The Hidden-C (Chlamydia) Test ® comprises a novel, alternative, non-invasive method for the detection of Chlamydia or other infectious microorganisms by real-time PCR in a few drops of menstrual blood. The detection of Chlamydia in menstrual material allows the diagnosis of both active and “silent” Chlamydial infections with extreme sensitivity even when the infection is contained (“hidden”) in the female upper genital tract in a dormant state and consequently it is difficult to detect by conventional methods of testing.

1.   What is the Hidden-C Test ^®

2.   The Hidden Chlamydia Hypothesis:

3.   Why chlamydia is a public health problem ^[1]

Genital infection with Chlamydia trachomatis (commonly known as ‘chlamydia’) is the most common bacterial sexually transmitted infection in many European countries ^[2]. Rates in sexually active young people are commonly between 5 % and 10 % (in the general population through conventional testing). The number of diagnosed cases is increasing, in part due to increased testing and the use of more sensitive tests.

People with genital chlamydia may experience symptoms of genital tract inflammation including urethritis and cervicitis, but the majority remains asymptomatic. Chlamydia is a significant public health problem because untreated chlamydia may lead to pelvic inflammatory disease, infertility and poor reproductive outcomes in some women. The cost of treating infertility due to chlamydia is high as it requires tubal surgery and in-vitro fertilization. In spite of the fact that inexpensive and effective treatment is available, control of chlamydia is challenging since most people are asymptomatic. Though the infection often causes no symptoms, it can have severe long-term consequences in a proportion of cases. In women, C. trachomatis that ascends from the endocervix to the upper genital tract can cause pelvic inflammatory disease (PID), which can result in scarring and adhesions in the Fallopian tubes and adnexae. This increases the risk of ectopic pregnancy, tubal infertility and chronic pelvic pain ^[3]. In the UK it has been estimated that 64,000 cases of PID and 3,000 ectopic pregnancies each year are attributable to chlamydial infection, although the evidence for these statements is weak. These complications cause considerable distress to the individuals and, in the case of infertility, have major cost implications for health services ^[4].

Chlamydia is likely to be the commonest preventable cause of such reproductive tract morbidity. Chlamydia has been reported to account for up to two thirds of cases of tubal infertility and a third of ectopic pregnancies ^[4]. Infection during pregnancy is associated with premature rupture of the membranes, low birth weight and miscarriage^[5]. Chlamydia can also be transmitted from mother to baby during labour, causing eye and respiratory infections ^[6]. 

In men, chlamydia can lead to acute genital inflammation (epididymitis, epididymo-orchitis) and occasionally to sexually-acquired reactive arthritis (SARA). 

More importantly, our studies have shown that in men intracellular Chlamydia infections (among other microorganisms) can compromise sperm quality, while the presence of pathogenic microorganisms inside spermatozoa and subsequently in the resulting zygote, make the developing embryos immunogenic and may initiate immunologic rejective procedures in the implantation site from the beginning of gestation leading to early  and recurrent miscarriages (early  miscarriages are often confused with inability to conceive) ^[7,8]. This may lead to male-factor infertility, early pregnancy failure or recurrent miscarriage (both after natural conception and assisted reproduction procedures) due to sperm-originating pathogens. For detection of intracellular pathogens in sperm we have recently developed a new patented test by the trade name SPI Test ™ (Sperm Pathogen Immunophenotyping), exclusively used by our lab which can distinguish and pinpoint intracellular infections inside spermatozoa of the male partner ^[7,8].

Finally, in men and women chlamydia may produce proctitis while individuals with chlamydia are at increased risk of acquiring or transmitting HIV ^[9]. 

Table1: Complications of Chlamydia ^[10]

 4.   Scientific data supporting the HIDDEN-C Test ^®

Use of high sensitivity diagnostics, is essential in order to accurately estimate the prevalence of Chlamydia at a local as well as at an international level. This becomes even more important when the selection of candidates for screening is based on prior positive diagnosis such as in the case of partner notification (In this case, a false negative for a woman could potentially equal to two or more missed positive diagnoses). 

In one international publication, we compared the sensitivity of 3 different diagnostic methods used for C. trachomatis detection, in a group of women with a background of infertility [11].  In the overall study group, the prevalence of C. trachomatis was 25.3% for the menstrual tissue samples using PCR (the HIDDEN-C Test ^®), 18.3% for cervicovaginal secretions using PCR and 13.8%, for cervicovaginal secretions by culture/DFA, respectively (Fig.1). A statistically significant difference was revealed between the two methods (PCR or culture/DFA) and between menstrual tissue and cervicovaginal secretions PCR for chlamydia. 

Clearly, highest positivity of Chlamydia was detected by the Hidden-C Test ^® when the women of the study group self-collected menstrual blood samples at home.

In the above publication, for the PCR protocol, commercially available C. trachomatis primers were used. Incredibly, when we performed the Hidden-C Test ^® with an in-house developed C. trachomatis detection kit, Chlamydia positivity increased to 37% for the same study group.  

Fig.1 C. trachomatis positivity by different specimen collection and C. trachomatis detection methods in a group of subfertile women.

Similar positivity (~39%) was observed when different groups of women (883 women in total) with infertility backgrounds, were tested by the method described above, over a period spanning May 2010 to Feb 2011 (Fig.2). Furthermore, the same positivity was observed in menstrual tissue specimens collected from UK infertility clinics during the same time period (of the 883 specimens tested, the 150 were sent from the UK).

Fig.2 Number of UK and Greek samples positive for C. trachomatis infection compared to total number of samples tested, using the HIDDEN-C Test ^®

 5.   The clinical significance of the Hidden-C Test ^®

According to recent data a number of commensal infectious agents (microorganisms) that until now, were traditionally considered obligatory pathogenic could be considered part of the normal flora of the human body. It seems that Chlamydia, which under specific circumstances can create health problems, are much more common than we originally thought. Furthermore, according to our clinical experience, although infrequent, Chlamydia infection is in some cases possible by non-sexual transmission as well.

Unfortunately, there are no official guidelines for estimation of the potential health risk of a Chlamydia infection according to sex, age or reproductive status, while the same is true for guidelines concerning recommendations on severity of treatment personalized for each patient. It is, however, safe to suggest, that Chlamydia testing should at least take place before any invasive procedure or salpingography to minimise the risk of spreading the infection throughout the genital tract. In addition, it is our strong belief that Chlamydia testing should be performed for preventive screening, before a chronic infection leads to irreversible or difficult to cure reproductive (or other) health problems.

Up until the development of the Hidden-C test ^®, there wasn’t a satisfactory diagnostic test for the detection of Chlamydia. Diagnostic approaches that indicated the presence of Chlamydia indirectly such as the Pap test or other traditional, low sensitivity tests, to a large extend misinformed the general public in regard to the prevalence of Chlamydia, due to a high occurrence of false negative results. After 18 years of experience in the field of reproductive medicine, we finally developed this test as the method of choice for the study of the female genital tract through microbial DNA detection by Real Time PCR in menstruation tissue samples. 

This new test has been greatly received by the general public both in Greece and internationally. Moreover, the Hidden-C ^® Test appears to be the test of choice for young women for infertility prevention reasons as well as part of infertility investigation testing for couples with difficulty of conception or recurrent miscarriages. 

The Hidden-C Test ® reveals a positivity rate (positive diagnosis) for the presence of Chlamydial infection of about 37% in samples investigated for infertility, in Greek and UK population.

Furthermore, we propose that men should also be tested for Chlamydia (among other microorganisms shown to adversely effect sperm quality), as it can lead to chronic epididymitis and to deterioration of sperm characteristics of the spermiogram. In these cases, the presence of Chlamydia in semen should be taken into account together with the parameters of a spermiogram, as far as antibiotic treatment is concerned. Moreover, the microbial factor should be tackled first during treatment of oligo-, astheno- or teratospermia, especially prior to surgical intervention such as a varicocele correction or aspiration of testicular parenchyma. Consequently it is imperative that men too should be tested through high sensitivity/high specificity methods. Addressing this need, we recently developed a novel, award winning, internationally patented test (the SPI Test ™) that for the first time allows, detection of harmful pathogens inside sperm cells, with unparalleled sensitivity and specificity. This test can explain bad spermiogram parameters and allow etiological treatment, specifically aiming at the problem. 

6.   The Hidden-C Test ™ Advantages: 

a) High Sensitivity

It is particularly important that the method is characterized by high sensitivity and specificity, which minimize false negative and false positive results, respectively. The sensitivity of cervicovaginal secretion culture/DFA, the conventional method of testing is very low (Table 2). The otherwise highly sensitive methods such as NAATs, have also smaller chance of detecting endometrial microbes when performed on cervical fluid specimens (45-60%). On the other hand, the correct specimen type used by the Hidden-C Test ^® combined with the extremely high sensitivity of state-of-the-art Real Time PCR, exceeds >95% sensitivity for screening the endometrium for the presence of microbes.

Table 2: Advantages and disadvantages of Chlamydia detection methods ^[12]

b) High quality, easy to collect and c) inexpensive to deliver samples

The Hidden–C Test ^® is a more sensitive method, compared to fine needle biopsy samples due to the fact that menstruation tissue is much more representative of the endometrium and it also contains cervical and vaginal material as it courses through the female genital tract. It is also very easy to extract sufficient quantity of material. In this aspect, this method is superior compared to other invasive techniques. Women, avoiding any gynecological examination stress can collect the sample themselves at home and repeat it if the first attempt fails. Obviously, a very important advantage of our method is that it can be easily repeated and thus antibiotic therapy results monitored. 

Specimens for the Hidden-C Test ^® are self-collected by patients and are then sent to the laboratory by post. This provides the most accessible and acceptable method for the patient, while it does not require expensive facilities or trained staff. Furthermore, specimen collection is completely passive (the blood drips into the container), always guaranteeing a good quality sample (the patient cannot make a mistake collecting the sample) and ensuring reproducibility of results.

The Hidden-C Test ^® is the only test that combines Sensitivity, Economy, Ease & Convenience, Speed and Zero Rejection due to specimen inadequacy.