RealLine Chlamydia trachomatis / Neisseria gonorrhoeae

Pathogen information:

Chlamydia trachomatis is a Gram-negative, ovoid, non-motile bacterium from the genus Chlamydia. It is an obligate intracellular parasite, for which humans are the only host [1]. Chlamydia is one of the most common sexually transmitted diseases worldwide. Infections are most common among those between the ages of 15 and 25 and are more common in women than men. The young women have the highest prevalence of genitourinary C. trachomatis infections. Most infected women (more than 70%) and about 50% of infected men are asymptomatic [2]. Women infected with chlamydia are up to three times more likely to become infected with HIV, if exposed [3]. In men, the most common manifestation of C. trachomatis infection is inflammation of the urethra (about 50% of cases) [4]. The untreated infection may result in epididymitis, which in some cases can lead to fertility problems. Neonates born of infected mothers can develop inclusion conjunctivitis, nasopharyngeal infections and pneumonia due to C. trachomatis [5]. Definitive diagnosis is very important for infected person and his/her partner(s). As many people in the population are infected and the significant proportion of infection is asymptomatic, the diagnosis is often established by screening, which is recommended to perform regularly in sexually active young women, in other people from higher risk groups, and at the first prenatal visit [6]. Most C. trachomatis strains have a multicopy extrachromosomal cryptic plasmid, which is widely used as the target for PCR testing. As some C. trachomatis isolates are reported not to carry cryptic plasmid or have deletion(s) in it [7], “RealLine Chlamydia trachomatis/ Neisseria gonorrhoeae” assay kit detects two DNA fragments from gyrA gene and cryptic plasmid, specific to C. trachomatis species.

Neisseria gonorrhoeae is a species of Gram-negative diplococci non-motile bacteria from the genus Neisseria. It causes a sexually transmitted disease, gonorrhea, which remains a major global public health concern since many identified strains of N. gonorrhoeae are resistant to most available antibiotics [8]. Infection generally localizes in the genital mucosa, but can be found also in ocular, nasopharyngeal and anal mucosa [9-11]. Symptoms include purulent exudates from penis and painful urination in men, and abnormal vaginal discharge in women, although often the infection is asymptomatic. Complications from untreated genital-tract-localized infection can cause pelvic inflammatory disease, ectopic pregnancy and infertility in women, and epididymitis and infertility in men [12, 13]. Rarely, disseminated gonococcal infection develops in untreated patients resulting in infectious arthritis and endocarditis [14]. Perinatal transmission may occur during childbirth leading to neonatal conjunctivitis and blindness [15]. In the past, gonorrhea was diagnosed by a Gram stain of the patient’s purulent exudate; nowadays, nucleic-acid-based assays are used, but often the diagnosis is confirmed by culture methods [8]. “RealLine Chlamydia trachomatis / Neisseria gonorrhoeae” assay kit detects a part of PivNG (pilin gene inverting protein homolog) gene sequence, specific to N. gonorrhoeae.

RealLine Chlamydia trachomatis / Neisseria gonorrhoeae” assay kit is intended for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in human specimens: urine, semen, prostate fluid, swabs of the epithelial cells (urethral, cervical, vaginal), using the method of real-time polymerase chain reaction (PCR) with fluorescence detection of amplified product.

The extraction of DNA from clinical materials can be performed using the extraction kits:

• RealLine DNA-Express (REF VBC8899-R)
• RealLine Extraction 100 (REF VBC8896-R)
• INSTANT Virus RNA/DNA Kit – IPC16 (Roboscreen GmbH, Germany)
• INSTANT Virus RNA/DNA Kit – FX (Roboscreen GmbH, Germany)
• VeriLab UMag (Laboveritas SIA)

When using DNA extraction kits of other manufacturers, it is highly recommended to use Internal Control sample (IC) (VBC8881, BIORON Diagnostics).

The Str-format Kit is intended for use with block-type PCR cyclers: iQ5 iCycler, CFX96 (Bio-Rad, USA), and different modifications of DT-96 / DTprime, DT-48 / DTlite (DNA-Technology, Russia), RealLine Cyclers (BIORON Diagnostics GmbH, Germany).

The Str-Format Kit contains 96 tubes (0.2 ml) in strips with lyophilized Mastermix. 50 μl of extracted DNA have to be pipetted into the tube and the ready mastermix is diluted. The kit contains reagents required for 96 tests, including control samples.

The use of:

!  Extraction Kits for nucleic acids from clinical specimen from other supplier

!  other real-time PCR devices

!  appropriate reaction volumes, other than 50 µl

has to be validated in the lab by the user. The special notes regarding the internal control IC have to be strongly followed.

RUO - For research use only. Not for use in diagnostic procedures.

RealLine DNA-Express (REF VBC8899-R)

RealLine Extraction 100 (REF VBC8896-R)

INSTANT Virus RNA/DNA Kit – IPC16 (Roboscreen GmbH, Germany)

INSTANT Virus RNA/DNA Kit – FX (Roboscreen GmbH, Germany)

VeriLab UMag (Laboveritas SIA)

The kit is intended for use with block-type PCR cyclers: iQ5 iCycler, CFX96 (Bio-Rad, USA), and different modifications of DT-96 / DTprime, DT-48 / DTlite (DNA-Technology, Russia), RealLine Cyclers (BIORON Diagnostics GmbH, Germany).

Analytical specificity

The analytical specificity of “RealLine Chlamydia trachomatis / Neisseria gonorrhoeae” assay kit is ensured by the specific primers and probes. The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis.

The analytical specificity of Chlamydia trachomatis and Neisseria gonorrhoeae DNA detection is determined using the Standard Reference Panel of negative DNA extracts as percentage of samples determined by the kit as negative. Analytical specificity equals 100%.

Analytical sensitivity

The analytical sensitivity of Chlamydia trachomatis and Neisseria gonorrhoeae DNA detection is determined on five samples containing 100 copies of Chlamydia trachomatis and Neisseria gonorrhoeae DNA per sample prepared from Standard Reference Samples (Chlamydia trachomatis DNA SRS and Neisseria gonorrhoeae DNA SRS) as percentage of samples determined by the kit as positive. Analytical sensitivity equals 100%.

Evaluation

Sensitivity of Chlamydia trachomatis DNA detection was evaluated on a collection of 415 clinical samples: 200 urine samples (123 from men and 77 from women), 133 cervical swabs and 82 urethra swabs from patients with STI characteristic symptoms. Sensitivity equaled 96.6%.

CE-marked reference kit confirmed the presence of C. trachomatis DNA in the samples.

Sensitivity of Neisseria gonorrhoeae DNA detection was evaluated on a collection of 228 clinical samples: 78 urine samples (55 from men and 23 from women), 129 cervical swabs and 21 urethra swabs from patients with STI characteristic symptoms. Sensitivity equaled 98.4%.

CE-marked reference kit confirmed the presence of N. gonorrhoeae DNA in the samples.

All discordant results obtained concern samples with low content of detectable DNA fragment that falls outside the reliable detection limit of the kits.

Specificity of C. trachomatis and N. gonorrhoeae DNA detection was evaluated on a collection of 200 urine samples and mucosal swabs from healthy donors. 120 Urine samples (30 from men and 90 from women) and 80 mucosal swabs (11 from men and 69 from women) were analyzed. Obtained results were in full agreement with CE-marked reference kits.

Specificity equaled 100%.

• Instruction for use
• Molecular Diagnostic Products