Sexually transmitted infections

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1. Neisseria gonorrhoeae (NG)

Gonorrhea neisseria bacterium commonly known as neisseria gonorrhoeae, is the pathogen of human gonorrhea, man is the only host of neisseria gonorrhoeae.

Neisseria gonorrhoeae mainly spread through sexual contact, urinary tract and genital tract The incubation period is 2~5 days. It causes human acute or chronic suppurative infection of the mucous membrane of the urogenital system, is the sexually transmitted disease of the highest incidence in China at present, also is one of the reasons cause infertility.

Early infections in adults generally cause preurethritis , women urethritis and cervicitis. Symptoms are painful urination, frequent urination, urethral discharge, the cervix with purulent secretion and so on. If it further spreads to the reproductive system, it could cause chronic infections, such as men appear prostatitis, seminal vesicle, funiculitis and epididymitis and women appear bartholinitis, pelvic inflammatory disease and so on. Mother suffering from vaginitis of sex of the ball or cervicitis, baby is susceptible to gonorrheal conjunctivitis when borns.

Human has not natural resistance to neisseria gonorrhoeae infection, most patients can be self-healing, and appear specific IgG, IgM and IgA, but the immune don't last. Reinfection and chronic patients are common.

Laboratory diagnosis of NG infection and the significance of fluorescent probes quantitative detection of NG-DNA:

(1) Smear: smearing with patients’ purulent secretion, gram staining and microscopy, if the gram-negative diplococcus are found in neutrophils, combined with clinical symptoms and medical history (contact), it has diagnostic value. Smear method for the typical male patients with gonorrhea, has high detection rate. But for female patients, due to the secretion of exfoliated cells and acid fast bacilli markings which makes the background difficult to identify, the detection rate is very low, prone to false negative results.

(2) Culturing: Culturing specimen in selective medium appears the typical colony. The oxidase test is positive. When bacterial smear made of typical colonies is visible to gram-negative diplococcus, ic could be diagnosed. But the resistance of neisseria gonorrhoeae is very weak, leaving the host will die easily. It is not easy to culture separately.

(3) the fluorescent probe PCR technology directly amplify the genes from patients with suspected of neisseria gonorrhoeae, pathogen can be detected quickly and accurately in a short time directly from very low level clinical specimens of all kinds of pathogenic bacteria. So this technology has important significance in the diagnosis and identification of various kinds of inflammation caused by suspected neisseria gonorrhoeae.

(4) in the transmission of neisseria gonorrhoeae, the neisseria gonorrhoeae infected persons with minor symptoms or no symptoms is the more important source of infection. The advantage of the fluorescent probe PCR detection is early diagnosis and differential diagnosis in patients with this part, is of great significance in the prevention and treatment of sexually transmitted diseases.

(5) In the same way, neisseria gonorrhoeae DNA tests can be used for curative effect appraisal. With effective antibacterial treatment, the specimens neisseria gonorrhoeae DNA copy number will be reduced and turn negative after curing.

2. Ureaplasma urealyticum (UU)

Ureaplasma urealyticum is a bacterium that is found in the urogenital tracts of humans. It stains gram negative, but that is because it lacks a cell wall. This organism can exist as normal commensal flora in the reproductive tract (especially in women) and remain undetected unless specifically tested for. Women with this infection often experience fertility problems and can even be rendered infertile. Infected mothers can also transmit the infection to their baby. Often the babies are born prematurely and are prone to diseases like pneumonia and meningitis. It can also affect the babies’ respiratory tract. U. urealyticum is also found in males, but is less common.

Ureaplasma urealyticum can be transmitted in various ways, including directly by sexual transmission through direct contact between couples, vertically from mother to offspring, or through hospital-acquired infections from transplanted tissues. Until a female is specifically tested for an infection, this microorganism can live as normal flora in her reproductive tract and remain undetected. Thus, the symptoms for this bacterium can vary from person to person. Some of the most common symptoms for women associated with this bacterium include infertility, recurrent pregnancy loss, pelvic pain, premenstrual symptoms like spotting between menstrual cycles, and vaginal symptoms like uterine infection. The bacterial infections that are caused by Ureaplasma urealyticum can also lead to fertility problems such as tubal disease, recurrent miscarriages, decreased sperm motility, and poor post coital tests. Furthermore, Ureaplasma urealyticum has been attributed to many diseases in the lower urogenital tracts of humans.

The significance of fluorescent probes quantitative detection of UU-DNA:

Etiology diagnosis of UU infection usually uses simple culture method and immunofluorescence antibody. But the positive rate is low, and it’s time consuming. Some nonpathogenic of U. Urealyticum or pollution in the process of or culture may result in false positive results. Fluorescent probe quantitative polymerase chain reaction (RT-PCR) directly detect UU-DNA in genital tract secretions or urine of suspicious patients, it is of high sensitivity and specificity, which improves the positive detection rate.

The diagnosis of pathogens just provide some basis for clinical diagnosis. After pathogen infection, the degree of disease and disease time are related to the number of pathogens, which requires quantitative results. Fluorescence quantitative PCR probe not only uses for the pathogen infection diagnosis, also monitors the disease progression through detecting the changes of UU-DNA copy number before and after medication. Reports showed that, reviewing the UU patients after treatment 2w, UU-DNA copy number of positive patients have a significant reduction, its negative conversion ratio was 44.7%. Viewing after treatment 3w, the negative conversion ratio was 76%. This implys than UU has certain resistance to the clinical treatment antibiotics used, the treatment should be continued over 3w. Using fluorescence probe quantitative PCR to detect UU-DNA has the advantages of rapidity, high specificity, quantity and accuracy, avoids blindly medicine, namely reduces patient pain, shortens the course of the disease, reduces the economic burden of patients and provides the basis for clinical evaluation of curative effect.


3. Chlamydia trachomatis (CT)

Chlamydia trachomatis strictly parasitize in cells, has a unique development cycle. It not only can cause eye and lung infection, also is the main pathogens of sexually transmitted diseases. In recent years in Europe, the United States and other countries, the infection rate and harmfulness of chlamydia trachomatis have exceeded neisseria gonorrhoeae and among the top of the sexually transmitted diseases.

After invading the body, chlamydia trachomatis mainly reproduce in mucosal epithelial cells, and inhibit the metabolism of host cells. The toxic metabolites generated in the breeding process of Chlamydia trachomatis can cause cell toxic reaction and allergic, leading to pathological damage.

After chlamydia trachomatis infecting the body, the immune response is not strong. The maintenance time so short that maks occult infection form easily and repeatedly. Genitourinary dysfunction caused by infection is mainly urethritis for male. Most men without treatment turn into a chronic infection, cyclical increase, or combination of epididymitis and prostatitis. For women, infection can cause urethritis, cervicitis, salpingitis and pelvic inflammatory disease concurrently, hinder the pregnancy and lead to reproductive damage. Perinatal transmission can cause neonatal pneumonia, and mother-son transmission rates of chlamydia trachomatis is as high as 30% - 70%.

Urinary tract infection caused by CT often merges with of neisseria gonorrhoeae infection. Neisseria gonorrhoeae has an activative and stimulative effect in the reproduction of chlamydia .

Laboratory diagnosis of CT infection and the significance of fluorescent probes quantitative detection of CT-DNA:

As to the diagnosis of chlamydia trachomatis, due to the constraints of economic factors at present, most hospital laboratories use gold immnnochromatography or ELISA method to detect corresponding antibody of chlamydia trachomatis. Although these methods are quick, easy and of high specificity, they actually test the human immune response state of chlamydia trachomatis. Immunological detection has a "window period", and the antibody detection can't judge whether it is current infection or previous infection. For example, CT-IgM positive can not be sure that CT must exist in the human body, and CT-IgM negative cannot rule out the infection of CT neither.

Fluorescence probe quantitative PCR can truly reflect the existence and reproduction of CT, more important is to enhance the detection rate of CT specimen. Theoretically, fluorescence probe quantitative PCR can detect one pathogen in the specimens, general laboratories can check out 10 CT-DNA gene copies, too.

Clinical data showed that using fluorescent probe quantitative PCR to detect urethral discharge of male urethritis patients with suspected CT infection, the CT positive rate was 25%. CT positive rate of women cervicitis patients with cervical secretions was as high as 40%, the average copy number reach 105 orders of magnitude. Fluorescent probe quantitative PCR can improve the positive detection rate of CT, realize fast, early and specific (specificity 100%) diagnosis, and also be used as a reliable indicator for curative effect observation.  


4. Herpes simplex virus (HSV)

Herpes simplex virus is a typical representative of the herpes virus. Causing a variety of infections and diseases, this virus has more and more attention. The Herpes simplex virus type 1 is an ubiquitous pathogen of humans that usually causes either asymptomatic infection or mild skin and mucosal diseases. Antibodies to HSV 1 occur in about 90% of adults. Normally HSV 1 is transmitted by oral secretions or open wounds prior to the age of five. Recently in adults primary infections were observed, too. After the primary infection some viruses establish a latent state in their host cells (mostly ganglial cells). The virus DNA is integrated into the genome of the host cell, where it remains until the infected person dies. After stimulation of the host cell, recurrent infection occurs, which is called an exacerbation, when clinical symtoms appear. The recurrence may be caused by different kinds of traumas, as fever or physiological changes and diseases. Immunosuppressed persons may show a severe clinical course. HSV 1 causes different clinical symptoms in about 10% of the primary infections. HSV 1 causes 85% and HSV 2 15% of oral primary infections. The major clinical manifestations associated with HSV 1 infections are gingivostomatitis, keratitis, conjunctivitis, vesicular eruptions of the skin, encephalitis, eczema and some lethal infections of newborns. In some cases HSV 1 infection leads to a meningitis with different neurological symptoms. Persons at an increased risk for serious or prolonged HSV infections are those with eczema, severe burns or a defect in their cell-mediated immunity.

Laboratory diagnosis of HSV infection and the significance of fluorescent probes quantitative detection of HSV-DNA:

The "gold standard" for diagnosis of HSV infection remains isolation of the virus in tissue culture. But it’s time consuming and cumbersome. For clinical diagnosis, it’s ussal to use ELISA and indirect immunofluorescence (IFA) to detect HSV specific antibody. But in the early phase of the disease (the "window period"), it is not easy to detect virus antibody in the blood. At this point, the appropriate choice is taking fluorescence quantitative PCR method for detecting probe. Research confirms that in genital ulcer healing process, fluorescent probe quantitative PCR detection is more sensitive to asymptomatic detoxification. Fluorescence quantitative PCR probe detection has important significance in early screening of HSV infection and preventing the happening of cervical cancer. What’s more, RT-PCR can be used for classification of HSV. At last, RT-PCR detection can be used for assessment of curative effect and prognosis, and carry on HSV molecular virology and epidemiological studies.


5. Treponema pallidum (TP)

Treponema pallidum is a spirochaete bacterium with subspecies that cause treponemal diseases such as syphilis, bejel, pinta, and yaws. The treponemes have a cytoplasmic and an outer membrane. In natural circumstances, treponema pallidum infect humans only, human is the only source for the spread of syphilis. Syphilis can be divided into congenital and acquired kinds, the former is transmitted through sexual contact and the latter is transmitted through the placenta from mother to fetus.

The acquired type virus is divided into three phases, which are characterized by repeated, latent and recurrence. At all phases of infection, syphilis patients can create two kinds of antibodies. One kind is anti-treponema pallidum antibody, another kind is anti-cardiolipin antibody, called reagin.

Laboratory diagnosis of TP infection and the significance of fluorescent probes quantitative detection of TP-DNA:

Serum tests of none treponema pallidum antigens include VDRL, USR, RPR and TRUST.And these tests appears false negative result easily. Serum tests of treponema pallidum antigens include FTA-ABS, TPHA and TP-ELISA. But the operation is cumbersome.
Fluorescent probe PCR detecting treponema pallidum DNA directly, has better sensitivity and specificity than serological methods, is the advanced method of diagnosis of treponema pallidum.
In the first and second phase, syphilis has certain secretiveness, which makes it easy to result in missed diagnosis and misdiagnosis. For early syphilis, using RT-PCR to detect TP-DNA is more sensitive than serological method. In addition, due to the IgG lasting a lifetime, it is difficult to evaluate the effect of the treatment. So RT-PCR detection provides the most objective basis for assessment of curative effect.


6. Human immunodeficiency virus (HIV)

HIV is a kind of virus that attacks human immune system. It puts T4 lymphocytes in human immune system as a target, devours and destructs T4 lymphocytes largely, leads to destroying the immune system, eventually makes the immune system collapse, make human body lose resistance to various disease, then appearsmorbidity and mortality.

Virus exists widely in infected blood, semen, vaginal secretions, saliva, urine, milk, cerebrospinal fluid, cerebral interstitial fluid of the nerve symptom, with the highest concentrations in blood, semen, vaginal secretions. People infected are of long incubation period and high mortality rate.

The gene structure of HIV is the same as that of other retrovirus and it is bimolecular which refers to electronic dimmer formed by 2 similar positive strand RNA sense complementary paired with parts basic groups. The viral genome is formed by 9,749 nucleotides and it includes 3 groups and 9 genes (gag, pol, env, tat, rev, nef, vif, vpu and vpr).

Laboratory diagnosis of HIV infection and the significance of fluorescent probes quantitative detection of HIV-RNA:

Currently there are more than 100 kinds of methods to detect HIV. Generally these can be divided into two major categories: antibody detection and virus detection. Virus detection includes cell culture (virus separation), p24 antigen detection and virus nucleic acid detection. Cell culture method is of complex operation and high cost. P24 antigen detection is easy to appears false positive result.

Virus nucleic acid detection usually reflects the viral load by detecting HIV RNA level, has high sensitivity. Using fluorescent probes quantitative polymerase chain reaction (RT-PCR), can detect virus nucleic acid in the first two weeks after HIV infection. Virus nucleic acid detection method can be used for the early diagnosis of HIV, such as window auxiliary diagnosis, therapeutic course monitoring, guiding treatment, efficacy measurement and disease processes prediction.


7. Human papillomavirus (HPV)

Human papilloma virus belongs to DNA tumor virus, has host and tissue specificity, infects human skin and mucosa epithelial cells only, and lead to different degrees of hyperplastic lesions. Humans are the only natural host of HPV. HPV Transmits primarily through direct contact with infected part, or indirect contact with contaminated by the virus. Genital infection is mainly transmitted by sexual contact. The newborn can be infected through the birth canal. HPV infection only stays in the local skin and mucous membrane, does not produce viremia.

There are more than 100 type of HPV, different types of HPV infringe different locations and the disease is not the same. HPV has been identified as the leading cause of cervical cancer in women, as well as a growing risk factor in orpharyngeal cancer. 99.7% cervical cancer can be found in the high-risk type HPV infection. Cervical cancer is one of the most common malignancy among women. Its incidence occupies the second place of women malignant tumor.There are about 500 thousands of new cases of HPV in the world each year, of which 200 thousands women died of cervical cancer. But cervical cancer is the only one currently that can be found and prevented in the early phase, can be cured 100%, is expected to wipe out cancer.

Laboratory diagnosis of HPV infection and the significance of fluorescent probes quantitative detection of HPV-DNA:

Fluorescent probe quantitative polymerase chain reaction (RT-PCR) detects HPV - DNA directly, has more sensitivity and specificity than the nucleic acid molecule hybridization techniques, is quite rapid and accurate. The method is simple, can achieve ideal result and improve the HPV detection level greatly. What’s more, RT-PCR can be used for classification of HPV. At last, RT-PCR detection can be used for assessment of curative effect and prognosis.