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TOPICAL PROBLEMS OF DIAGNOSIS AND Ws TREATMENT OF UROGENITAL INFECTIONS f
LABORATORY DIAGNOSTICS OF REPRODUCTIVE INFECTIONS: PROBLEMS AND WAYS OF SOLVING THEM
A. Savicheva (savitcheva@mail.ru)
D. O. Ott Research Institute of Obstetrics and Gynecology RAMS, St. Petersburg, Russia.
■ The paper is devoted to the main problems of microbiological diagnostics of infections that influence reproduction. Long-term investigations in the Laboratory of Microbiology at the Ott Institute and the joint Russian-Swedish project “The improvement of quality in diagnostics and treatment of sexually transmitted infections (STIs)” carried out in St. Petersburg and the Leningrad Oblast allowed an algorithm of the microbiological examination of patients with urogenital and transplacental infections.
Of special importance here is the use of microscopic investigation by a physician in the outpatient setting with the aim of early diagnostics and timely treatment of some urogenital infections.
Introduction
The demographic situation in Russia remains difficult. The latest documents of the Russian Federation Government and of the Ministry of Public Health and Social Development indicate infectious morbidity, especially with urogenital infections, is the main cause of deterioration of the population’s reproductive health. Qualified and timely diagnostics of not only urogenital infections but also all other infections that influence reproductive health are one way of reducing the morbidity rate. Reproductive infections include genital infections with most frequently occurring STIs and transplacental infections that are of importance only in pregnant women.
Up-to-date etiologic diagnostics of reproductive infections is the basis for early therapy and for informing patients about peculiarities of disease spreading and their consequences for both sexual partners, and in case of pregnancy, about a possible disease in the fetus or newborn infant.
In the diagnostics of reproductive tract infections microbiological investigations are of utmost importance. The successful treatment of these infections is highly dependent on the accuracy of the etiological diagnosis. Microbiological methods of diagnostics facilitate correct diagnosis if a physician is aware of the disease to be diagnosed, properly collects adequate material for investigation, knows the composition of normal urogenital flora and is knowledgeable on how to assess the results.
During the past decade (1998-2007) in the Leningrad Oblast and during the past 6 years (2001-2007) in St. Petersburg, a Russian-Swedish project (“The improvement of quality in diagnostics and treatment of sexually transmitted infections, STIs)” has been running. The WHO Centre on STI at Uppsala University (Sweden) and the D. O. Ott Research Institute of Obstetrics and Gynecology of the RAMS (St. Petersburg) have participated in this project. The research is carried out under the aegis of the Public Health Committees of the Leningrad Oblast and St. Petersburg.
Within the framework of the project, a methodology of a two-level diagnostics of reproductive infections has been developed. In the first level a physician evaluates the physiologic state of the patient, examines and microscopically investigates vaginal, cervical, urethral (female and male) smears. The second level involves a laboratory investigation aimed at specific diagnosis by determining the most likely etiologic agent of the infection.
The first level is very important for both the physician and the patient. At the first patient visit, the physician in the patient’s presence assesses the state of urogenital tract mucosa using elementary skills in microscopic study and determines the strategy of the patient’s management (i.e. prescribes treatment and, if necessary, sends the clinical material to the laboratory for special investigation to reveal definite causative agents).
The aim of the present paper is to review the main methods of microbiological diagnostics of reproductive infections.
Materials and Methods
To reveal the presence of urogenital infection agents the material is taken from the urethra in males and from the vagina, urethra, cervix in women: the first portion of first void urine mostly in males. In order to look for transplacental infections venous blood is studied for the presence of specific antibodies.
The main methods of investigation include microscopic, cultural (inoculation), nucleic acid amplification tests (NAAT: PCR and others) and serological (ELISA).
Results
Normal microflora of the urogenital tract.
Out of the great number of various microorganisms in male and female genital tracts, only a few actually cause disease. Pathogenic microorganisms, such as Treponema pallidum, Neisseria gonorrhoeae, Haemophilus ducrei, Chlamydia trachomatis and Mycoplasma genitalium, as well as protozoon Trichomonas vaginalis upon their detection, require the specific therapy to be initiated. There is a large group of facultative pathogenic microorganisms that can cause disease under specific conditions: these are mycoplasmas (Mycoplasma hominis, Mycoplasma fermentans), ureaplasmas (Ureaplasma urealyticum, Ureaplasma parvum), Enterococcus, enterobacteria, staphylococci, streptococci, Gardnerella vaginalis, yeast-like fungi (Candida spp.), etc. The same microorganisms may be part of genital normal flora.
Microscopic methods of diagnostics. The diagnostics of reproductive tract infections should begin with the assessment of genital microbiocenosis and the quantity of leucocytes, epithelium, the ratio of leucocytes to epithelium, mucus and bacterial morphotype. Optimally, the clinician who collects the genital material from the patient and performs pelvic examination should perform the microscopy as well. A specially trained physician using the method of bedside microscopy of native and stained smears can estimate an inflammatory reaction: find the “clue cells”, mycelium of yeast-like fungi and determine motile Trichomonas vaginalis [1, 2, 3]. The physician’s appraisal of inflammatory reaction on the basis of a physical examination and the microscopic study allows him/her to take adequate material and direct it to the laboratory for more specific testing.
Diagnostics of bacterial vaginosis, candidiasis and trichomoniasis. Bacterial vaginosis is diagnosed according to Amsel’s criteria on the basis of the clinical and microscopic pattern. During the microscopic examination, attention is drawn to numerous cast-off cells of the multilayer epithelium with bacteria adherent to them (“clue cells”) and to a low number of leucocytes (the ratio of leucocytes to epithelial cells is < 1:1). It is important to keep in mind that the association of microorganisms that can be found in bacterial vaginosis may include spp. of Gardnerella, Mycoplasmas, Ureaplasmas, Mobiluncus genus vibrio, anaerobic cocci and anaerobic bacteria [6, 8, 12].
Urogenital Candida infection has a typical clinical presentation and its detection in the laboratory is mainly done by microscope, i.e. detecting mycelium and/or budding forms of yeast-like fungi.
Microscopic and cultural methods are used to diagnose T. vaginalis infection, which are identified as motile forms that are easily observed when studying native (wet) smears [6, 8, 12].
Diagnostics of gonorrhea. At present, microscopic examination of women using Gram-stained materials taken from the urethra or cervical canal is not sufficient to diagnose gonorrhea. The reason for this is that the sensitivity of this method is only 30 - 50 %. However, in males with clinical signs of urethritis this method is highly sensitive and specific. Our investigations demonstrated that there is a need in revising the list of methods recommended for gonorrhea diagnostics, especially regarding the screening of this infection. Moreover, improvement of the quality of growth media being used for the cultural diagnostics of gonorrhea is required [13].
Diagnostics of genital chlamydial infection. For the diagnostics of infection caused by Chlamydia trachomatis cultural, direct immunofluorescent (DIF) and NAATs are used. These techniques are intended to isolate the etiological agent or find its antigens or DNA/RNA.
For a long time, the cell culture (CC) method for the diagnostics of chlamydial infection was considered the “gold standard” in comparison with the immunofluorescence method. Presently, the method of cell culture remains the “gold standard” by its specificity; however, it is less sensitive than molecular-biological methods. According to our data, the sensitivity of the cultural method is 40 - 60 %. In order to avoid disputable cases when the method of cell culture gives a negative result while another method gives a positive result, some authors suggest the use of one additional method — extended “gold standard”. The result is considered positive if the cultural method or two methods show positive results [11, 12].
Recently, new methods, such as real-time PCR and Nucleic Acid Sequence-Based Amplification (NASBA) in real time [5] have been introduced to diagnose urogenital chlamydial infections.
The detection of genital mycoplasmas. To reveal genital mycoplasmas it is necessary to use the cultural method and real-time PCR. These methods allow the determination of the number of mycoplasmas in the material being studied (for Ureaplasma spp. and Mycoplasma hominis). It should be emphasized that exclusively NAATs (PCR and real-time PCR) are able to detect Mycoplasma genitalium. Concentration of mycoplasmas more than 104 microbial bodies in 1 ml or 1 g of the material may be of diagnostic value, particularly in the presence of a concrete disease such as vulvovaginitis, cervicitis in females and urethritis in males. Lower concentrations should not be taken into account because a low number of mycoplasmas (besides pathogenic species) may be found in healthy persons. It is inadmissible to use DIF for detection of genital mycoplasmas and serology for the detection of antigens.
The detection of human papilloma virus (HPV). The problem of inclusion of the HPV test into the pri-
mary screening of cervical carcinoma is discussed as the primary cytological screening underestimates the severity of a disease in 30 % of women. All recommendations on using the test for human papilloma virus in cervical carcinoma screening are based on the fact that cervical lesions of high degree and cancer do not develop in the absence of the virus. The necessary condition for the development of cervical carcinoma is a lasting persistence of a high viral load with virus of a particular type. For the detection of HPV and virus concentration, real-time PCR is used [4].
Diagnostics of transplacental infections. Causative agents of transplacental infections are Listeria monocytogenes, Treponema pallidum, Toxoplasma gondii and viruses (rubella, cytomegalia, 1st, 2nd type herpes, 3rd type herpes — Varicella zoster, measles, parvovirus B19 and others). It should be noted that the detection of this or that infection in pregnant women does not always mean it will be transmitted to the fetus. It is known that Listeria is transmitted to the fetus in 100 % of primary infections during pregnancy, C. trachomatis in 50 - 70 %, group B streptococci in 37 %, Mycoplasma spb. in 30 % and cytomegalovirus in 50 % [7, 10].
For diagnostics of these infections, there are known methods as described above. For diagnostics of infections such as toxoplasmosis, cytomegalia and rubella, these methods use the detection of antibodies of different classes in maternal blood serum. The detection of acute infection arising for the first time during pregnancy is of great importance for pregnant women. Therefore, when planning pregnancy a woman should be examined for the presence of only specific IgG showing the state of immunity. At the first visit of a pregnant woman in the women’s dispensary as well as in the case of contact with these infections, blood serum should be examined for the presence of IgG, IgG-avidity and IgM [9].
Conclusions
To diagnose reproductive infections different microbiological methods are used. For this it is important to choose a method that satisfies all quality standards, is exceedingly sensitive and highly specific. Parameters of diagnostic sensitivity and specificity should be more than 95 %.
Results of our investigations on optimization of microbiological diagnostic methods for reproductively important infections allowed us to produce an algorithm of research where the use of microscopic and molecular-biologic methods was included. Using these methods of diagnostics of reproductive tract infections rationally, it is possible to make a quick and qualitative diagnosis and to begin timely treatment.
The algorithm of examination of pregnant women and of those who are planning pregnancy aimed to di-
agnose acute infection that appeared for the first time and latent chronic (anamnestic) infection will allow us to make a correct prognosis for the course and outcome of pregnancy.
In conclusion, it is necessary to emphasize the importance of setting up an independent reference laboratory that, based on the experience of research activity, could carry out the validation, standardization of the methods being used in diagnostics of reproductive infections, elaborate standards of diagnostics and introduce quality control of laboratory activities.
References
1. Manual for physicians: STIs. Methodological materials for diagnosis, treatment and prevention of most common STIs. / Savicheva A., Sokolovskiy E., Domeika M. [et al.]. — St. Petersburg: N-L, 2002. — 112 p. [in Russian]
2. Savicheva A. Short manual for microscopic diagnosis of STIs / Savicheva A., Sololovskiy E., Domeika M. — SPb: Foliant, 2004. ■— 128 p. [in Russian].
3. Manual for physicians: STIs / Sokolovskiy E., Savicheva A., Domeika M. [et al.]. — Moscow: MEDpress-Inform, 2006. — 256 p. [in Russian
4. ShipitsinaE. V. Papilloma virus infection: risk factors of the cervical neoplastic progression / Shipitsina E. V., Babkina K. A., Orzheskovskaya E. A., Savicheva A. M // Z. Akus. Zen. Bolezn. — 2004 — Vol. LIII, N 3. — P. 38-45. [in Russian]
5. ShipitsinaE. V. A method of nucleic acids amplification NASBA (Nucleic Acid Sequence-Based Amplification) and possibilities of its use in obstetrical-and-gynecological practice / Shipitsi-na E. V., Budilovskaya O. V., Savicheva A. M. // Z. Akus. Zen. Bolezn. — 2005. — Vol. LIV, N 2. — P. 83 - 89. [in Russian]
6. European STD Guidelines // International J. of STD&AIDS. —, 2001. —Vol. 12, Suppl. 3.— 102 p.
7. Gilbert R. Toxoplasmosis // in “Congenital and prenatal infections” Cambridge university press. — 2000. — p. 305 - 320.
8. Guidelines for Treatment of Sexually Transmitted Diseases // CDC MMWR Recommendations and Reports — 1998 — Vol. 47, N RR-1. — 117 p.
9. Naot Y. Method for avoiding false-positive results occurring in immunoglobulin M enzyme-liked factor and antinuclear antibodies / Naot Y., Barnett E. V., Remington J. S. // J. Clin. Microbiol. — 1985. — Vol. 21. — P. 931-935.
10. Peckman C. S. Cytomegalovirus infection, congenital and neonatal disease / Peckman C. S. // Scand. J. Infect. — 1991. — Vol. 78. — p. 82-87.
11. Ridgway G. L. Current problems in microbiology: 1. Chlamydial infections: which laboratory test? / Ridgway G. L., Taylor-Robinson D. // J Clin Pathol. — 1991. — 44. — P. 1-5. 128.
12. Sexually Transmitted and Other Reproductive Tract Infections. A guide to essential practice // WHO, 2005. — 186 p.
13. Laboratory Diagnosis of Neisseria gonorrhoeae in St. Petersburg, Russia: inventory, performance characteristics and recommended optimization / Unemo M., Savicheva A., Budilovskaya O. [et al.] // Sex Transm Infect. — 2006. — Vol. 82. — P. 41- 44.
