gonorrhea
Introduction
Introduction to gonorrhea Gonorrhea is a purulent infection of the genitourinary system caused by Neisseria gonorrhoeae (referred to as Neisseria gonorrhoeae). It can also invade the eyes, pharynx, rectum and pelvis, as well as disseminated infections. It is one of the common sexually transmitted diseases. . Gonorrhea is transmitted through sexual intercourse, and a small number can also be transmitted through the patient's pus contaminants, which can be transmitted to the baby through the birth canal. basic knowledge The proportion of illness: 0.012% Susceptible people: mostly occur in young men and women Mode of transmission: sexually transmitted contact Complications: Prostatitis, seminal vesiculitis, epididymitis, urethritis, urethral stricture, pelvic inflammatory disease, pelvic abscess, abdominal pain
Cause
Cause of gonorrhea
Morphology and staining (25%):
Neisseria gonorrhoeae are oval or bean-shaped. The length of the bacteria is 0.6-0.8 m and the width is about 0.5 m. They are often arranged in pairs, and the adjacent faces are flat or slightly concave, like two beans paired together, no flagella, no spores. In the body of acute infection, its morphology is more typical, mostly in the cytoplasm, in the chronic phase is outside the cell, the gonococcal Gram stain is negative, when stained with alkaline methylene blue, the bacteria are blue, stained with Pappehheim Saathof At the time, the cells are red and the background is sky blue, which is very clear. Neisseria gonorrhoeae has five different colony forms on artificial medium, namely T1, T2, T3, T4, T5, of which T1 and T2 are virulence, T3 and T4 are sterile hair, and can not infect humans.
Cell structure (20%):
The pathogenicity of Neisseria gonorrhoeae is closely related to the structure outside the cells. The outer structure of Neisseria gonorrhoeae is the outer membrane. The main components of the outer membrane are membrane proteins, lipopolysaccharide and pili, and membrane proteins can adhere to the human mucosa. Through cell phagocytosis into human cells, multiplication in the cells, leading to cell disintegration, gonococcal spread to the submucosa to cause infection, pili tend to adhere to the surface of the uterine cavity and oral epithelial cells, pathogenic and infectious.
Resistance (25%):
Neisseria gonorrhoeae is more delicate, most afraid of dryness, suitable for growth under humidity, temperature of 35 ~ 36 ° C, containing 2.5% ~ 5.0% carbon dioxide, the optimum pH for growth is 7.0 ~ 7.5, the physicochemical factors of Neisseria gonorrhoeae The resistance is quite poor. It will die within 1~2h in a completely dry environment. However, if it is attached to the underwear and the bedding, it can survive for 18 to 24 hours. It can survive for several days on thick pus or wet objects. 50 ° C can only survive for 5 min, Neisseria gonorrhoeae is very weak against commonly used mucobacterial fungicides, it is especially sensitive to soluble silver salts, 1:4000 silver nitrate solution can cause it to die within 7min, so that Neisseria gonorrhoeae in pus 2min Within the death, 1% phenol (carbonic acid) solution can kill it within 3min. In addition to the resistant strain, Neisseria gonorrhoeae is sensitive to antibiotics, but the minimum inhibitory concentration of antibiotics is gradually improved.
The pathogen is neisseria gonorrhoeae, also known as gonococcus, referred to as Neisseria gonorrhoeae, belonging to the family Neisseria, Neisseria.
1. Found that in 1879, Neisser isolated gonorrhea from 35 secretions of acute urethritis, vaginitis and neonatal acute conjunctivitis. In 1885, Bumm was used in human, bovine or sheep coagulation serum medium. The cultivation of Neisseria gonorrhoeae was successful, and the same symptoms could be produced by inoculating the strain in the urethra of healthy people. At this point, the conclusion that Neisseria gonorrhoeae is the pathogen of gonorrhea has been established.
2. Culture and biochemical characteristics grow well on medium containing animal protein such as blood, serum, egg yolk, ascites, etc. The suitable temperature for culture is 35-36 ° C, suitable pH is 7.5, and it needs to be cultured in 5% carbon dioxide environment. .
In the human urethra, glucose can be fermented, but fructose can not be fermented. Therefore, this characteristic can be used as a glucose fermentation test to identify with meningococcus, which does not produce a sputum matrix and hydrogen sulfide. The production of oxidases has a certain significance in early identification.
3. Nutrient classification According to the amino acid and nucleic acid nutrition foundation required for gonococcal culture, the Neisseria gonorrhoeae can be divided into 35 nutrient types, among which Arg, Hyx, Ura type Neisseria gonorrhoeae can cause asymptomatic gonorrhea and disseminated gonorrhea. Resistant to serum, but sensitive to penicillin and other drugs.
Pathogenesis
(1) Affinity to the epithelium: Neisseria gonorrhoeae has a special affinity for the columnar epithelium and the transitional epithelium. The male and female urethra, the female cervix covers the columnar epithelium and the transitional epithelium, so it is susceptible to gonococcal invasion, while the male scaphoid and female vagina are The stratified squamous epithelium is covered with strong resistance, generally not invaded, or the inflammation is very light, so gonococcal vaginitis is rare in adult women. Because the vaginal mucosa is columnar epithelium, the young girl is susceptible to infection and the skin is not easily gonococcal. Infection, rare cases of primary gonococcal skin infection, humans have no innate immunity to gonococcal bacteria, re-infection can occur after recovery.
(2) Adhesion: The specific receptor on the gonococcal pili can bind to the corresponding part of the mucosal cell; the outer membrane protein II can mediate the adhesion process; it can also release the IgAl decomposing enzyme to resist the rejection of the cell, thus, Neisseria gonorrhoeae and epithelial cells rapidly adhere to each other, and the pH in the microenvironment, ion bridge, hydrophobic structure and sex hormones can also promote the adhesion process.
(3) Invasion and infection: Neisseria gonorrhoeae adsorbs on the microvilli of epithelial cells, and the outer membrane protein I is transferred to the cell membrane, and then Neisseria gonorrhoeae is phagocytosed into the cells, and the Neisseria gonorrhoeae can be adsorbed on the sperm, which can be quickly Ascending to the cervical canal, the mucus of the cervical can temporarily prevent Neisseria gonorrhoeae to the uterine cavity, and in the columnar epithelial cells of the cervix, it will cause disease. Once the gonococcal cells invade the cells, they will proliferate and damage the epithelial cells. Cocci to the submucosal space, causing infection of the submucosa.
(4) Formation of lesions: Neisseria gonorrhoeae invade the submucosal layer and continue to proliferate. It will multiply in the first generation within 36 hours. Through the synergistic action of endotoxin lipopolysaccharide, complement and IgM, it will form an inflammatory reaction, causing mucosal redness and swelling, and at the same time, due to white blood cells. Aggregation and death, necrosis and shedding of epithelial cells, pus appeared, and lesions were most severe in glands and crypt openings.
(5) spread spread: inflammation caused by gonococcal infection can spread along the urinary tract, genital tract spread, in males can be extended to the prostate, seminal vesicles, vas deferens and epididymis, in women can spread to the uterus, fallopian tubes and pelvis, serious Neisseria gonorrhoeae can enter the bloodstream and spread to various tissues and organs throughout the body, leading to disseminated infection.
Prevention
Gonorrhea prevention
Prevention of gonorrhea should also be noted that patients with gonorrhea should be prohibited from sleeping with children, especially girls, baths or public baths, bath towels, etc. In areas with high prevalence, all newborns should be treated with silver nitrate solution or other effective antibiotics. Eye drops.
1. Promote knowledge of sexually transmitted diseases, promote noble morality, and prohibit prostitution.
2. Use a condom to reduce the incidence of gonococcal infection.
3. Prophylactic use of antibiotics can reduce the risk of infection. You can take norfloxacin or amoxicillin before and after sexual intercourse, which can effectively prevent sexually transmitted infections.
4. Sexual partners are treated at the same time.
5. Patients pay attention to personal hygiene and isolation, not to bed with family, children, especially girls, and bath.
6. Implement a system of neonatal silver nitrate solution or other antibiotic droplets to prevent gonococcal ophthalmia.
Complication
Gonorrhea complications Complications Prostatitis Seminal vesiculitis Epididymitis Urethritis Urethral stricture Pelvic inflammatory disease Pelvic abscess Abdominal pain
Male gonorrhea complications
1, gonorrhea combined with prostatitis symptoms: 1 day or half a day before the onset of acute prostatitis often stop pus or pus reduction, patients have high fever, frequent urination and pain, rectal examination showed prostate enlargement, tenderness, urine Liquid turbidity, if the treatment is not timely, the prostate can form abscesses, patients with chronic prostatitis generally have no obvious symptoms, the first urination after waking up, the urethral opening has a sealing phenomenon, a small amount of white secretions are discharged when the penis is squeezed, secretions Examination can reveal epithelial cells, a few pus cells and gonococcus.
2, gonorrhea combined with seminal vesiculitis symptoms: acute fever, frequent urination, dysuria, terminal urine turbidity and blood, rectal examination can touch the swollen seminal vesicle and severe tenderness, chronic symptoms, no rectal examination It shows that the seminal vesicle is hard and fibrotic.
3, gonorrhea with epididymitis symptoms: generally combined with acute urethritis, unilateral, mostly low fever, epididymal swelling and tenderness, ipsilateral groin and lower abdomen have reflex pain, palpation shows epididymal swelling, there are Intense tenderness, urinary turbidity.
4, male gonorrhea can be combined with urethral stricture: repeated gonorrhea can cause urethral stricture, a small number of vas deferens or even infarction, and then secondary semen cyst and infertility.
Female gonorrhea complications
Female gonorrhea and symptoms of gonococcal pelvic inflammatory disease, including acute salpingitis, endometritis, secondary fallopian tube ovarian abscess and pelvic abscess caused by rupture, peritonitis, etc., due to treatment, mistreatment, female gonorrhea patients, extreme It is easy to develop from genitourinary infection to pelvic and accessory infections, causing serious consequences such as infertility, abdominal pain, and even life-threatening.
Symptom
Symptoms of gonorrhea Common symptoms Lack of back pain, white mucus, urine, nausea, libido, severe pain, inflammation, loss of appetite, anal itching
The clinical manifestations of gonococcal infection depend on the degree of infection, the sensitivity of the organism, the virulence of the bacteria, the location of the infection and the length of the infection. At the same time, it is related to the health of the body, whether sexual life is excessive, and alcoholism. According to clinical manifestations, gonorrhea can be divided into no gonorrhea and comorbid gonorrhea; asymptomatic and symptomatic gonorrhea; disseminated gonorrhea and acute and chronic gonorrhea.
First, no complication gonorrhea
(a) male no complication gonorrhea
Acute gonococcal urethritis (acute gonorrhea): The incubation period is 1-14 days, often 2-5 days. From the beginning, acute urethritis, redness, itching and slight tingling of the urethra, followed by thin mucus effluent, causing urination discomfort. After about 2 days, the secretion became sticky, the urethra overflowed, and the pus was deep. Yellow or yellow-green, accompanied by increased symptoms of urinary tract discomfort, redness and swelling developed throughout the penis glans and part of the urethra, frequent urination, urgency, dysuria, difficulty urinating, inconvenient movement, and frequent penile erections at night. There may be swelling of the inguinal lymph nodes, redness and pain, and may also purulent. The first week of acute symptoms is the most serious. If left untreated, the symptoms gradually decrease or disappear after about one month. Two weeks after the onset of acute anterior urethritis, about 50-70% of patients have gonococcal invasion of the posterior urethra, which is characterized by urinary distress, frequent urination, and acute urinary retention. The characteristic of dysuria is that the pain or pain is exacerbated at the end of urination, and it is acupuncture-like. Sometimes there is a perineal pain, and there may be no hematuria. After 1-2 weeks, the symptoms gradually disappeared. Systemic symptoms are generally mild, a few may have fever up to 38 ° C, general malaise, loss of appetite and so on.
Chronic gonococcal urethritis (chronic gonorrhea): Symptoms lasting more than 2 months are called chronic gonococcal urethritis. Because the treatment is not thorough, Neisseria gonorrhoeae can be concealed in the urethral body, paraurethral glands, and urethral crypts to make the disease course chronic. If the patient's constitution is weak, suffering from anemia, tuberculosis, the condition begins with a chronic process, mostly for the anterior and posterior urethra combined infection, good invasion of the urethral bulb, membrane and prostate. Clinical manifestations of the urethra often have itching sensation, burning sensation or mild tingling, fine urine flow, urination weakness, drips. Most patients have a small amount of serous sputum seal in the urinary tract in the early morning. If the genital or penile roots are squeezed, the thin mucus overflows. The urine is basically clear, but there is silk.
(2) Women without complication gonorrhea
The main part of female primary gonococcal infection is the cervix. Neisseria gonorrhoeae can adhere to the stratified squamous epithelium. The infection site of Neisseria gonorrhoeae is observed at the junction of the squamous-columnar epithelium of the cervix. Patients with gonorrhea cervicitis often have no symptoms at an early stage, so the incubation period is difficult to determine. Cervical congestion, tenderness, increased purulent secretions, often genital itching and burning sensation, occasionally lower abdominal pain and low back pain. These atypical symptoms make patients often do not go to the clinic, so they become the main source of infection; gonococcal urethritis often occurs 2-5 days after sexual intercourse, urethral congestion, tenderness and purulent secretions, mild Frequent urination, urgency, dysuria, burning sensation during urination, purulent discharge in the urethra; gonococcal vestibular gland inflammation often unilateral, redness and swelling in the opening of the gland, severe pain, severe abscess formation. There are fever and other systemic symptoms; gonococcal vaginitis is less common, the symptoms of the elderly are mild, some patients have abdominal swelling, back pain, vaginal discharge, some patients have lower abdominal pain and menorrhagia; women gonococcal vulva Vaginitis, manifested as vulvar and vaginal inflammation. There are more vaginal purulent secretions, sometimes yellow and green secretions in the vagina and urethra, painful urination, and redness in the vulva. Secretions can flow to the anus, causing irritation. In severe cases, it can infect the rectum and cause gonococcal proctitis.
Second, there are complications of gonorrhea
(a) Men have comorbid gonorrhea:
Gonorrhea urethritis has various comorbidities, mainly prostatitis, seminal vesiculitis, epididymitis.
1. Prostatitis: Acute prostatitis is caused by the discharge tube and gland of Neisseria gonorrhoeae entering the prostate. There are fever, chills, perineal pain and urinary tract infections accompanied by dysuria. The prostate is swollen and tender when examined. However, Neisseria gonorrhoeae is not a common cause of acute prostatitis. The prostate caused by Neisseria gonorrhoeae is mainly characterized by chronic lesions. The symptoms are mild, there is perineal discomfort, penile pain, there is a "mouth" phenomenon in the morning urethral opening, and the lymph in the urine is seen. The prostate massage fluid has pus and lecithin. , smear or culture to find gonococcus, anal examination can touch small nodules on the prostate, and there is discomfort or pain, drainage of pus near the drainage tube to form scar contraction affects ejaculation, resulting in infertility.
2. Epididymitis: usually occurs after acute urethritis, mostly on one side. There is low fever, epididymis swelling pain, ipsilateral groin and lower abdomen have reflex pain, the beginning and testicular boundaries are clear, gradually unclear, testicular tenderness, swelling, severe tenderness. The urine is often cloudy. There can be prostate and seminal vesiculitis at the same time.
3. Seminal vesiculitis: fever, frequent urination, urgency, dysuria, no urinary turbidity and blood. Rectal examination can touch the swollen seminal vesicle and have severe tenderness. Chronic seminal vesiculitis generally has no symptoms. Rectal examination shows that the seminal vesicle is hard and fibrotic.
4. Urethral gland inflammation: occurs in the perineum or around, there are finger size nodules, pain, acute suppuration and ulceration, compression of the urethra and dysuria, may have systemic symptoms such as fever, slow progress.
5. Urethral stricture: repeated authors can cause urethral stricture, a small number of vas deferens or obstruction can occur, dysuria, thinning of the urinary tract, and severe urinary retention. Secondary vas deferens, seminal vesicle cysts and infertility.
(2) Women have comorbid gonorrhea
The main complication of female gonorrhea is gonococcal box inflammation, such as acute salpingitis, endometritis, secondary fallopian tube ovarian abscess and pelvic abscess caused by rupture, peritonitis. Sudden onset after menstruation, there are high fever, chills, headache, nausea, vomiting, lower abdominal pain, increased purulent leucorrhea. Both sides of the attachment are thickened and tender.
Third, other parts of gonorrhea:
1. Gonorrhea conjunctivitis: Newborns appear more than 2-3 days after birth, mostly bilateral, eyelid swelling, purulent secretions, adults mostly self-inoculation, often unilateral, showing the same newborn. Due to the pus overflow, if the "pust eye" is delayed, the cornea is steamy and the cornea is perforated. Lead to blindness.
2. gonococcal pharyngitis: mainly seen in oral sex, the so-called "mouth" Western homosexual or heterosexuals are more common, manifested as acute pharyngitis or acute tonsillitis, occasional fever and cervical lymphadenopathy. There are symptoms such as dry throat discomfort, sore throat, sore throat.
3. Neisseria gonorrhoea: mainly seen in male homosexuality. Women are mostly caused by vaginal infections. The table is urgent and heavy, there are pus and blood, anal canal mucosa congestion, purulent secretion, gonococcal culture positive.
Fourth, disseminated gonococcal infection
The disseminated gonococcal infection is due to the spread of gonococcal bacteria through the bloodstream to the whole body, resulting in a more serious systemic infection. The incidence rate is about 1% of gonorrhea patients. Most of the strains causing disseminated gonococcal infection are AHU-nutritive. The strain has stable resistance to normal human serum. In addition, normal human serum has IgM antibody against gonococcal lipopolysaccharide. With the help of complement, it is large. Most gonococcus have a bactericidal effect, and patients lacking C5, C6, C7 and C8 complement components are prone to gonococcal sepsis or gonococcal meningitis.
(1) gonococcal sepsis: the patient begins to have fever, the body temperature can be as high as 40 ° C, but usually between 38 ° C and 40 ° C, chills are not common, some patients have skin papules, ecchymoses, pustules, hemorrhagic Or necrotizing skin damage, some skin lesions have pain symptoms. In the lesions, fluorescent immunostaining can be used to detect the growth of Neisseria gonorrhoeae or cultured Neisseria gonorrhoeae, and PCR to detect Neisseria gonorrhoeae DNA positive. The pathological tissue of the lesion is superficial ulcer with pus formation, diffuse inflammation of the dermis and subcutaneous tissue, polymorphonuclear leukocyte infiltration, involvement of small blood vessels, thrombosis and localized necrosis.
(B) gonococcal arthritis: joint swelling, pain, for one or several septic arthritis. Generally asymmetrical, rarely involving the hip, shoulder and spine joints. The joint fluid test has the presence of gonorrhea, which can lead to fibrosis caused by bone destruction and rigidity of the bones and joints.
(C) gonococcal keratosis: may be due to gonococcal or its toxins, can not find gonococcal lesions in the skin lesions, often with gonococcal arthritis, skin lesions are good for the hands and feet, ankle heel and waist . Patches or plaques that are usually flattened and slightly swelled, conical, yellow, or copper red or grayish white. The skin lesions of the palmar sputum showed horny hyperplasia and large keratinization.
(D) gonococcal endocarditis: gonococcal is the main pathogen of endocarditis in the first few decades of antibiotic use, gonococcal endocarditis is almost invisible, gonococcal endocarditis and other types Endocarditis has the same clinical manifestations. Endocarditis often involves aortic or mitral valve, subacute or acute endocarditis due to rapid destruction of the valve, leading to death.
(5) gonococcal meningitis: uncommon, may be associated with arthritis and a typical rash to distinguish meningococcal meningitis.
Fifth, the impact of gonorrhea on pregnancy and newborns
When a woman goes gonorrhea with salpingitis, it can lead to infertility. The incidence of infertility caused by female gonorrhea is about 20%, and the incidence of infertility increases with the increase in the number of infections. For women infected with gonorrhea more than three times, the incidence of infertility can reach 70%. Cervical gonococcal inflammation can lead to early rupture of membranes, intra-amniotic infection, intrauterine infection, intrauterine growth retardation, and premature delivery. Newborns have high morbidity and mortality due to premature birth, low body weight and sepsis. Upstream infection of gonococcal bacteria after birth can cause endometritis, puerperal fever, severe postpartum sepsis, neonatal gonococcal conjunctivitis and vaginal vaginal vulvitis.
Examine
Gonorrhea examination
Laboratory inspection:
Neisseria gonorrhoeae laboratory tests include smear, culture to check Neisseria gonorrhoeae, antigen detection, drug susceptibility test and PPNG determination, and genetic diagnosis.
(1) Smear examination:
Take the patient's urethral secretions or cervical secretions for Gram stain and find Gram-negative in polymorphonuclear leukocytes
Diplococcus, smear for patients with simple gonococcal anterior urethritis with a large number of purulent secretions, the positive rate of this method is about 90%, can be initially diagnosed, female colon secretions, more bacteria, poor sensitivity and specificity The positive rate is only 50-60%, and there is a false positive. Therefore, the World Health Organization recommends using a culture method to check female patients. Chronic gonorrhea has fewer positive gonococcal bacteria in the secretions, so the positive rate is low. Therefore, the prostate massage solution should be taken. Increase the detection rate.
The pharyngeal smear found that Gram-negative diplococcus could not diagnose gonorrhea, because other Neisseria species are normal in the pharynx, and the smear positive for atypical symptoms should be further examined.
(2) Training and inspection:
Neisseria gonorrhoeae culture is an important evidence for diagnosis. The culture method is a sensitive method for males with mild or asymptomatic symptoms. As long as the culture is positive, the diagnosis can be confirmed. Before the genetic diagnosis, the culture is recommended by the World Health Organization. The only way to screen for gonorrhea is to use modified Thayer-Martin(TM) medium and New York City (NYC) medium. The domestic use of chocolate agar or blood agar medium contains antibiotics, optionally Inhibit the growth of many other bacteria, culture at 36 ° C, 70% humidity, 5% -10% CO2 (candle) environment, 24-48 hours observation, colony morphology, Gram stain, oxidation Identification by enzyme test and sugar fermentation test, the positive rate of culture is 80%-95% for males and 80-90% for females.
(three) antigen detection
1. Solid phase enzyme immunoassay (EIA): It can be used to detect gonococcal antigens in clinical specimens. It can be used in women's populations in areas with high prevalence rates and cannot be cultured or specimens need to be sent for a long time. To diagnose gonococcal infections.
2. Direct immunofluorescence assay: direct detection by detection of monoclonal antibodies to the outer membrane protein I of Neisseria gonorrhoeae
Epidemic fluorescence test, but currently the sensitivity of male and female bipolar specimens is not high, the specificity is poor, and the level of judgment of the experimenter, so the experiment can not be recommended for the diagnosis of gonococcal infection.
(four) genetic diagnosis
1. Gene probe diagnosis of Neisseria gonorrhoeae
The probes for gonococcal gene probes are: plasmid DNA probes, chromosomal gene probes and rRNA gene probes.
(1) Plasmid DNA probe
1 Concealed plasmid DNA probe, Neisseria gonorrhoeae plasmid is divided into three types: zygosity plasmid, the largest molecule, 36 kb DNA; drug-resistant plasmid consists of two plasmids, DNA length 5.6 kb and 7.1 kb, respectively; concealed plasmid 4.2 kb, The cryptic plasmid is present in 96% of clinical isolates of Neisseria gonorrhoeae. Other Neisserias do not contain this plasmid, so its sequence can be used as a specific DNA probe to detect Neisseria gonorrhoeae. Torres uses nucleic acid hybridization technology to detect Neisseria gonorrhoeae. In order to conceal the plasmid, the probe was used to detect 134 strains of Neisseria gonorrhoeae and 131 related strains. 124 strains of Neisseria gonorrhoeae were positive for hybridization, accounting for 93%. They could also cross-react with other Neisseria species. The sensitivity test of the needle showed that 102 CFU of Neisseria gonorrhoeae could be detected. It was proved that the CPPB gene sequence in the cryptic plasmid was in all the gonococcal chromosomes (including strains without the plasmid), so the CPPB gene probe has good specificity and sensitivity. Sex, Torres et al. used the CPPB gene probe to detect 201 clinical specimens using a non-radioactive digoxin labeling system with sensitivity and specificity of 95% and 98%, respectively.
2 drug-resistant plasmid DNA probe
Neisseria gonorrhoeae resistant plasmids can be divided into:
1 The toxin-producing gonococcal bacterium (PPNG) is positive for -lactamase;
2 has a high level of plasmid-mediated resistance to tetracycline gonococcal (TRNG).
The PPNG strain was first isolated in the laboratory in 1976. The strain contains a gene encoding a serotonin-producing enzyme. The gene can be integrated into the chromosome or in the plasmid DNA, and the latter is mostly called the blue. Toxin enzyme plasmid, two kinds of plasmids, 7.4 kb and 5.3 kb, respectively. Pescador designed a specific probe for detecting gonococcal -lactamase gene in 1998, which was labeled by enzyme chemiluminescence, liquid phase hybridization. The photometric measurement is the amount of specific hybrids. 104-105 CFU of PPNG strain can be detected within 4 h. Although TRNG strain is resistant to tetracycline, it is usually sensitive to -lactamase and quinolone antibiotics. Therefore, in the experiment The susceptibility test can be classified as sensitive bacteria. Pescador uses an oligonucleotide probe against the tetracycline gonococcal (TRNG) tetm gene, which is mediated by tetracycline, labeled with enzyme chemiluminescence, and hybridized in liquid phase. 1.5×104 CFu of Neisseria gonorrhoeae containing the tetm gene was directly detected from clinical specimens.
(2) Chromosome probe
Chromosomal probes include known gene probes, such as pili DNA probes and paI gene probes, which play important roles in the process of gonococcal infection of human cells; gene probes of unknown function, these probes The sequence is complementary to the specific sequence of the chromosome, but the function of these gene sequences is not known at present. The above two chromosomal probes have low detection number due to the low copy number of the complementary sequence in Neisseria gonorrhoeae, so the detection sensitivity is low, so it is generally not used unless There are special research purposes.
(3) rRNA gene probe
The rRNA gene probe uses a DNA complementary to rRNA as a probe, and the target sequence of the probe is an rRNA sequence, and the gene probe of the rRNA is characterized by:
1 can increase the sensitivity of probe detection, rRNA gene probe can simultaneously detect rRNA molecules and DNA molecules;
2rRNA is evolutionarily conserved;
3 hybridization method is simple and rapid;
4 Because the content of rRNA is high, the specimen does not need to be enriched. The gonococcal detection probe PACE C produced by Gen-Probe in the United States uses rRNA and its gene as the detection target sequence, and is radioactively labeled and can be detected within 2 hours. Peter used this probe to detect 395 clinical specimens. The sensitivity and specificity were 92.9% and 99.4%, respectively. He believed that the PACE C system was a reliable method for screening gonococcal bacteria in clinical specimens. The probe can also detect Asymptomatic gonococcal infections, which are currently difficult to achieve.
2, gonococcal gene amplification detection
The probe technique described above detects the gonococcal method, although it has a great improvement in sensitivity, specificity and convenience than the culture method, but it still has certain limitations, such as the Neisseria gonorrhoeae that requires specimens in most cases. The concentration is very high, and the appearance of PCR technology and ligase chain reaction further improves the sensitivity of detecting Neisseria gonorrhoeae. It has the advantages of rapid, sensitive, specific and simple, and can directly detect a very small amount of pathogens in clinical specimens.
(1) Extraction of Neisseria gonorrhoeae DNA
1 DNA extraction from culture
The cultured Neisseria gonorrhoeae was dissolved in an alkaline lysate at a concentration of 102 cfu/ml. The lysate consisted of 1 M NaCl 1 M NaOH and 1% Sodium dodecyl sulphate. The lysate was mixed and boiled for 1 min, then 100 l. The alkaline lysate was neutralized with 1 M Tris pH 7.0, extracted once with Tris equilibrium phenol, extracted once with phenol-chloroform, and then precipitated with absolute ethanol or isopropanol. The extracted DNA was dissolved in 30 l of distilled water or TE buffer. In the liquid.
2 clinical cotton swab specimen extraction
The cotton swabs with the secretions were squeezed in 2 ml of sterile physiological saline or PBS buffer for 1 min to dissolve the specimens in the solution as much as possible, and the cotton swabs were discarded, and the suspension was at 2-3000 r/ Centrifuge for 5 min at min, aspirate the supernatant, and re-dissolve the cells in 100 l of 1× PCR buffer containing 0.4% Tween 20 and 200 g/ml proteinase K. The cell suspension was warmed at 50-60 ° C for 1 h, then heated at 95 ° C for 10 min. The inactivated proteinase K was centrifuged at 12000 r/min for 10 min, and the supernatant contained a DNA template.
(2) Design of PCR primers
Since the CPPB gene of Neisseria gonorrhoeae is present in the gonococcal chromosome and in the 4.2 kb cryptic plasmid, and in 96% of Neisseria gonorrhoeae, many PCR primers are designed in the CPPB gene region.
Target gene primer sequence fragment length (bp)
CPPB NG1 5'GTT TGG CTG GTT GAT TCA AG 3' 633
NG2 5'GCA AGA TTT CCG ATTT GGC G 3'
CPPB HO1 5'GCT ACG CAT ACC CGC GTT GC 3' 390
HO2 5'CGA AGA CCT TCG AGC AGA CA 3'
rRNA Primer 1 5'-AGG CTG TTG CCA ATA TCG GC-3' 206
Primer 2 5'-ACA CTC GAG TCA CCC AGT TC-3'
CPPB GC1 5'CTT ATC GTT TGG CTG GTT GAT TC 3' 435
GC2 5'ACC AAG ACC AAA GGT TTG ACA CTG 3'
GC3 5'ATT TTC CAG TGT CAA AC 3' 241
GC4 5'TAT TCA AGC CCT ATC TG 3'
(3) PCR amplification
Take 2 l of Neisseria gonorrhoeae DNA extract and add 28 l of the reaction solution. The final PCR reaction solution contains 100 mol/L of dNTPs, 0.5 mol/L of primers, 1 U of Taq DNA polymerase, 1.5 mmol/L of Mg2+, and sterile paraffin oil. 30 l, 1000 r / min centrifugation for 30 s, PCR amplification cycle, reaction conditions: 94 ° C denaturation 1 min, then 94 ° C 30s, 57 ° C 1min, 72 ° C 1min, a total of 30 cycles, and finally 72 ° C extension 5min.
The amplified product was electrophoresed on a 2% agarose gel for 30 min, stained with ethidium bromide, and the amplified DNA fluorescent band was observed under ultraviolet light. The size of the amplified molecule should be consistent with the size of the amplified target sequence.
(4) Sensitivity and specificity of PCR
Since CPPB also contains the CPPB gene on the gonococcal chromosome that does not contain the cryptic plasmid, 96% of the gonococcal bacteria will have a cryptic plasmid. Therefore, the primers using CPPB as the target sequence have extremely high sensitivity. Experiments show that the general tradition One-step PCR (GC1-GC2) method can detect three Neisseria gonorrhoeae, while single-tube nested PCR (GC2-GC4) can detect 0.3 Neisseria gonorrhoeae (9 CPPB genes). These primers were tested by specificity. It can amplify the DNA of Neisseria gonorrhoeae, but does not produce specific products for N. gonorrhoeae.
(5) Single-tube nested PCR method
Based on the traditional nested PCR, two pairs of PCR primers were specially designed. The two outer primers (GC1, GC2) were 25b, the annealing temperature was relatively high (68°C), and the nested inner two primers, GC3-GC4. For the 17b annealing temperature is lower (46 ° C), the other components of the PCR reaction solution are the same as the general PCR, so that the outer primer is amplified first by controlling the annealing temperature (68 ° C), after 20-30 cycles (first time) PCR), and then reduce the annealing temperature (46 ° C) to make the inner primers use the first PCR product as a template for nested amplification, the sensitivity of the PCR can reach 0.3 Neisseria gonorrhoeae.
(6) Neisseria gonorrhoeae ligase chain reaction (LCP) detection method
At present, the method for PCR detection of Neisseria gonorrhoeae is widely used, and its specificity and sensitivity are continuously improved. At the same time, another genetic diagnosis technique, ligase chain reaction (LCP), is also applied to it with its high specificity and high sensitivity. In the detection of cocci, LCP differs from PCR in that LCP uses four pairs of primers, the enzyme used is ligase, ligase can connect two adjacent primers, and the two primers can be used as templates for the other two primers. The latter can be ligated under the action of ligase and can be used as a template for 30-40 cycles. The template treatment method used for LCP is equivalent to the preparation of PCR template. The probe used for LCP can be designed on the CPPB gene. Designed on chromosomal gene sequences, such as the opa-1 gene, Abbott Laboratories designed four LCP probes in the 48 bp region of the opa-1 gene, since the opa-1 gene is 11 times in the gonococcal chromosome. Repeat, therefore, the set of LCP probes has high sensitivity and specificity, LCP reaction process:
The template was added to the LCP reaction solution, LCP reaction solution: 20 mmol/L Tris-HCl pH 7.6; 100 mmol/L KCl 10 mmol/L MgCl2; 1 mmol/L EDTA; 10 mmol/L NAD+; 10 mmol/L DTT , two adjacent probes with labeling each 40fmol / L, unlabeled probe each 40fmol / L, 15U heat-resistant ligase, reaction conditions: 97 ° C 1s, 55 ° C 1s, 62 ° C 50s, 40 cycles 100 l of the reaction product was added to the microplate of the microplate, and the color reaction was carried out. Finally, the light value was read by a microplate reader. According to Buimer's experiment, the sensitivity of the LCP in detecting the male urethral cotton swab specimen was 100%, and the urine was obtained. The specimen was 88.9%, the female cervical swab specimen was 95.4%, and the specificity of the LCP method was as high as 100%, which was significantly higher than the specificity of PCR and avoided the occurrence of false positives.
3. Precautions for clinical genetic diagnosis of Neisseria gonorrhoeae
At present, the genetic diagnosis method for clinical detection of Neisseria gonorrhoeae mainly adopts the PCR method, but this method should pay attention to several problems in clinical detection.
(1) Primer design In addition to the gonococcal PCR primers listed above, it can also be designed from other genes, but
It is the primer sequence that should be specific, because the chromosome of the bacteria is large, many gene sequences are not clear; at the same time, there is a certain homology between the bacteria, and there is also homology between the plasmid sequences contained in the bacteria. Therefore, primers must be designed for comparative analysis of gene databases, and specificity and sensitivity experiments should be carried out, and primers should be selected for clinical detection.
(2) Clinical specimen processing For clinical specimens, the purer the PCR template requirements, the better the position should be taken when collecting specimens. For asymptomatic patients, appropriate sampling should be performed to ensure the collection of pathogenic bacteria. In addition, due to the complexity of the clinical specimens, sometimes the PCR amplification of the specimens is not ideal. This may be due to excessive impurities, which requires further purification. For example, purification by phenol-chloroform extraction will improve. This purification method is cumbersome, and a commercially available DNA purification kit is currently available for relatively easy extraction of high-purity DNA from clinical specimens.
(3) Detection method of PCR products Recently, clinical PCR detection of Neisseria gonorrhoeae almost all uses electrophoresis to identify PCR products. This method has many problems, such as false positive and false negative results due to subjectivity of macroscopic observation. Hybrid colorimetry is currently used instead of electrophoresis to improve the specificity and sensitivity of the results.
In conclusion, the PCR method and the LCP method have greatly improved sensitivity and specificity compared with the traditional culture method, and the time is also greatly shortened. With the continuous improvement of the gene diagnosis technology, the PCR method and the LCP method are detected in the gonococcus Will become a routine detection method.
(5) Susceptibility test: further susceptibility test after culture positive, sensitivity test by paper diffusion method, or minimum inhibitory concentration (MIC) by agar plate dilution method to guide the selection of antibiotics.
(6) PPNG detection : -lactamase, using paper acidity quantitative method, using Whatman I filter paper PP-NG, the strain can make its color change from blue to yellow, positive for PPNG, negative for N-PPNG.
Diagnosis
Diagnosis of gonorrhea
1. Diseases identified by men and women with gonorrhea
(1) Non-specific urethritis: There are often obvious causes of the disease, such as the insertion of the catheter, urethral probe, genitourinary tract or inflammation of adjacent organs, and the secretion smear stain can detect Gram-positive cocci.
(2) NGU: clinical symptoms are similar to gonorrhea, but lighter, the incubation period is longer than gonorrhea, up to 1 to 3 weeks, the secretion is small and serous, dysuria is rare, no systemic symptoms, and secretions are taken for examination. Chlamydia or mycoplasma can often be found.
(3) soft squat: damage is generally located in the external genital area, occurs in the urethra, the scapular fossa has purulent secretions, urethral redness and severe pain, ulcers can detect Ducrey Haemophilus.
(4) Genital herpes: caused by HSV, the damage is cluster blister, usually located in the external genitalia, perianal, occasionally in the cervix, occurs in the urethra, there may be a small amount of secretions and urethral tingling or discomfort, often accompanied by local burning sensation The pain is obvious and can be repeated.
(5) fixed drug eruption: there is a history of medication or injection medication, mostly caused by sulfonamides, barbiturates, salicylates or tetracyclines, often occur in the genital skin mucosa junction erythema, edema, blisters, Erosion and exudation, etc., with itching or pain, no redness and valgus in the urethra.
(6) tuberculous urethritis: may have urethral tingling and a small amount of secretions, but no history of sexual contact, can not find gonococcal, can be found tuberculosis, accompanied by other parts of the symptoms of tuberculosis.
2. Diseases identified by male gonorrhea
(1) Foreskin balanitis: common in foreskin is too long or phimosis, mostly caused by smegma stimulation and secondary pyogenic infection, intracapsular inner leaf, glans and coronary sulcus redness, more dirt and purulent secretions , no urinary tract symptoms and redness and swelling of the urethra, pus and so on.
(2) Urine turbidity: The urine is milky white, and there is salt crystallized sediment after standing, without accompanying symptoms.
3. Diseases identified by female gonorrhea
(1) Candida vaginitis: genital and vaginal opening itching, more vaginal discharge, such as watery or curd-like, vaginal mucosa redness, erosion, may have a white film attached to the vaginal wall, take a white film smear microscopic examination visible bacteria Silk and spores.
(2) Trichomonas vaginitis: consciously vaginal pruritus in the vagina, vaginal secretions often pink foam, vaginal mucosa more bleeding, cervical can have strawberry-like protrusions and punctiform hemorrhage, vaginal secretions Trichomonas can be detected.
(3) Bacterial vaginosis: mainly refractory leucorrhea, grayish white, non-suppurative, with a "fishy-like" odor, Gardner bacilli and anaerobic bacteria can be detected from vaginal secretions.
(4) young female Staphylococcus aureus vulvitis: more common in preschool or school-age girls, occur in summer, vulva, perianal redness, pustules or purulent secretions, accompanied by pain or itching, secretion smear Gram-positive cocci were detected.
4. Diseases identified with GPID
There are acute appendicitis, infectious abortion, pelvic endometriosis, ectopic pregnancy, ovarian cyst pedicle torsion or rupture.
