SEXUALLY TRANSMITTED DISEASES (STDs) are major public health problems in developing countries. The prevalence of HIV acquired through heterosexual contact is increasing in sub-Saharan Africa, including Tanzania. 1,2 STDs, and in particular genital ulcer diseases (GUDs), have been associated with an increased risk for HIV infection and transmission. 3–13
The three most common causes of GUD in developing countries are Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus type 2 (HSV-2). 5,14,15 Gold-standard laboratory tests for these organisms 14,16,17 are not often available in areas where patients with GUD are often encountered. For example, in Tanzania, only the rapid plasma reagin (RPR) card test for syphilis detection is routinely and widely available. Thus, the diagnosis of GUD is based primarily on the clinical presentation of ulceration. Since clinical diagnosis is an unreliable means of distinguishing between different GUDs, 18 accurate laboratory tests are needed to ensure correct diagnosis for selection of appropriate treatment strategies.
Studies in Tanzania have shown that effective treatment of GUD and other STDs reduced the incidence of HIV infection. 19,20 Therefore, recognition and control of chancroid, syphilis, and herpes may be important for HIV prevention. However, this requires knowledge of GUD etiology in specific settings, since the etiology of GUD varies geographically. In Tanzania, scant information exists regarding the relative distributions of H ducreyi, T pallidum, and HSV-2 infections. Most of the available data are based on seroprevalence studies of syphilis, HSV-2, and reported STD episodes. 21–23 In view of the high prevalence of HIV infection among GUD-infected individuals, 10–12 efforts should be made to use appropriate diagnostic techniques to determine the prevalence of GUD in Tanzania. The accumulated information would facilitate the management and treatment of GUD patients in Tanzania.
Use of the polymerase chain reaction assay (PCR) facilitates the laboratory diagnosis of fastidious microorganisms, thus reducing the need for expensive laboratory facilities and tests that would be required to identify these infectious agents. A PCR (M-PCR) assay has been developed that allows the simultaneous amplification of DNA from three pathogens from a single swab specimen of genital ulcer secretions. 15 Unfortunately, this M-PCR assay is not commercially available. We recently developed a PCR method that can detect all of the above-mentioned GUD pathogens. In this study, we apply this PCR methodology to identify the etiology of GUDs.
The aims of this study were to determine the etiology of GUDs and the prevalence of HIV antibodies in GUD patients at two urban STD clinics in Tanzania.
Patients and Methods
This study was conducted at two sites, Dar es Salaam and Mbeya in Tanzania. Both Dar es Salaam and Mbeya are urban municipalities; Dar es Salaam is the commercial and administrative former capital city of Tanzania. Mbeya is situated in the southwest of Tanzania, where the main transportation roads from Zambia, Malawi, Burundi, and Zaire cross, and because of the lorry drivers this involves the market for prostitution. In 1999, the prevalences of HIV-1 infection among antenatal clinic attendees in Dar es Salaam and Mbeya were 15.3% and 18%, respectively. 24
The study was conducted with the approval of the ethical committees in Tanzania. The STD clinic in Dar es Salaam is attended by patients with STD infections, whereas in Mbeya the two clinics are primary healthcare centers that also serve STD patients. A total of 134 patients with GUD attended the clinic in Dar es Salaam during the recruitment period (24 February to 20 May 1999), and 64 GUD patients attended the clinics in Mbeya during the study period there, February 1999. Enrollment was offered only in new cases of GUDs involving patients who had not taken antibiotics for at least 10 days before participation. The participants either presented with a genital ulcer at the STD clinic in Dar es Salaam or GUD was discovered during examination by a clinician at the primary healthcare centers in Mbeya, after which they were referred to the GUD research team. After informed consent was obtained, interviews were conducted with use of standardized questionnaires. Experienced STD care providers examined the patients, and clinical diagnoses were noted on the basis of the physical examination. Treatments for only bacterial STDs were provided to the patients, according to the Tanzania STD syndromic treatment guidelines. All patients were offered pre-HIV-test counseling, and those who agreed to be HIV-tested and to be given their results were given posttest counseling.
A total of 102 consecutive patients were enrolled in this study at the Ilala Municipality Central STD Clinic in Dar es Salaam (n = 52) and at the two STD/primary healthcare centers, Igawilo and Kiwanja Mpaka, in Mbeya (n = 50). Ninety-three age-matched subjects, 52 from Dar es Salaam and 41 from Mbeya, who had genital discharge but no visible genital ulcers were also enrolled at the study sites and underwent syphilis and HIV serology tests.
Blood samples were drawn from all patients, and genital ulcer specimens were collected from GUD patients. The area of ulceration was cleaned with saline, and two swab specimens were collected from the base of the biggest ulcer when multiple ulcers were present. For PCR analysis, one swab was inserted into a vial containing 1 mL PBS, compressed, and then discarded. A duplicate swab was used to inoculate a chocolate agar plate for isolation of H ducreyi and processed as previously described. 25 Specimens were stored and transported at −20 °C and analyzed by PCR at the Department of Medical Microbiology and Immunology, University of Göteborg, Sweden, for the DNA of H ducreyi, T pallidum, and HSV-2.
Preparation of Specimens for PCR Analyses
A pilot trial was performed involving the sample preparation. Clinical specimens for PCR analysis were prepared by means of two different methods. After thawing, each sample was divided into two aliquots. One aliquot was boiled for 10 minutes, cooled, and centrifuged, and the supernatant was retained. The other aliquot was treated with the reagents in the Amplicor kit (Roche Diagnostics, Branchburg, NJ) in order to extract the DNA. Samples for PCR were prepared according to the manufacturer's instructions. The results obtained with the Amplicor preparation are presented in this study report.
Two assays were used for the detection of H ducreyi, T pallidum, and HSV-2. The first PCR assay detected the target DNA of individual organisms, with use of specific primer pairs (designated S-PCR). The second PCR assay was recently developed in-house: the three primer pairs were combined for simultaneous detection of the three DNA pathogens, designated as (T-PCR). The latter assay was optimized before the clinical samples were tested. The oligonucleotide primers for H ducreyi detection amplified a 758-bp fragment of the 16S rRNA, 25,26 while the T pallidum primers amplified a 658-bp fragment of the gene encoding the 47-kDa lipoprotein 27 and the HSV-2 primers amplified a 100-bp fragment of the glycoprotein G 28 gene. All primers were purchased from Cyber Gene AB (Huddinge, Sweden), and the PCR master mix reagents were from Promega (Madison, WI). The primer sequences, the PCR mixture concentrations, and the cycling conditions used have been described previously. 25–28
All PCR reactions were performed in a final volume of 50 μl. Each PCR run included a negative control sample containing the reagent mixture and sterile PBS. The positive controls contained DNA from HSV-2 strain 333, 29H ducreyi strain CCUG 7470, and the plasmid pMN23, which encodes the 47-kDa immunogen of T pallidum30 (kindly provided by Dr. M. V. Norgard). Amplification was performed in a thermocycler (TOUCH Gene; Techne, Cambridge, UK). The PCR cycles consisted of denaturation for 60 seconds at 95 °C, annealing for 75 seconds at 60 °C, and extension for 60 seconds at 72 °C. After 40 cycles, the reactions were incubated at 72 °C for an additional 10 minutes and then stored at 4 °C until analyzed. PCR products were electrophoresed in 2% agarose gels stained with ethidium bromide and visualized with UV light. Specimens that were positive in one PCR reaction and negative in another were reanalyzed. A specimen was considered positive if the two PCR assays produced positive DNA signals or if two separate positive results were obtained from PCR with the single primer set. Since a PCR internal control for inhibition was not used, the first 20 GUD samples from each site were sent for PCR analysis at the routine laboratory of the Department of Clinical Bacteriology, University of Göteborg.
HIV antibodies in serum samples were screened with a rapid latex agglutination test (Capillus HIV-1/HIV-2; Capillus, Galway, Ireland). Reactive sera were confirmed by means of ELISAs (Behring Enzygnot anti-HIV 1/HIV 2, Behring, Marburg, Germany; and Ortho HIV-1/HIV-2, Ortho-Clinical Diagnostics, NJ). For syphilis serology, all sera were tested by RPR (BioMerieux, Marcy L’Étoile, France) and the passive particle agglutination test for the detection of T pallidum antibodies (Serodia-TPPA; Fujirebio, Tokyo, Japan).
Descriptive statistical and correlation analyses using the chi-square test were performed with the Quest computer program (University of Umeå, Sweden). To calculate the sensitivity and specificity values for the diagnosis of etiological agents, the results from the single-DNA-detection PCR were used as the reference standards.
The group of GUD patients in Dar es Salaam consisted of 38 men and 14 women with a mean age of 29.2 years (range, 18–52 years). The Mbeya group included 16 men and 34 women with a mean age of 25.6 years (range, 14–40 years). Approximately 92% of the GUD patients had received primary education, but only 6% of the Mbeya patients and 25% of the Dar es Salaam patients had received secondary education. Among men, circumcision was more common in Dar es Salaam than in Mbeya; the circumcision frequencies were 90% and 13%, respectively. In Dar es Salaam the percentages of men and women with multiple ulcers were 75% and 69%, respectively, and in Mbeya, 84% and 50%, respectively. In men ulcers were most commonly situated on the penile shaft and glans, and in women on the labia majora and perineum. In both study sites, only 7 (6%) of the 102 patients reported always using a condom, and almost half of the patients reported occasional condom use. The patients with STD but not GUD numbered 52 in Dar es Salaam (24 men and 28 women; mean age, 27.7 years) and 41 in Mbeya (12 men and 29 women; mean age, 26.5 years).
DNA Detection in Samples From GUD Patients
PCR testing by means of the two different methods revealed that 63 (62%) of the 102 ulcer specimens contained either single or multiple DNAs of HSV-2, H ducreyi, and T pallidum (Table 1). The most commonly detected pathogen in ulcer specimens from the two study sites was HSV-2. In Dar es Salaam, 10 of the 14 women (71%) and 23 of the 38 men (61%) had ulcers that were positive for HSV-2 DNA, whereas in Mbeya, 6/34 women (18%) and 11/16 men (68%) had ulcers that contained HSV-2 DNA.
The H ducreyi infection rates among women and men in Dar es Salaam were 7% and 16%, respectively; the corresponding rates in Mbeya were 3% and 25%, respectively. This result indicates that significantly more men than women were infected with H ducreyi in both cities (P ≤ 0.001). The PCR assays successfully identified five H ducreyi-positive culture specimens and seven negative ones. However, some of these negative culture plates contained contamination, particularly in Dar es Salaam, which may have caused an underestimate of the culture results.
Among the 102 genital ulcer specimens, only one sample was positive for T pallidum. Overall, 39 (38%) of the 102 ulcer specimens were negative in the PCR assay for DNA of all three pathogens.
HIV-1 Seroprevalence and Its Association With HSV-2 Infection
Serum antibodies to HIV were detected in 50 (48%) of the 102 GUD patients. The HIV infection rate in Dar es Salaam was significantly higher among women (11/14 [78%]) than among men (13/38 [34%]) (P = 0.004). The opposite pattern was observed in Mbeya, where 14 of 34 women (41%) and 12 of 16 men (75%) (P = 0.026) were HIV-infected. Tendencies similar to those mentioned above were observed among the non-GUD patients from the cities: a higher percentage of women than men were HIV-positive (11/28 [39%] and 7/24 [29%], respectively) in Dar es Salaam. In Mbeya, 13 of 29 women (45%) and 6 of 12 men (50%) were HIV-seropositive.
A higher percentage of noncircumcised men were infected with HIV than circumcised men (70% versus 30%;P = 0.003). HIV seropositivity was more common among GUD patients in Dar es Salaam and Mbeya than among the non-GUD patients (46% versus 35% and 52% versus 45%, respectively), although this finding was not statistically significant. HSV-2 was detected at significantly higher rates among HIV-seropositive than among HIV-seronegative patients with GUD in both Dar es Salaam and Mbeya (71% versus 57% and 46% versus 20%, respectively;P < 0.003) (Table 2). Furthermore, women with HSV-2 in Dar es Salaam were significantly more likely to have HIV infection than men with HSV-2 (6/10 [60%] and 9/23 [39%], respectively;P ≤ 0.006). For Mbeya, the respective figures were 4/6 (67%) and 6/11 (55%).
Syphilis serology of the GUD patients revealed that 21/102 had reactive RPR tests, and 15 of these cases were confirmed by the TPPA test. Positive TPPA results in the absence of reactive RPR test were found for 12 (26%) of the remaining 81 sera tested. Thus, 9/52 (17%) and 18/50 (35%) of the subjects with genital ulcers in Dar es Salaam and Mbeya, respectively, had serologically confirmed syphilis. The corresponding figures for the non-GUD patients were 10/52 (19%) and 6/42 (14%), respectively.
Comparison of the Two PCR Assays
Of the 102 specimens run in parallel for DNA detection by S-PCR and T-PCR, DNA was detected by both methods in 49 HSV-2 infection cases, 10 H ducreyi infection cases, and 1 T pallidum infection case. However, the T-PCR failed to detect H ducreyi DNA in two specimens and HSV-2 in one specimen, although these cases were correctly identified in the S-PCR reaction. Thirty-nine specimens were negative for all three DNA species in both PCR assays.
The PCR results for the 40 specimens sent to the routine laboratory were consistent with the S-PCR results except for one sample, which was negative in both PCRs but considered indeterminate in the routine PCR.
Comparisons Between Clinical Diagnosis and PCR Analysis Results
The clinical diagnosis and PCR results corresponded poorly for cases of genital herpes, chancroid, and syphilis (Table 3). In Dar es Salaam, 20/28 clinically diagnosed cases of herpes (71%) were confirmed by PCR, and 13 clinically diagnosed cases of chancroid or syphilis were PCR-positive. Only 2/17 chancroid cases (12%) were positive by PCR for H ducreyi. In addition, five cases that were diagnosed as herpes or syphilis were also positive by PCR for H ducreyi. Only 1/7 clinically suspected cases of syphilis (14%) was positive by PCR. In Mbeya, only 33% of clinically diagnosed herpes cases were detected by PCR. Furthermore, HSV-2 was detected by PCR in 14/16 cases (87%) in which a mixed infection with chancroid and syphilis was suspected (Table 3).
In this study, HSV-2 was found to be the most common cause of GUD among the Tanzanian patients, as detected by PCR. Genital herpes has been reported as an important cause of GUD in studies conducted in sub-Saharan Africa and South Africa, 6,13 which is in agreement with our current study. The high seroprevalence of HSV-2 among STD patients in Tanzania has been reported previously. 21 A high seroprevalence of HIV was found also in this study, which is in accordance with reports from many large cities in East and South Africa, where the HIV prevalence among STD patients currently exceeds 50%. 1,13
Patients with GUD have an increased risk for the transmission and acquisition of HIV infection. 3,7,9,11,31 Epidemiologic evidence of the role of HSV-2 infection in increased HIV infection susceptibility comes from several studies of heterosexual acquisition of HIV in African countries and in Thailand. 6,7,12,32–34 Genital herpes may act as an opportunistic pathogen in HIV-infected persons. Moreover, it has been shown that HIV infection enhances the genital shedding of HSV-2, even in the absence of clinical lesions. 35,36
A strong association was found in this study between the presence of HSV-2 DNA and HIV seropositivity. Unrecognized lesions due to herpes might allow HIV access to susceptible target cells. In the current study in Dar es Salaam, GUD due to HSV-2 was detected more frequently in females than in males. Furthermore, the prevalence of HIV infection was higher among females than males. This correlation was not observed for the Mbeya patients. Fewer HSV-2 and HIV infections were detected in women in Mbeya than in those in Dar es Salaam. However, in both cities women who had DNA to HSV-2 were more often HIV-seropositive. Several studies have previously documented a higher seroprevalence of HSV-2 infection in women than in men. 13,37,38
In Mbeya, the STD clinics were integrated with a primary healthcare facility that included antenatal care. Most of the patients attending the clinic were women, some of whom probably could not be classified in the high-risk STD group, and this might have biased the gender-related prevalence of HSV-2 and HIV infection seen in Mbeya. The low prevalence of HIV seropositivity found among these women might decrease their risk of HSV-2 acquisition or recurrent infection. Also, poor sampling might have occurred and influenced the outcome of the results, since the samples were collected by two different clinicians from the two primary healthcare clinics in the city. On the other hand, low rates of male circumcision might play a role in the higher prevalence of HIV and HSV-2 among men in Mbeya. Studies of the link between male circumcision and the risk of HIV-1 infection in men in sub-Saharan Africa suggested that noncircumcised males had an increased risk for HIV-1 infection. 39
The trend in which HSV-2 has replaced chancroid as the dominant cause of GUD has been reported in more recent studies in many African countries where the seroprevalence of HIV has increased over time. 6,8 The increase in genital HSV-2 may be due to increased rates of recurrences among HIV-infected individuals or to increased transmission of HSV-2 from HIV-infected individuals to uninfected individuals. On the other hand, decreases in the prevalence of bacterial GUD—in particular, chancroid—may be due to improved bacterial STD management as well as improved HSV-2 diagnostic tools, which may suggest a relative increase in HSV-2 as a cause of GUD.
Earlier studies of GUD patients in African countries indicated that chancroid was the most prevalent cause. DNA from H ducreyi was detected by PCR in 33% of 25 GUD patients in Mwanza, Tanzania, 40 and in 36% of patients in Antananarivo, Madagascar, 5 and 56% of those in Lesotho. 13 In the current study, the prevalence of this infection was found to be lower than that reported earlier. Both by culture and PCR analysis, proven chancroid was found more frequently in men than in women. The use of culture to diagnose chancroid, on the other hand, may lead to underdiagnoses; 33% of PCR-identified H ducreyi infections were missed by culture, because H ducreyi is difficult to grow in culture and contamination of culture plates hampers findings.
We were unable to detect T pallidum DNA in ulcer specimens by PCR, except in one case, in which antibody to T pallidum also was detected. Twenty-five of the 26 T pallidum antibody-positive specimens from patients with GUD were negative by PCR analysis. We confirmed that our PCR assay could detect the DNA of T pallidum by analyzing smears laid on T pallidum antigen prepared for the use of syphilis- and T pallidum-positive clinical samples from the routine laboratory. Although the high level of seroprevalence for syphilis could have been due to instances of treated syphilis or latent syphilis infection, it is also possible that some of these seropositive ulcer specimens may contain very few organisms that the PCR assays were not able to detect, in which case the detection level of the PCR assay may need improvement. On the other hand, we could not rule out that inhibition of the PCR reaction might have occurred. Although we did not use PCR internal controls, in almost half of the GUD samples tested in another laboratory by PCR the detection level was similar.
However, the current study has limitations, because the “gold standard” laboratory technologies such as tissue culture and dark-field microscopy for diagnosis of herpes and T pallidum infection were not used, owing to lack of local technical facilities. However, PCR and M-PCR demonstrated better results in the diagnosis of GUD in several studies in which both techniques were compared. 18,26,40
The T-PCR assay was almost as sensitive as the S-PCR assay for the detection of HSV-2 and T pallidum DNA, but it was less sensitive for the detection of H ducreyi. Currently, we have no explanation for this phenomenon. Although highly sensitive diagnostic techniques such as PCR were used, 38% of all ulcer specimens were found to be negative for all three etiological agents tested. The percentage of PCR-negative specimens was particularly high in Mbeya. The PCR-negativity of specimens may have been due to the fact that (1) the ulcers were caused by other, nontest pathogens, such as the Chlamydia trachomatis causing lymphogranuloma venereum; (2) the sample collection was inadequate; (3) the pathogens had already been eradicated by treatment; or (4) superinfecting pathogens were involved. Clinical diagnosis proved to be unreliable, and the local laboratory lacked appropriate laboratory diagnostic capabilities, which are both expensive and technically demanding.
In conclusion, the etiologies of GUD in the two cities in Tanzania showed similar patterns, i.e., genital ulcers were predominantly due to HSV-2 infection. The prevalence of HIV was high among the STD patients. HSV-2 was detected at significantly higher rates among HIV-seropositive than among HIV-seronegative patients with GUD in both Dar es Salaam and Mbeya. In addition, HIV infection was more common among noncircumcised male patients. The very high prevalence of HIV infection in patients with GUD in both Dar es Salaam and Mbeya indicates the need for urgent action. The treatment of bacterial infections with drugs that are widely used for syndromic treatment of GUD will not be effective in areas where HSV-2 is the predominant cause of GUD; therefore, efforts should be made to consider treatment for HSV-2 infection. In addition, GUD should be managed by education, encouragement of condom use, and use of algorithms based on the local prevalence and observed etiology of GUD.
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