Development of multiplex Real Time PCR and high-resolution melting assay for detection the sexually transmitted pathogens in a sample of Iraqi patients

Abstract

Sexually transmitted infections consider the main general health trouble in the world which occasion of severe sickness, long-period complications, medical, mental sequels, infertility, and mortality. The development of new methods for detection bacteria and viruses related with sexually transmitted diseases (STDs) is very important for control and management of these infections. The collection of study samples has taken place at the period collected between September and November 2021 in Baghdad, Iraq, from two of specialized Hospitals and one private hospital, it has included 150 clinical specimens from married couples patients (75 cervical swabs from females and 75 seminal fluids from males), also 50 samples from healthy control (25 couples), during this period, from both gender with age ranging from 18 to 45 years.  The current study was conducted to detect sexually transmitted infectious agents (bacteria and viruses) by developing HRM (high resolution melting) assay with multiplex real-time PCR using of set primer of each pathogen that was designated by professional software program (Beacon Designer™ automates the design of real time primers and probes) for the accurate detection of these infections in Iraqi patients. This study conducted for detection seven different sexually transmitted pathogens (3 bacteria, 3 viruses, and one parasite) among 150 suspected patients as married couples. The results of RT-PCR with HRM assay of the positive controls  demonstrated different melting curves, where each melting peak represent the amlification of one of the seven pathogens, also the melting peakof the internal control (housekeeping gene). This experiment included three panels, the panel one represent the optimal melting peak for the positive control of Trichomonas vaginalis, Mycoplasma hominis, Cytomegalovirus the melting temperature each of them was )80.0℃, 84.90℃ and 88.0℃) respectively. The panel 2 ncluded the melting peak for the positive control of Human Papilloma Virus, Neisseria gonorrhoeae, and Herpes simplex virus, the melting temperature for each of them was) 79.6℃, 84.75℃ and 87.75℃) respectively. The panel 3 showed the melting curve for the positive control of Chlamydia trachomatis and internal control (Homo sapiens hemoglopin subunit beta (HBB) gene), where the melting temperature was) 79.35℃, and 88.65℃) respectively. The results of STDs detection revealed that the frequency of the sexually transmitted infections (bacteria, viruses and T. vaginalis) among 150 of married couple's patients in some of Baghdad hospitals was 108 from 150 (72%).  The virus's infections were predominant in comparison with other infectious agents. Out of 108 infections, the outcomes showed the prevalence of virus infections in STDs patients was 63% (68/108), while the bacterial pathogens was 31.5% (34/108) and T. vaginalis infections was 5.5% (6/108). The predominant virus in all patients was CMV (31.5%), followed by HSV (24.1%), while HPV was only 7.4%. In bacteria, the high prevalent species was C. Trachomatis with 14.8%, followed by M. hominis (9.3%) and N. gonorhoeae (7.4%). According to the age groups, it was found that the viral infection frequency was higher than other agents in all age groups, where the age group 36-45years was the highest percentage (27.8%). The age group 36-45years was also exhibited the highest frequency for all agents with (41.7%).