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.

The Hidden-C test ® can be used by couples facing difficulty of conception, early and recurrent miscarriages and infertility of unknown etiology with very good results.  

  • Sample is representative of the whole endometrium 
  • Easy, non-invasive self-collection of sample
  • Safe storage and transport of samples by post for testing 
  • Extremely high sensitivity and specificity

The determination of endometrial status by testing menstruation tissue is an internationally patented clinical diagnostic test (European Patent 1395670). LOCUS MEDICUS SA, Athens, Greece is the only clinical laboratory authorized by the proprietor Dr. Vassilis Tsilivakos for its use.


1.   What is the Hidden-C Test ®

The Hidden-C (Chlamydia) Test ® is the molecular detection of Chlamydia or other microorganisms by real-time PCR in a few drops of menstrual blood (containing shed endometrial cells or tissue). The detection of Chlamydia in menstrual material allows the diagnosis of infection even when it is contained (“hidden”) in the upper female genital tract and as a result difficult to detect by conventional methods of testing.
Conventional testing methods for Chlamydia are characterized by very low sensitivity. At the same time, more advanced high-sensitivity, PCR-based testing methods are used on cervical/vaginal swab samples and are thus limited in testing for the presence of Chlamydia or other infections in these areas. As a result, Chlamydia infections that are localized in the upper genital tract where they are often asymptomatic can easily remain undetected which can lead to serious long-term health problems. If left untreated, chronic Chlamydia infection may lead to pelvic inflammatory disease, tubal obstruction, infertility and poor reproductive outcomes in some women. On the other hand, if detected early, inexpensive and effective treatment is available. 
The Hidden-C test ® comprises a novel, alternative, non-invasive method for investigating infectious agents on specimens representative of the whole endometrium including small quantities of lower tract mucous, which allows evaluation of both active as well as “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.
The Hidden-C test ® provides:
  • Samples representative of the whole endometrium as well as traces of the infectious and microbial load of cervical and vaginal mucous
  • Easy and convenient, non-invasive sample collection that can be performed by the patient at home
  • Samples that can be easily stored in the fridge for a long time and send by post for testing when convenient, without alteration of the result due to storage
  • Most importantly this test guarantees extremely high sensitivity and specificity
The Hidden-C test ® can be used in cases of couples with difficulty of conception, early and recurrent miscarriages and infertility of unknown etiology with very good results. The connection between infertility and Chlamydia may include tubal obstruction and inflammation, endometritis and problems of immunological etiology. Moreover, the Hidden-C test ® can be used for preventive medicine of reproductive health, while we consider its use imperative before any kind of invasive procedure or salpingography to prevent spreading of infection further up the genital tract.
Apart from detection of Chlamydia, menstrual tissue testing can also be used for detection of Mycoplasma hominis and Ureaplasma urealyticum in menstrual blood (3 in 1 test) or for microorganism detection panels relative to endometritis as well as infertility (18 in 1 test). State of the art real-time PCR testing offers extreme sensitivity and specificity for the detection of these microorganisms compared to conventional testing.
Treatment of detected infections using specialized yet simple antibiotic therapy usually results in significantly better reproductive outcomes both in cases of natural conception as well as in cases where IVF or other assisted reproduction techniques are used.

2.   The Hidden Chlamydia Hypothesis:

During the last 18 years, in the fertility clinic of LOCUS MEDICUS SA, the correlation between infectious agents and infertility is being investigated through a wide array of cutting edge diagnostic tests including reproductive immunology and molecular microbiology techniques (Real-Time PCR, Microarrays, DNA sequencing, Flow cytometry etc.). Since 1996 and after more than 15,000 cases of Chlamydia studied, among the results of this effort was an international patent for the investigation of the endometrial status through the study of shedding endometrium tissue. Part of this patent is the molecular detection of microorganisms like Chlamydia in menstruation material or “period blood”: The Hidden-C Test ®.
The discovery of this novel test was based on the following clinical observations:

C. trachomatis infections can be localized in the upper female genital tract (i.e. the endometrium and the Fallopian tubes) in which case conventional testing using cervical/vaginal swab specimens may miss such an infection unless it is also present in the cervix or the vagina.

Cells of the shedding endometriumfound in menstruation material (period blood) will contain Chlamydia originating from an upper tract infection as well as from the lower genital tract as the material flows through, so a PCR detection test performed on menstruation blood specimens will detect infections of Chlamydia (or any other clinically relevant microorganism) of both the upper and the lower genital tract.
The above observation also led to the assumption that the use of this new test would reveal a much higher Chlamydia prevalence which up until its development was concealed due to false negative results. Indeed this new diagnostic test revealed higher positivity rates for Chlamydia both in Greece and the UK than that previously indicated due to false negative results of conventional testing methods.
The advantages of the Hidden-C Test ™ include:
  • More Sensitivity
  • Economy
  • Accessibility to the population
  • A non - invasive, more acceptable specimen collection method
  • Less susceptibility to inaccurate specimen collection


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]

Women (Infection at any time)

Women (Infection in pregnancy)


Pelvic inflammatory disease



Tubal infertility

Premature rupture of membranes


Ectopic pregnancy

Low birth weight

Sexually-acquired reactive arthritis

Sexually-acquired reactive arthritis

Transmission from mother to baby leading to ophthalmia neonatorum and atypical neonatal pneumonitis

Low sperm quality, male-factor infertility,early pregnancy failure and recurrent miscarriage [7,8]


 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.

The positivity for C. trachoma-tis in cervical-vaginal swab specimens was 13% by culture/DFA or 18% for the same specimens analyzed by real-time PCR.  The highest positivity was observed when the same women collected menstrual tissue samples themselves, followed by real-time PCR analysis using a commercial C. trachomatis detection kit (25%), or an in-house PCR protocol (37%).

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 ®

Out of 883 UK and Greek women with infertility backgrounds tested for C.trachomatis using PCR testing on menstruation tissue (HIDDEN-C Test ™), 347 (39%) were found positive.


 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]




Diagnostic method

·       Nucleic acid amplification tests (NAATs)

·       polymerase chain reaction (PCR)

·       Strand displacement amplification [SDA]

·       Transcription-mediated amplification [TDA]

·       High sensitivity (90–95 %)

·       Can be used on urine samples and vulvovaginal swabs (including self administered tests)

·       Validated for extragenital sites, including rectum

·       Expensive

·       False positive results may be a problem in some settings

·       Not licensed for extragenital sites

·       EIA (enzyme-linked immunosorbent assay)

·       Can be adapted for point-of-care tests

·       Cheap

·       Low sensitivity (40–70 %)

·       Not appropriate for urine and  self-collected swabs

·       Cell culture

·       Can be used on all specimen types

·       High specificity

·       Low sensitivity (60–80 %)

·       Expensive – requires technical

expertise and is labour intensive

·       Not suitable for large through-put

·       Direct fluorescent antibody

(DFA) tests

·       Can be used on all specimen types

·       Rapid turnaround time

·       Low sensitivity for urine

·       Labour intensive

·       Requires expertise


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. 




Specimen collection

·       Clinician-obtained

·       Ability to obtain good quality sample, e.g. endocervical swab which may increase sensitivity

·       Less acceptable to some patients

·       More expensive in staff time

·       Self-collected

·       More acceptable to some patients

·       Less clinical facilities required

·       May be less sensitive


·       Point-of-care tests (administered by healthcare professionals)

·       Treatment can be offered at same time as diagnosis, so no need for patient recall

·       Currently all EIA-based, therefore less sensitive than NAATs

·       Over-the-counter, self-administered tests

·       May be more acceptable and accessible for some groups

·       Reliability of method needs to be assured — currently few quality controls

·       Postal tests

·       Patients can take samples in their homes

·       Tests are carried out by laboratory

·       Good regulation and quality control


·       Need to be linked to access to treatment

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.



  1. Adapted from ECDC TECHNICAL REPORT, Review of chlamydia control activities in EU countries, Stockholm, May 2008 and ECDC GUIDANCE, Chlamydia control in Europe, Stockholm, June 2009)
  2. Low N. Current status of chlamydia screening in Europe. Euro Surveill. 2004;8.
  3. Cates W, Wasserheit JN. Genital chlamydial infections: epidemiology and reproductive sequelae. Am J Obstet Gynecol. 1991;164:1771-81.
  4. Adams EJ, Turner KM, Edmunds WJ. The cost effectiveness of opportunistic chlamydia screening in England. Sex Transm Infect. 2007;83:267-74.
  5. Peipart JF. Clinical practice. Genital chlamydial infections. NEJM. 2003;349:2424-30.
  6. Hammerschlag MR. Chlamydial infections in infants and children. In: Holmes KK, Mardh PA, Sparling PF, editors. Sexually transmitted diseases. 3rd ed. New York: McGraw Hill; 1999. p. 593.
  7. AD Gritzapis et al., J Antivir Antiretrovir 2014, 6:2 (4th World Congress on Virology October 06-08, San Antonio, USA, 2014
  8. AD Gritzapis et al., Andrology October 2014, 2:2 (Proceedings of the 8th congress of  European Academy of Andrology), ECA Barcelona, Spain 2014
  9. Fleming DT, Wasserheit JN. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect. 1999;75:3-17.
  10. Adapted from ECDC GUIDANCE, Chlamydia control in Europe, Stockholm, June 2009
  11. Michou et al. (2014). Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility. J Obstet Gynaecol Res. Jan;40(1):237-42. doi: 10.1111/jog.12165. Epub 2013 Oct 7
  12. Tables 2a, b and c were adapted from European Centre for Disease Prevention and Control (2009) ECDC Guidance: Chlamydia control in Europe, Stockholm, June 2009, ISBN 978-92-9193-165-1, doi 10.2900/1136
Corresponding author Dr. Panagiotis Venieratos PhD, Molecular Biologist, Locus Medicus SA, Athens, Greece
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The successes of the department of Investigation on Female and Male Infertility established, early on, this particular department and then the gynaecological department of the center.


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