Establishment of a two-dimensional PCR method for simultaneous detection of nine sexually transmitted disease pathogens: insights into coinfection rates and epidemiological trends in HPV screening

Abstract

This study developed a two-dimensional PCR (2D-PCR) method for simultaneous detection of nine sexually transmitted disease pathogens (STDPs) in cervical brush samples collected after human papillomavirus (HPV) screening, aiming to evaluate co-infection rates and epidemiological trends. The 2D-PCR assay was optimized under single-tube closed conditions, with sensitivity and accuracy validated. The prevalent nine STDPs were assessed in 2,193 females undergoing routine gynecological inspections and HPV screening. Statistical analysis revealed correlations between HPV genotypes and identified pathogens. Results showed detection limits (LODs) of 10²-10³ copies/μL for STDPs, with high concordance to triplex real-time PCR (Kappa = 0.90). Overall, 48.6% of samples tested positive for ≥1 pathogen, with 36.02% positive for ≥1 STDP. The most prevalent STDPs were Ureaplasma parvum/Ureaplasma urealyticum (27.04%), Mycoplasma hominis (3.42%), and Trichomonas vaginalis (0.23%). HPV-positive individuals exhibited higher STDP infection rates (46.32%) than HPV-negative counterparts (32.86%). Significant associations were observed between HPV infection and U. parvum/U. urealyticum or M. hominis. HPV 52 was the predominant genotype in STDP-infected individuals (6.2%), with genotypes 52, 53, 6, 11, 42, 43, and 61 significantly more prevalent in STDP-positive groups. The 2D-PCR method proved sensitive and specific for multiplex STDPs detection, while epidemiological data highlighted significant STDP-HPV correlations.

Importance: This study introduces the first single-tube 2D-PCR method for efficient, high-throughput detection of nine STDPs, addressing a critical gap in co-infection diagnostics. The high prevalence of STDPs and their strong association with human papillomavirus (HPV) infection underscore the clinical relevance of co-pathogen screening, particularly in HPV-positive populations. The significant linkage between U. parvum/U. urealyticum, M. hominis, and HPV infection suggests potential synergistic mechanisms influencing HPV persistence or progression. Notably, the predominance of HPV 52 and increased prevalence of genotypes 53, 6, 11, 42, 43, and 61 in STDP-infected individuals highlight how pathogen co-infections may modulate HPV genotype distribution. These findings provide a robust tool for integrated STDPs/HPV screening and offer epidemiological insights to guide targeted prevention strategies, ultimately enhancing management of sexually transmitted infections and HPV-related cervical disease.

Keywords: 2D-PCR; HPV; co-infection; epidemiology; sexually transmitted diseases.

Fig 1

(A-C) Melting curves from mixed plasmids in FAM (A), HEX (B), and ROX (C) detection channels. (D-F) Clinical samples show co-infections with M. hominis & T. vaginalis (D), C. trachomatis (E), and N. gonorrhoeae & U. parvum/U. urealyticum (F).

Fig 2

Melting curves of pathogen-specific plasmids: M. hominis (A), M. genitalium (B), N. gonorrhoeae (C), T. vaginalis (D), HSV-1 (E), C. trachomatis (F), HSV-2 (G), U. parvum / U. urealyticum (H) at serial dilutions (10⁵–10¹ copies/μL).

Fig 3

(A) Pie chart showing distribution of pathogenic microorganisms in 790 STDPs-positive patients. (B) The age distribution curves of the three most prevalent STDPs (U. parvum/U. urealyticum, M. hominis, and T. vaginalis) and HPV-positive individuals.