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Understanding how our armoury works in the molecular diagnosis of sexually transmitted diseases


21 July, 2010  

Early and rapid diagnosis of STIs increases the chances to limit the effects of the disease, so a thorough knowledge of the different laboratory testing techniques is vital in planning eradication campaigns against these scourges

Jordi Bosch and Jordi Vila
Department of clinical microbiology
Hospital Clinic, IDIBAPS
School of Medicine
University of Barcelona
Barcelona
Spain

Sexually transmitted diseases (STDs), also known as sexually transmitted infections (STIs), present a major public health concern in both industrialised and developing countries. However, information about infection rates is hard to collect, especially for many developing countries. No single organisation regularly collates STI statistics worldwide and different countries have different types and levels of reporting systems. It is thought that many reports substantially underestimate the number of new STI cases because social stigma and other factors prevent people seeking healthcare.
A World Health Organization (WHO) report published in 2001 provides estimates of the extent of the world STI epidemics as they were in 1999 (previous reports were published in 1990 and 1995). As of early 2007, there are no recent international estimates. WHO estimates that 340 million new cases of syphilis, gonorrhoea, chlamydia and trichomoniasis occurred throughout the world in 1999 in men and women aged 15-49 years. The largest number of new infections occurred in the region of south and south-east Asia, followed by sub-Saharan Africa and Latin America and the Caribbean. The highest rate of new cases per 1,000 population occurred in sub-Saharan Africa.
Infection rates can vary enormously between countries in the same region and between urban and rural populations. In general, however, the prevalence of STIs tends to be higher in urban residents, unmarried individuals, and young adults. Infection caused by Chlamydia trachomatis is the most common treatable bacterial STI. It can cause serious health problems, such as pelvic inflammatory disease, ectopic pregnancy and infertility, if not treated. It is estimated that around 92 million chlamydia infections occurred worldwide in 1999, affecting more women (50 million) than men (42 million). In the 1990s, rates among pregnant women in Europe ranged from 2.7% in Italy to 8.0% in Iceland, while studies in South America found rates of 1.9% among teenagers in Chile and 2.1% among pregnant women in Brazil. Rates among pregnant women tend to be much higher in Asia: up to 17% in India and 26% in rural Papua New Guinea. In Africa, studies among pregnant women have revealed rates from 6% in Tanzania to 13% in Cape Verde.
Neisseria gonorrhoeae can infect the cervix, urethra, rectum, anus and throat (Table 1).
Gonorrhoea is a curable STI, but, if left untreated, can cause serious health problems such as infertility, meningitis and septicaemia. An estimated 62.35 million cases of gonorrhoea occurred in 1999, affecting more women than men.
Studies of pregnant women in Africa have found rates for gonorrhoea ranging from 0.02% in Gabon to 3.1% in Central African Republic and 7.8% in South Africa. In the Western Pacific in the 1990s, the highest prevalence rates (3% or greater) were in Cambodia and Papua New Guinea. Other areas such as China, Vietnam and the Philippines had rates of 1% or less. From 1995 to 1999, a significant increase in gonorrhoea incidence occurred in eastern Europe, with the highest rates in Estonia, Russia and Belarus.

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Syphilis is a bacterial infection caused by Treponema pallidum that is usually sexually transmitted but may also be passed from an infected mother to her unborn child. Syphilis is a curable STI which, if left untreated, can eventually lead to irreversible damage to the heart and nervous system. An estimated 12.22 million cases of syphilis occurred worldwide in 1999 – slightly below the 1995 estimate. In contrast to a decline in rates observed in western Europe, the 1990s saw an alarming increase in syphilis infections in the newly independent states of the former Soviet Union. There, the incidence increased from 5-15 per 100,000 in 1990 to as high as 120-170 per 100,000 in 1996.
Rates of syphilis may vary greatly within just one region. Studies of pregnant women in Africa have revealed rates of 17.4% in Cameroon, 8.4% in South Africa, 6.7% in Central African Republic and 2.5% in Burkina Faso. In the Western Pacific, relatively high rates of 8% in the South Pacific, 4% in Cambodia and 3.5% in Papua New Guinea have been reported. In 1997, studies among pregnant women in the eastern Mediterranean region showed syphilis infection rates of 3.1% in Djibouti, 3.0% in Morocco and 2.4% in Sudan.

Classical microbiological diagnosis of STI and other genital infections
N. gonorrhoeae is a cause of urethritis and cervicitis and can be detected also in the urine, pharynx or anus (Table 1). Gram stain for urethral secretions and growth in selective mediums, such as Thayer-Martin, for all samples are the techniques of choice, since ‘in vitro’ susceptibility studies are necessary due to the increase in resistance to penicillin and quinolones. C. trachomatis is also a cause of urethritis and cervicitis and can be equally detected in the urine, pharynx or anus. L1, L2 and L3 serovars cause lymphogranuloma venereum.
Cellular culture is the reference method, but it is laborious. Antigen detection techniques such as direct immunofluorescence, enzyme immunoassay or immunochromatography are easy to carry out but show poor sensitivity.
Genital mycoplasmas play a controversial role in STI. Mycoplasma genitalium can cause urethritis and cervicitis, and its growth in culture is very slow and difficult. Mycoplasma hominis can be isolated in the urethra and vagina, but its implication in urethritis and vaginitis is not clear, even though it can be a cause of salpingitis and endometritis. Ureaplasma urealyticum can be implicated in both urethritis and vaginitis and can also cause chorioamnionitis and endometritis. Both microorganisms can play a role in bacterial vaginosis. M. hominis and U. urealyticum are easily isolated in specific agar and broths and in commercial kits. Gardnerella vaginalis and Mobiluncus species (spp.) are implicated, together with different anaerobic species, in bacterial vaginosis. The cause of this dysbacteriosis is their proliferation and the decrease in normal vaginal microbiota like Lactobacillus spp. Even though a predominant vaginal culture of G. vaginalis can be significant, Gram staining is the recommended method for achieving the diagnosis of bacterial vaginosis.
T. pallidum can be detected in primary chancre and in mucocutaneous secondary lesions with dark-field microscopy or direct immunofluorescence. Nonetheless, serology continues to be the most commonly used method for the diagnosis of syphilis. Haemophilus ducreyi is the cause of soft chancre or chancroid. Its diagnosis with direct Gram stain or selective culture is difficult with little sensitivity. Klebsiella granulomatis causes inguinal granuloma, and for diagnosis, the visualisation of Donovan corpuscles in specific stains is still necessary, since the culture is very difficult.
Trichomonas vaginalis is implicated in vaginitis and urethritis. Wet mount is easy but has little sensitivity, especially if the sample has been taken some time ago and culture is expensive and slow. Candida albicans and other species of candida are responsible for vulvovaginitis and, less frequently, for balano-urethritis. Yeasts and hyphae can be observed in wet mount, but selective culture is more sensitive, and they allow the identification of species more resistant to antifungal agents, such as C. glabrata and C. krusei.
Finally, the herpes simplex virus causes genital ulcers, and can also be detected in the urethra, cervix, vagina, anus or pharynx (Table 1). Type 2 is the most frequently isolated in genital samples, but type 1 can be also isolated. Antigen detection with direct immunofluorescence or enzyme immunoassay has little sensitivity, and cellular culture is necessary, although it requires complex technology and posterior type identification.

Molecular tools applied to the detection of microorganisms causing STI
Early and rapid diagnosis of STIs increases the chance to limit the effects of the disease. Left untreated, STIs such as gonorrhoea, syphilis, chlamydia, genital herpes and human papillomavirus can lead to devastating and sometimes long-term complications, as mentioned above. Rapid and accurate diagnosis can be achieved using molecular biology methods. Most of these tools are based on gene amplification, and among these the ‘conventional’ polymerase chain reaction (PCR) and real-time PCR are the most frequently used. Some kits also use multiplex PCR to detect several microorganisms in the same reaction (Table 2).

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There are several DNA-based methods to detect microorganisms causing STI (Table 2). Overall, detection of N. gonorrhoeae by molecular biology techniques provides a presumptive diagnosis and requires confirmation by culture in areas with high rates of resistance, since antimicrobial susceptibility should be determined. These methods are considered to be the most sensitive and specific procedures for diagnosis of C. trachomatis infections. M. genitalium infection by culture is very slow and, consequently, molecular techniques are the only procedures that can provide relevant diagnostic information. For T. pallidum, molecular techniques can provide direct benefits in the diagnosis of infection, since they present a high sensitivity. Molecular methods are advisable in H. ducreyi, because of the difficulties of culture and its low sensitivity.
In genital herpes, molecular techniques have also been recommended for routine diagnosis. For other genital infections, bacterial vaginosis, vulvovaginal candidosis and trichomoniasis, diagnosis by molecular methods is poorly established (Table 2).