32-17.46).

Conclusions: The fact that STD syndromic treatment impacts very little in reducing genital HIV shedding underscores the need for appropriate validation of STD syndromic diagnosis and management to control heterosexual transmission of HIV.

Introduction

Several studies have observed increased genital HIV shedding in patients with sexually transmitted diseases (STD) [^1-6] and treatment resulted in a dramatic reduction in viral shedding [^3-6]. The World Health Organization (WHO) recommends syndromic treatment of STD in developing countries at the primary health care level [⁷]. Treatments of STD in the previous interventional studies was, however, based on pathogen-specific laboratory diagnosis. Although improved syndromic treatment of STD has been shown to reduce HIV incidence [⁸], there is lack of information on its effect on genital HIV shedding. The aim of the present study was, therefore, to assess the impact of syndromic management of STD on genital HIV burden. Also, we evaluated the impact of treatment in those in whom the WHO STD syndromic treatment algorithm was not successful.

Patients and methods

Study participants

Study subjects were prospectively recruited at Tekle-Haymanot Health Center, Addis Ababa, Ethiopia between June and September 2001. Women presenting to the health center were offered voluntary HIV counselling and testing. After consent, a detailed history was obtained using a standard questionnaire. Subjects were eligible if they present with symptoms suggestive of STD and if they have not taken antibiotic treatment for at least 2 weeks prior to presentation. All patients were antiretroviral treatment naive. The project was approved by Institutional and National Ethical Clearance Committees.

At entry visit, Dacron swabs were used to collect cervical secretions that were immediately placed in 1 ml of RNA/DNA stabilization reagent and placed in ice before transfer to the Ethio-Netherlands AIDS Research (ENARP) laboratory within 3 h of collection. Samples were then stored at -80°C until analysis.

Women with symptoms suggestive of STD were treated according to a syndromic approach. A programme to improve syndromic case management in Addis Ababa health centers was launched by the Regional Health Bureau in collaboration with Médecins Sans Frontièrs Belgium [⁹]. The protocol was adopted based on the WHO syndromic treatment algorithm [⁷]. Drugs were dispensed in the form of prepackaged treatments, known as syndrome selective packaging, containing the full course treatment, condoms, and instruction leaflet and a partner referral card.

Women presenting with genital discharge syndrome (GDS), and a negative risk assessment were treated with metronidazole (single oral dose 2 g) and nystatin (intravaginal 100 000 IU for 14 days). Those with GDS, but a positive risk assessment, were treated additionally with doxycycliline (oral 200 mg divided in two doses for 7 days) and spectinomycin (2 g single intramuscular injection). Women with genital ulcer syndrome (GUS) were treated with benzanthine benzyl penicillin (single intramuscular injection) and erythromycin (1.5 g divided in to three doses daily for a total of 7 days). Patients were checked, on average, 2 weeks after treatment to assess compliance and clinical outcome, and for repeat sampling. Women who were not cured after syndromic STD management and those with infections that were not recognized at enrollment received pathogen-specific treatment, and were asked to return 2 weeks following re-treatment.

Laboratory methods

HIV screening was performed by enzyme-immunoassay and reactive samples were confirmed by Western blot. HIV-1 load in plasma and cervical swab was quantified by NucliSens assay (bioMerieux Inc., Durham, North Carolina, USA) that reliably quantifies the predominant Ethiopian HIV-1 subtype C [¹⁰]. HIV RNA levels below the lower detection limit of the assay were considered to be 80 copies/ml. CD4 T-cell counts were determined by FACScan (Becton Dickinson, San Jose, California, USA).

In addition to the syndromic diagnosis of STD, pathogen-specific diagnosis of certain STD was performed. Serology for syphilis was done using rapid plasma reagin test and Treponema pallidum passive particle agglutination assay. Those reactive to both were considered as having active syphilis. Neisseria gonorrhoeae was detected by Gram stain confirmed by culture and/or by ligase chain reaction (LCR). Chlamydia trachomatis was detected by ligase chain reaction. Trichomonas vaginalis was detected by wet-mount and/or by culture using InPouchTV (BioMed Diagnostics, San Jose, California, USA), and bacterial vaginosis (BV) was detected by quantitative morphology of Gram-stained slides based on the Nugent's criteria [¹¹]. Results of the syndromic diagnosis were compared to the pathogen-specific diagnosis. All laboratory assays were done at ENARP, except the Chlamydia trachomatis ligase chain reaction (Lcx PCR assays) that was done at Hadassah AIDS Center in Jerusalem.

Statistical analyses

Proportions were compared using χ² test. Median CD4 cell counts and HIV load between the different groups were compared using Mann-Whitney U test. Individual changes in CD4 cell counts and HIV load from baseline were compared using Wilcoxon signed rank test. Logistic regression analysis was also used to identify predictors of persistent illness or HIV shedding. A P value < 0.05 was considered indicative of statistical significance. All statistical analyses were performed using the Stata Statistical Software package (SPSS Inc., Chicago, Illinois, USA).

Results

A total of 236 women were screened; 112 (48%) did not have STD and were excluded. Of the remaining 124 women, 60 (48%) were HIV-1 infected (age range, 18-45 years; median age, 26 years). There were 43 (72%) women presenting with GDS and 17 (28%) with GUS. The performance of the syndromic diagnosis was poor either missing or over diagnosing many infections (Table 1). In addition, 11 HIV positive women without symptomatic STD were included as controls. Of these, one woman had serology-confirmed syphilis and four women had indeterminate score for BV [¹¹]; these were not excluded. Baseline characteristics of the women who were excluded from the study and the controls were not significantly different from those of the cohort with STD. The median time of follow-up was 17 days (range, 14-32 days) after completion of treatment.

Table 1

Cervical HIV-1 shedding was significantly higher among women with STD compared to controls [median, 3.15; interquartile range (IQR), 1.90-3.34 versus median, 1.90; IQR, 1.90-2.19 log₁₀ RNA copies/swab, respectively; P = 0.024]. Those with STDs were also more likely to have detectable cervical HIV RNA (68% versus 27%; P = 0.016). Cervical HIV load was also significantly higher in women with GUS than in those with GDS (Fig. 1a).

Fig. 1

Of the 71 women enrolled, 94% (56 STD positive, 11 controls) returned for follow-up and re-sampling. There were no significant differences in CD4 T-cell counts or plasma HIV-1 RNA at entry compared with those measured after treatment (data not shown). Although there was a trend towards reduced shedding of HIV-1 related to overall syndromic treatment of STD, there was no significant reduction in genital HIV shedding after treatment of GDS or GUS taken separately (Fig. 1a). In addition, we compared the concordance between syndromic STD treatment and laboratory-based diagnosis of STD and the impact on genital shedding of HIV-1 (Table 1). In almost 40% of the cases, the syndromic treatment did not cover laboratory-diagnosed infection and no significant reduction was noted on genital shedding of HIV-1.

To evaluate whether the outcome of syndromic STD treatment impacts on persistence of HIV shedding, we compared cervical HIV RNA levels based on the outcome status. Of the 56 women presenting with STD and evaluated at follow-up visit, 32 (57%) had clinical improvement while 24 (43%) had persistent symptomatic STD despite treatment. The median genital HIV load decreased significantly only in the subgroup of women with clinical improvement (Fig. 1b); these women had a mean decrease of 0.43 log₁₀ copies from baseline and more than 56% demonstrated decreases in cervical HIV RNA. In addition, 31% and 25% of the women with improved syndromic treatment outcome had a decrease in cervical HIV load of more than 0.50 and 1.0 log₁₀ copies per swab, respectively. In contrast, there was no significant change in cervical HIV load between entry and follow-up visit in the subgroups of women with persistent illness.

We found no association with age, baseline plasma HIV RNA, CD4 T-cell counts or persistent illness (data not shown). However, having GUS was significantly associated with treatment failure (odds ratio, 4.79; 95% confidence interval, 1.32-17.46), independent of plasma HIV load and CD4 T-cell count.

Discussion

This study demonstrates that symptomatic STD are associated with significant increases in genital HIV shedding, extending the findings of previous investigations [^1-6], and strengthening the notion that both ulcerative and non-ulcerative STD are important cofactors in promoting HIV transmission [¹²].

The WHO recommends syndromic STD treatment at the primary health care level in developing countries [⁷]. Improved syndromic treatment of STD has been shown to reduce HIV incidence [⁸] but the mechanism is not understood. In many developing countries the quality of syndromic STD treatment is very low [^7,13]. In this study, therefore, we examined among women the impact of syndromic management algorithm of STD on the genital shedding of HIV.

We found that the performance of the syndromic management algorithm was poor- it missed several infections and resulted in either under treatment or over treatment of STD. No previous studies have evaluated the impact among patients in whom treatment was not successful. Although syndromic treatment led to a modest decrease in HIV genital shedding related to overall treatment, there was no significant reduction in HIV genital shedding after treatment of GDS and GUS taken separately. In this study, we have also demonstrated that significant reductions in cervical HIV load were limited to only those women with clinical improvement. In this subgroup of women, cervical HIV RNA returned toward baseline levels over an average of 2 weeks after syndromic treatment. Women who failed to respond to syndromic treatment demonstrated persistent elevation of cervical HIV RNA levels, and some experienced further increases in genital shedding. The results support the contention that the effects of syndromic STD treatment on genital shedding of HIV depend on the prevalence of treatable STD in a given population. Thus, syndromic STD treatment in a population with higher prevalence of non-treatable STD or poor performance of syndrome treatment algorithm may explain for the modest effects on HIV transmission as seen in the Rakai trial when compared to the Mwanza trial [^8,14].

The lack of effect of syndromic treatment of GUS that we noted in this study is in contrast with that reported previously. Dyer et al. [⁶] documented that men with GUS had increases in seminal HIV RNA load, which declined after a single dose of benzanthine penicillin and azithromycin. The discrepancy between our results and those of Dyer et al. [⁶] could be ascribed to the differences in the type of the syndromic treatments, sex of the patients or drug susceptibility of the pathogens. The underlying reasons for the lack of a reduction in HIV genital shedding in the subgroup of women (n = 8) with GDS who were also seropositive for syphilis is not clear. Many STD control programs do not include syphilis serology in syndromic management [¹⁵]. Thus, a significant proportion of women with latent syphilis may remain unrecognized. However, it has been demonstrated that syphilis can enhance HIV replication in vitro [¹⁶]. Its effect in vivo has not been documented. The observation in the present study that untreated syphilis, without symptoms of genital ulcerations, results in persistence of genital shedding of HIV might be attributable to the systemic effects of latent syphilis. Thus, syphilis-induced increase in plasma HIV load may account for the increase in the HIV RNA levels in the genital secretions.

The fact that 43% of the women did not have any improvement is related in part, at least for women with GDS, to the poor syndromic treatment provided. For GUS, the current treatment guideline does not include the treatment of genital herpes simplex virus type-2 (HSV-2), which is also the case in most African countries. Therefore, the failure to cure GUS (46% in this study) could be due to the presence of genital herpes that was not treated. HSV-2 is recognized as a common cause of GUS in sub-Saharan Africa. Therefore, in line with the WHO's recent recommendation, acyclovir should be included into the current GUS management algorithm; however, access to the drug in Africa is limited because of the high cost and a concerted effort is needed to increase access to it by implementing mechanisms as has been done for antiretroviral drugs.

Several studies have demonstrated increased genital shedding of HIV with increasing plasma HIV load [^17-20]. However, the multivariate analysis in this study demonstrated a lack of effect of syndromic treatment of STD on cervical HIV RNA load independent of plasma HIV RNA load or CD4 cell counts. The persistence of certain genital infections due to failure to recognize them and/or treat them in the women non-responsive to syndromic therapy may have contributed to the lack of any effect on levels of HIV in genital secretions and, may, thereby, continue to amplify transmission of HIV infection.

Taken together, the data suggest that in addition to the poor syndromic treatment provided, untreated infections, such as herpetic ulcers, might be responsible for the persistence of genital shedding of HIV-1 that we observed in our study. The WHO recommends periodic assessment of syndrome aetiology in order to guide therapy [⁷]. Thus, improving the quality of the current syndromic management approach to treatment of STD is urgent in order to impact on control of transmission of both STD and HIV. Effective treatment regimens are available for the traditional STD, and when promptly applied along with other STD control measures, such as condom use and safe sex counselling, might be expected to have an impact on both STD and HIV control. The recognition of treatable co-factors associated with the sexual transmission of HIV has important implications for HIV control programs.

Acknowledgements

We thank the study participants for their kind collaboration and the laboratory staff of ENARP and Microbiology Section of EHNRI for their technical assistance.

Sponsorship: ENARP is a collaborative research project between EHNRI, the Amsterdam Municipal Health Service, the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, and the Academic Medical Center of the University of Amsterdam. ENARP is a bilateral project financially supported The Netherlands Ministry of Foreign Affairs and the Ethiopian Ministry of Health. S.M. has also received financial support from the MASHAV, Jerusalem, Israel.