HIV-1 CURRENTLY INFECTS more than 36 million people worldwide, including over 25 million living in sub-Saharan Africa. 1 HIV can be transmitted through contaminated blood and blood products, from mother to baby, and through sexual intercourse;2 the latter route of transmission is responsible for about 90% of new infections in sub-Saharan Africa. 3
It is now clear that many cofactors can influence the sexual transmission of HIV, including the virus type, the concentration in genital secretions, male circumcision, hereditary resistance factors, and the presence of sexually transmitted diseases (STDs) that produce genital ulcerations or mucosal inflammation. 4
Mucosal inflammation facilitates transmission of HIV by increasing viral shedding in infected people and increasing the number of cells receptive to HIV. 5 Though estimates vary, STDs increase the overall risk for transmission of HIV by about twofold to fivefold. 5,6 Accordingly, recognition and treatment of STDs are critical components of virtually every HIV control program, especially where the prevalence of STDs is high. 7
In the sub-Saharan country of Malawi, we have previously demonstrated that urethritis greatly increases the concentration of HIV in semen, and this increase can be gradually reversed with appropriate antibiotic therapy. 8 Although in our earlier study we found Neisseria gonorrhoeae to be the predominant cause of urethritis, many men were also infected with Trichomonas vaginalis alone or as a copathogen. 9 Indeed, this pathogen is the most common nonviral sexually transmitted organism worldwide. 10
T vaginalis appears to be highly prevalent in men and women elsewhere in sub-Saharan Africa 11–13 and in other developing countries. 10,14 T vaginalis is associated with an increase in the excretion of HIV in semen in some subjects, 8,9 and HIV-negative women infected with T vaginalis were observed to have nearly twice the median concentration of endocervical HIV-receptive CD4 lymphocytes. 15
Metronidazole, the drug used in treatment for trichomoniasis, is used in syndromic management of vaginal discharge in many settings. However, perhaps surprisingly, metronidazole is not used in the syndromic management of urethritis in men in Malawi, despite up to 25% to 30% of men treated for gonococcal and chlamydial urethritis continue to have symptoms after empirical therapy. 16
In an effort to understand these unacceptable treatment failure rates, we conducted a single-masked, randomized clinical trial to determine the effects of adjunctive metronidazole on the syndromic management of male urethritis and the elimination of infection with T vaginalis. Seminal plasma viral loads in a subgroup of patients with trichomoniasis were compared with those of STD-free controls to clarify the potential role of this pathogen in the shedding of HIV.
Study Site and Population
We conducted the study in the STD clinic of Lilongwe Central Hospital (LCH), in Malawi. Men older than age 18 years residing within 30 km of the LCH and presenting with symptoms of urethritis were considered for this study (Figure 1). Consecutive HIV-positive attendees to the LCH dermatology clinic without an STD were asked if they would be willing to enroll as controls for seminal HIV RNA concentration comparisons.
The current algorithm for urethritis in Malawi identifies men presenting because of urethral discharge and/or dysuria who have had sexual activity within the previous 2 weeks. A study nurse explained the rationale for the study to eligible men. The potential benefits and discomforts of participation were explained before enrollment. Men who consented and were able and willing to return for follow-up after 7 days were enrolled. The University of North Carolina at Chapel Hill and the Malawi Institutional Review Boards independently approved the study.
Malawian clinicians conducted all interviews and examinations in the local language (Chichewa). Each patient's demographic characteristics, recent sexual history, and selected medical history were obtained. During the physical examination, the clinician collected a urethral swab specimen for T vaginalis culture (InPouch TV; BioMed Diagnostics, San Jose, CA). A second urethral swab was used for Gram stain slide preparation to determine white blood cell counts per high-power field (WBCs/hpf) and to detect gram-negative intracellular diplococci. First-catch urine was also collected and cultured for T vaginalis. Polymerase chain reactions (PCRs) were performed on urine sediment to detect T vaginalis 17 and N gonorrhoeae DNA (Roche Applied Science, Amplicor CT/NG, Indianapolis, IN).
Ligase chain reaction (Abbott LCx, Abbott Park, IL) was used to detect Chlamydia trachomatis in 289 urine specimens; 122 urine specimens were not tested for C trachomatis. Subjects were considered positive for T vaginalis infection if culture or PCR was positive, and they were considered positive for gonorrhea if the presence of gram-negative intracellular diplococci was noted or PCR was positive. Blood was drawn for determination of HIV status by rapid test with ELISA confirmation (100 Capillus HIV test, Cambridge Diagnostics, Ireland; and Genetics Systems HIV-1 peptide EIA, BioRad, Hercules, CA) and for syphilis serology by rapid plasma reagin test (RPR; Becton Dickinson, Franklin Lakes, NJ). Men who were positive by HIV rapid test and negative by ELISA were considered to be of indeterminate status and were asked to return for retesting in 3 months.
After sample collection, each man with urethritis received treatment in accordance with Malawian syndromic management of STD guidelines: a single intramuscular dose of 240 mg gentamicin and 100 mg doxycycline twice daily for 7 days. Men with urethritis and genital ulcers at presentation did not receive doxycycline but were treated with a single intramuscular dose of 2.4 MU benzathine penicillin and 500 mg erythromycin four times daily for 7 days, a regimen complying with Malawian syndromic management guidelines for genital ulcers concurrent with urethritis.
All subjects were then randomized to receive either metronidazole (2 g in a single oral dose) or placebo. All men were asked to refrain from sexual activity, were given partner notification cards and condoms, and were asked to refer their sex partners to the clinic for treatment even if the partner was asymptomatic.
We asked the participants to return at 7 days for follow-up. The follow-up was designed to determine the effects of therapy on urethritis and infection with T vaginalis. Three measures of persistent urethritis were recorded: patient-reported symptoms, clinician-observed discharge, and WBCs/hpf on a Gram-stained urethral smear.
The follow-up questionnaire included current symptoms, sexual behavior, treatment compliance, and use of any additional medicines. The genital examination was identical to that at baseline.
Men whose InPouch culture at baseline was positive had an additional swab, semen, and urine culture performed for the detection of T vaginalis. Because PCR data were not available for clinical interpretation, men with T vaginalis detected at baseline by PCR only did not provide additional culture specimens. Men who reported sexual activity and remained infected were treated as per the Malawian guidelines, with the same treatment they had received at baseline. Men with persistent signs and/or symptoms of urethritis or persistent gonorrhea without evidence of reinfection were treated with ciprofloxacin (500 mg in a single oral dose).
Semen was collected from men with urethritis at the follow-up visit if they were HIV-positive and culture-positive for T vaginalis and had not received metronidazole. All of these men at baseline were treated as per the Malawi syndromic guidelines described above, and all men received metronidazole for their trichomoniasis immediately after the collection of semen. Semen was also collected from HIV-positive male attendees to a dermatology clinic without symptoms of an STD who were culture-negative for T vaginalis.
Both groups of men, from the STD and dermatology clinics, were asked to return in 2 weeks to give another semen sample. HIV RNA concentrations in semen were determined by PCR (Nuclisens HIV-1 QT; BioMeriéux, Durham, NC).
The primary outcome was persistent urethritis, defined by the observation of >3 WBCs/hpf on a Gram-stained urethral smear at follow-up. Secondary outcomes included patient-reported discharge or dysuria and clinician-observed discharge at follow-up.
On the basis of previously observed urethritis cure rates in Malawi, 16 we predicted that enrolling 200 men in each study arm would afford us 95% power, at a significance level of 0.05, to detect a drop in persistent urethritis from 25% to 10% with the addition of metronidazole. We took into consideration a 10% loss-to-follow-up rate.
The proportion of men with persistent urethritis in each treatment group was compared with chi-square tests or Fisher exact tests in situations with small cell sizes. Risk ratios with 95% confidence intervals were also calculated. Adjusted estimates of the risk ratios were determined in multivariate analyses with use of generalized linear models with a binomial distribution and log link function. Variables used in the modeling procedures were determined a priori or were associated with the outcome in bivariable analyses. The relationships between HIV infection and other STDs at baseline were described with prevalence ratios and 95% confidence intervals.
Analyses comparing HIV RNA concentrations in semen at baseline and follow-up were conducted by means of Wilcoxon rank sum tests. To account for the lack of independence between observations in individual subjects, the change in HIV RNA concentrations between baseline and follow-up in the control and urethritis groups were compared with use of generalized estimating equations with a time-interaction term.
Statistical analyses were performed with SAS (version 8.e; SAS Institute, Cary, NC) and Stata (version 7.0; College Station, TX). Data were double-entered and validated with Epi Info Software (version 6.04; Centers for Disease Control and Prevention, Atlanta, GA).
Description of Participants
Of the 411 men included in the study, 206 were randomized to receive placebo and 205 to receive metronidazole (Figure 1). The mean age of participants was 26.4 years (range, 18–63). A history of urethral discharge or genital ulceration was noted for 34%. Approximately two thirds of the men reported “ever using a condom,” and about 30% reported more than one sex partner in the month preceding enrollment in the study. Many married men (40.2%) did not live full-time with their spouses.
Demographic and risk behaviors were comparable in the metronidazole and placebo groups (Table 1). However, 28.8% of men in the metronidazole group at baseline reported previous urethral discharge, as compared with 18.0% of the men in the control group (P = 0.01). Also, more men in the metronidazole group were infected with T vaginalis (45/205 [22.0%] versus 26/206 [12.6%];P = 0.01).
An etiologic agent of urethritis was identified in 79.8% of the participants (Table 2). N gonorrhoeae was detected in 73.5% of the participants and was the sole pathogen detected in 62.5% of the subjects (257/411). T vaginalis was detected in 71 subjects (17.3%), of whom 45 (11.0%) were coinfected with N gonorrhoeae. Of the 71 men with T vaginalis, 35 were positive by PCR only, 6 by culture only, and 30 by both culture and PCR. Nineteen men were Trichomonas culture–positive but negative for gonorrhea or chlamydia. Of these, 16 (84.2%) had >4 WBCs/hpf in Gram stains; in comparison, of the 83 men who were negative by Trichomonas culture and for gonorrhea or chlamydia, only 42 (50.6%) had >4 WBCs/hpf (P = 0.01).
Of the 289 men tested for C trachomatis, two were positive (0.7%), both of whom were coinfected with N gonorrhoeae. We did not detect T vaginalis, C trachomatis, or N gonorrhoeae in 60 of 289 men (20.8%) who were tested for all 3 pathogens. Genital ulcers were more common in men without gonorrhea or trichomoniasis than in men with these infections (34/83 versus 75/328;P < 0.001).
At baseline, 178 subjects (43.1%) were HIV-positive (Table 2). The RPR test for syphilis was positive for 21 participants (5.1%). HIV infection was more common among subjects with a positive RPR (prevalence ratio [PR], 1.72; 95% CI, 1.28–2.30;P = 0.007). HIV infection was present with similar frequency among persons with gonorrhea alone (PR, 1.12; 95% CI, 0.83, 1.53;P = 0.44) or trichomoniasis alone (PR, 0.60; 95% CI, 0.28–1.27;P = 0.15) but was more common among those coinfected with T vaginalis and gonorrhea (PR, 1.61; 95% CI, 1.13–2.30;P = 0.01).
Three hundred seventy-three men (90.8%) returned for follow-up, with a mean return time of 8.1 days. Men who did not return for follow-up were not significantly different than those who completed the trial (Table 1). At follow-up, 58 of 366 men (15.9%) had persistent urethritis (Table 3). In addition, 110 of 373 (29.5%) still reported symptoms (dysuria and/or urethral discharge), while 59 of 371 (15.9%) had clinician-observed urethral discharge upon examination. A total of 156 subjects (42.2%) had at least one of these three indications of urethritis at follow-up.
Effect of Treatment on Persistent Urethritis and Trichomoniasis
A key rationale for this study was the possibility that effective treatment of trichomoniasis would reduce persistent urethritis in some men. However, the proportions of men with persistent urethritis at follow-up were not different in the metronidazole and control arms of the trial (Table 3). Of those who received metronidazole, 16.2% (29 of 179) had persistent urethritis, versus 15.5% (29 of 187) who received placebo (risk ratio [RR], 1.04; 95% CI, 0.65–1.68;P = 0.96).
No difference was observed in reported symptoms (RR, 1.05; 95% CI, 0.77–1.44;P = 0.76) or clinician-observed discharge (RR, 0.90; 95% CI, 0.56–1.43;P = 0.64) between the two groups at follow-up. Among those with persistent urethritis, inflammation tended to be more pronounced in the placebo group, although the difference was not statistically significant (P = 0.10).
In multivariate analysis, adjustment for age, HIV status, previous medications, previous discharge, duration of discharge at baseline, type of discharge at baseline, WBCs/hpf at baseline, trichomoniasis at baseline, gonorrhea at baseline, RPR status, and interim sexual activity did not alter the efficacy of metronidazole on persistent urethritis (RR, 0.92; 95% CI, 0.56–1.50;P = 0.73).
Treatment with metronidazole was effective in eliminating T vaginalis. Among the subset of 36 men who were culture-positive for T vaginalis at baseline, 32 (88.9%) returned for follow-up. Of these men, 7 (21.9%) remained culture-positive (Table 3). Six of the men (46.2%) were in the placebo group and only one (5.3%) was a metronidazole recipient (P = 0.006). The latter individual reported unprotected sex after administration of treatment, suggesting the possibility of reinfection. Among those men with T vaginalis infection at baseline (as determined by PCR and/or culture) who returned for follow-up (n = 65), treatment did not affect their persistent urethritis status (Table 3).
Although elimination of T vaginalis from the urethra did not prevent persistent urethritis, detection of T vaginalis by culture at baseline was associated with an increased risk of persistent urethritis. Among men with trichomoniasis detected by culture, 32.3% had persistent urethritis, compared with 13.4% of men with no trichomoniasis by culture or PCR (RR, 2.41; 95% CI, 1.33–4.35;P = 0.006). In contrast, among men with trichomoniasis detected by PCR only, 10.0% had persistent urethritis (RR, 0.75; 95% CI, 0.24–2.27;P = 0.60).
In multivariate analysis, this relationship persisted after adjustment for treatment group, age, HIV status, previous medications, previous discharge, duration of discharge at baseline, type of discharge at baseline, WBCs/hpf at baseline, gonorrhea at baseline, RPR status, and interim sexual activity (culture-positive RR, 3.73; 95% CI, 2.08–6.69;P < 0.001; PCR-only RR, 0.80; 95% CI, 0.26–2.48;P = 0.70).
Trichomonas and HIV Viral Burden
Semen specimens were collected from men with trichomoniasis and from control subjects without an STD. Three of the five men with trichomoniasis who volunteered samples before and after treatment with metronidazole were coinfected at baseline with N gonorrhoeae; initial semen collection occurred 1 week after their treatment for gonorrhea but before treatment with metronidazole. As shown in Figure 2, men with urethritis had more HIV in their semen than control subjects (median, 35,000 versus 1800 copies/ml;P = 0.06).
Approximately 2 weeks after treatment, the median values in the urethritis group (13,000 copies/ml) and the control group (4,600 copies/ml) were comparable (P = 0.84). This change in RNA level was different for the urethritis group than for the men without urethritis (P = 0.052), implying an important treatment effect. Seven HIV-positive men in the control group had asymptomatic infection with T vaginalis. Equivalent initial HIV RNA concentrations were noted in the semen of asymptomatically infected men (median, 1700 copies/ml) and control subjects without infection (P = 0.60).
Urethritis is a common, global problem with important health consequences that include pain, epididymitis, and infertility. Infected men can transmit urethral pathogens to female partners, leading to pelvic inflammatory disease, compromised pregnancy, and neonatal health and infertility. 18 In addition, urethritis has been shown to increase the concentration of HIV in semen, as well as increase the infectiousness of and susceptibility to HIV infection. 5,6 Treatment of urethritis is usually successful if knowledge of endemic pathogens and their antibiotic susceptibilities is accurate. Effective treatment of urethritis is a public health priority in its own right but has become even more significant in light of its role as a cofactor in facilitating the transmission of HIV. 4
As a result, increasing attention has been focused on strengthening and improving all STD management schemes, especially in areas where the HIV epidemic is severe. Because a variety of microorganisms can cause urethritis, syndromic therapy tailored to endemic pathogens is critical, especially in sites where laboratory testing is limited. In Malawi, urethritis in men is managed with combination therapy directed against N gonorrhoeae, C trachomatis, Ureaplasma, and Mycoplasma species; empirical treatment for T vaginalis infection has not been established.
In the United States, chlamydia and gonorrhea are the dominant causes of urethritis, and T vaginalis is recovered from fewer than 5% of patients. 19 However, T vaginalis is more commonly recovered from men with urethritis in sub-Saharan Africa and elsewhere. 9,11–13,20–22 This organism was the most common pathogen recovered from a population of asymptomatic men in rural Tanzania, 12 and it is often second only to gonorrhea among men attending STD clinics in Africa. 21,22 In these latter studies, T vaginalis was detected without chlamydia or gonorrhea coinfection in 3% to 10% of all subjects. In 1996, 20.8% of men (37 of 178) in urban Malawi with symptoms of urethritis were infected with T vaginalis, and 7.8% (14 of 178) had no other pathogen detected.
The current study was conducted to examine three questions: (1) whether metronidazole would eliminate T vaginalis infection; (2) whether the addition of metronidazole to the current treatment regimen in Malawi would reduce persistent urethritis, which might be caused by untreated infection with T vaginalis; and (3) what the effects of therapy would be on the concentration of HIV in the genital tract. Subjects were randomized to receive metronidazole or placebo. In this study, in all infected men but one (who reported sex without a condom between visits) T vaginalis infection was cleared within 1 week after therapy with metronidazole. We also noted spontaneous clearing of 54% of culture-positive infections in the placebo group.
Kreiger reported that in 35.7% of male STD patients in the United States (5 of 14), T vaginalis infection cleared spontaneously. 23 Although detection methods varied considerably, other reports show spontaneous clearance rates ranging from 17% to 57% over the course of weeks to months. These results must be viewed with some caution, because up to 30% of Malawian men have been shown to harbor T vaginalis solely in the semen, 24 and semen is not typically collected to detect this pathogen.
Despite effectively clearing infection, the addition of metronidazole had not affected persistent urethritis at the 1-week follow-up. These results suggest that an infection or condition other than trichomoniasis accounts for persistent urethritis or that the clinical salutary effect(s) of metronidazole cannot be expected to occur only 1 week after therapy.
Persistent urethritis has often been ascribed to infections with Chlamydia, Mycoplasma, or Ureaplasma species. 25 However, Chlamydia was rarely detected in this or an earlier study in Malawi. 9 Poor adherence to doxycycline used as empirical therapy could have left ureaplasma or mycoplasma infections unresolved. However, self-reported treatment adherence did not influence the likelihood of persistent urethritis in this study.
In addition, several investigators have tried unsuccessfully to identify heretofore unrecognized organisms causing urethritis or to modify treatment regimens to deal with this problem. 25,26 In an earlier study of urethritis we detected evidence of persistent inflammation in the genital tract even 3 weeks after successful antibiotic therapy, which was ascribed to slow reduction in critical cytokines in the genital tract. 9
Consistent with the importance of unresolved inflammation, we found that men with trichomoniasis identified by culture at baseline were more than twice as likely to have persistent urethritis than men who were positive by PCR alone or negative for trichomonas by all methods, independent of other risk factors (RR, 2.52; 95% CI, 1.27–5.00;P = 0.008). We believe this finding is associated with the fact that the burden of Trichomonas was probably greater in those men who were culture-positive, leading to a more vigorous inflammatory response (i.e., a greater number of WBCs/hpf on urethral gram stain).
We and others have demonstrated that urethritis increases the concentration of HIV in semen and that pathogen-specific treatment can slowly reduce excretion of HIV. 8,27,28 In the current study, men with trichomoniasis had greater concentrations of HIV in their semen than control subjects before treatment. As expected, treatment of urethritis and/or trichomoniasis reduced excretion of HIV in semen to levels equivalent to those in uninfected control subjects.
This study was designed to determine whether metronidazole should be added to the regimen for urethritis to treat undiagnosed infection with T vaginalis in high-prevalence areas. The absence of an affect of this therapy on persistent urethritis is disappointing. However, metronidazole successfully eliminated trichomonads from the urethra and (in conjunction with syndromic management of urethritis) reduced the excretion of HIV in semen. Data from mathematical modeling suggest that the level of HIV excretion in our subjects with trichomoniasis results in a per-sex-act probability of transmission of approximately 0.009. 29 This represents a greater than 10-fold increase over the predicted probability of transmission among our controls subjects.
Given these results and the high prevalence of T vaginalis in men and women in Malawi, the addition of empirical treatment for men would likely reduce the burden of this infection and perhaps reduce the spread of HIV. The addition of metronidazole to the treatment protocol for urethritis in men in Malawi would increase the cost per patient from US $1.17 to $1.22. We believe that administration of metronidazole should be strongly considered as a part of syndromic management of urethritis in men in Malawi and in other countries where T vaginalis infection is common.
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