Introduction Syphilis is an important public health problem in Paraguay. A 2006 study found high levels of syphilis (19.0%) infection and unprotected sex with most recent client (also 19.0%) and moderate level of HIV infection (1.8%) in female sex workers (FSW). Several HIV and STI prevention interventions have been put in place since then. A recent study sought to assess current infection and risk behaviours.

Methods 432 FSW were recruited from sex work locations in the greater metropolitan area of Asuncion using time-location sampling between January and May, 2012. A face-to-face survey and rapid and confirmatory biological tests in accordance with national algorithms assessed risk behaviours and syphilis and HIV infections. Weighted statistical analysis accounted for the sampling strategy and adjusted for clustering by recruitment venue. Adjusted 95% confidence intervals (CI’s) for the 2006 and 2012 studies were compared to identify significant trends.

Results Median age of participants was 25 years (interquartile range (IQR), 21–32). Median age of first sex work was 20 years (IQR 18–24). Differences in VIH prevalence among 2006 (1.8%) and 2012 (2.5%%) studies weren´t significant (P = 0.4). Prevalence syphilis (25.3%, IC 21.3%–29.8%) were higher than 2006 estimates (P < 0.05)).Median number of clients during the past week was 15 (IQR 8–25). 40.5% (CI 35.8–45.3) reported drinking at last sex with a client. Condom use with last client was 98.1% (IC 96.2%–99.1%), increased over 2006 (P < 0.05) Among 59.3% who had a stable partner, 77.3% (IC 71.4–82.0) had not used a condom at last sex with a stable partner.

Conclusion Increasing levels of syphilis infection suggest a need to reevaluate and intensify STI prevention and control interventions, with greater emphasis on diagnosis and treatment, reducing drinking with clients as well as providing alternatives to sex work for FSW.