bubble_chart Overview

Gonorrhea is a purulent infection of the genitourinary system caused by Neisseria gonorrhoeae (commonly known as gonococcus). It can also affect the eyes, throat, rectum, pelvic cavity, and lead to hematogenous disseminated infections. It is one of the common sexually transmitted diseases.

bubble_chart Epidemiology

Gonorrhea is a sexually transmitted disease that is widely prevalent around the world. Before the liberation of our country, the spread of gonorrhea was extremely severe. In the initial stage [first stage] after liberation, gonorrhea ranked second among sexually transmitted diseases. By the intermediate stage [second stage] of the 1960s, gonorrhea was nearly eradicated in our country. However, with the reform and opening-up policies in the 1980s, gonorrhea was reintroduced and spread from coastal cities to inland areas, with a rapid annual increase in incidence. Gonorrhea now ranks first among sexually transmitted diseases in terms of prevalence. Our Genetic Diagnosis Center's sexually transmitted disease specialist clinic treats over 20,000 patients annually, with gonorrhea accounting for about 60% of cases, predominantly affecting men more than women. Many female patients are sex workers. Gonorrhea exhibits clear seasonal patterns, with the highest incidence occurring between July and October and the lowest between December and March. Currently, the incidence is declining among high-income groups but rising among middle- and low-income groups. Infections are gradually decreasing in large cities but increasing in small and medium-sized cities, with gonorrhea spreading from urban to rural areas, leading to a rise in rural cases.

The prevalence of gonorrhea worldwide is highest in some countries in Europe, America, and Africa. In the United States, the incidence was 473 per 100,000 in 1975 and 300 per 100,000 in 1988. In Kampala, Uganda, the rate was as high as 10,000 per 100,000. Asian countries also report alarming rates, with Singapore at 630 per 100,000 in 1980 and Thailand at 408 per 100,000 in 1985. In the U.S., gonorrhea incidence is highest among men, while the carrier rate is highest among women. The highest infection rates are observed among sexually active individuals, adolescents, the impoverished, Black populations, those with less education, and unmarried individuals, who play a significant role in spreading the disease. Since 1985, the incidence among White populations in the U.S. has steadily declined, but no significant decrease—or even an increase—has been observed among Black populations. Some transmissions are linked to drug use among sex workers, where women or sex workers engage in transactional sex under the influence of drugs. The gonorrhea infection rate among sex workers is 8.5% in Singapore and Taipei, 31% in Colorado, U.S., and 51% in Bata, Africa.

Transmission routes:

Humans are the only natural hosts of gonococcus, and gonorrhea patients are the primary source of transmission. Gonorrhea is mainly spread through unprotected sexual intercourse, but non-sexual contact routes also exist. Sexual contact is the primary mode of transmission, with almost all adult cases resulting from sexual activity. A man has a 25% chance of infection after a single sexual encounter with an infected woman, and the risk increases with more encounters. Non-sexual transmission occurs through contaminated clothing, bedding, towels, bathtubs, toilets, etc. Neonatal gonococcal conjunctivitis is often caused by infection during delivery through the birth canal of an infected mother. Pregnant women with gonorrhea can transmit the infection to the amniotic cavity and fetus. Additionally, iatrogenic infections can occur through healthcare workers' hands or medical instruments. Mild or asymptomatic gonorrhea patients are significant sources of transmission.

bubble_chart Pathogen

The pathogen of gonorrhea is the gonococcus, which belongs to the Neisseria genus. In 1879, Neisser first isolated the gonococcus, so it is also called Neisseria Gonorrhoeae. In 1885, Bamm successfully cultured the gonococcus on a solidified serum medium.

The morphology of the gonococcus resembles that of the meningococcus, appearing round, oval, or kidney-shaped, often arranged in pairs with adjacent surfaces flattened or slightly concave, measuring 0.6μm × 0.8μm. It is Gram-negative, staining pink, and appears blue with methylene blue staining. In acute cases, gonococci are commonly found within the white blood cells of secretions, whereas in chronic cases, they are mostly outside the white blood cells. The gonococcus has weak resistance, fearing dryness and thriving in moist environments with temperatures of 35–36°C and 2.5–5% carbon dioxide. In completely dry conditions, it can survive only 1–2 hours, but in slightly damp clothing, towels, or bedding, it can survive for 18–24 hours. At 50°C, it survives for only 5 minutes. The gonococcus is highly susceptible to common disinfectants, being killed by 1:4000 silver nitrate in 7 minutes and dying within 3 minutes in 1% phenol.

The outer membrane of the gonococcus consists of lipopolysaccharides, outer membrane proteins, and pili, which have adhesion and pathogenic effects.

Pili are composed of polypeptides and are antigenic. The terminal amino acid sequence is relatively constant, while the middle and carboxyl-terminal sequences often vary, determining the antigenic diversity of pili among different strains. Pili are involved in the adhesion of gonococci and also inhibit phagocytosis by white blood cells. Colonies cultured for 20 hours have pili on their cell surfaces and are virulent. As colonies age, pili disappear, and inoculation into the urethra does not cause urethritis.

The outer membrane proteins of gonococci include at least three types, with Protein I being the major protein, accounting for 60% of the outer membrane proteins. The antigenicity of Protein I varies among different gonococci. Due to its stable antigenic properties, monoclonal antibodies can be produced for serotyping gonococci. It is expressed in two forms, PIA and PIB, and forms pores in the cell membrane, allowing water-soluble substances, other metabolites essential for bacterial metabolism, and certain antibiotics to enter the cell. Protein II is involved in the adhesion of gonococci to human epithelial cells and white blood cells, as well as intercellular adhesion, and exhibits heat modifiability. Protein III, also known as Rmp, has reducible modifiability and strong immunogenicity, cross-reacting with other Neisseria species and blocking the bactericidal effects of other antibodies. Recently, an iron-regulated protein called Frp has been identified, which expresses iron receptors under iron-deficient conditions.

Lipopolysaccharide is one of the important surface structures of gonococci, serving as an endotoxin. It collaborates with submucosal and systemic complement to induce inflammatory reactions and is associated with the virulence, pathogenicity, and immunogenicity of gonococci. Six antigenically distinct gonococcal lipopolysaccharides have been identified.

bubble_chart Pathogenesis

Normally, urine should be sterile. Due to the continuous flushing of the urethra by urine, it is difficult for invading microorganisms to colonize the urinary tract. However, Neisseria gonorrhoeae can easily adhere to the urinary tract {|###|}Chinese Taxillus Herb{|###|}, primarily because the gonococcus possesses pili, which make it highly sensitive to single-layer columnar epithelial cells and transitional epithelial cells, such as those in the anterior urethra, cervix, posterior urethra, and bladder mucosa. This allows the gonococcus to easily adhere to these cells. Gonococci thrive poorly in acidic urine (pH<5.5）很快殺死，因而膀胱和腎臟不易被感染，而前列腺液含有精胺及鋅，故可受淋球菌感染。

), and the {|###|}Chinese Taxillus Herb{|###|} flora in the urethra and vagina have a certain inhibitory effect on gonococcal growth. The presence of these flora provides some natural resistance to the body. The mucosal surface contains lactoferrin, and iron is essential for the growth and reproduction of gonococci. When environmental iron levels are low, gonococcal growth is restricted. Gonococci exhibit varying sensitivity to different cells, being most sensitive to the columnar epithelial cells of the anterior urethral mucosa. Therefore, the anterior urethra is most susceptible to infection. The posterior urethra and bladder mucosa consist of transitional epithelium, to which gonococci are less sensitive than columnar cells, making them less likely to be infected compared to the anterior urethra. The navicular fossa mucosa is composed of stratified squamous cells, which are less prone to gonococcal infection. Gonococci rapidly adhere to urethral and cervical epithelial cells via pili, protein II, and IgA protease. The outer membrane protein I of gonococci transfers to the epithelial cell membrane of the urethra, after which the gonococci are engulfed by columnar epithelial cells and then translocated beneath the submucosa. There, through the synergistic action of endotoxin lipopolysaccharide, complement, and IgM, they induce an inflammatory response. Within about 30 hours, widespread mucosal edema and adhesion occur, accompanied by purulent discharge. During urination, the adhered urethral mucosa stretches, stimulating local nerves and causing pain. Due to the inflammatory response and mucosal erosion and shedding, typical urethral purulent discharge forms. The inflammatory stimulation leads to spasmodic contraction of the urethral sphincter, resulting in urinary frequency and urgency. If small mucosal blood vessels rupture simultaneously, terminal hematuria may occur. Once bacteria enter the urethral glands and crypts, they can also invade the submucosal layer from the mucosal layer, blocking glandular ducts and crypt openings, leading to localized abscess formation. During this process, the body produces both local and systemic antibodies. The immune response to gonococci manifests in various ways, with host defense primarily relying on IgG and IgM, while IgA also plays a preventive role on mucosal surfaces. In males with gonococcal urethritis, urethral secretions often exhibit an antibody response to the infecting gonococci, known as the mucosal antibody response. These antibodies include not only IgA but also IgG and IgM. In terms of serum antibody response, after gonococcal infection, serum levels of IgG, IgM, and IgA increase. IgA, as a secretory antibody, enters the bloodstream from mucosal surfaces and plays a crucial role in serum antibody-complement-mediated bactericidal activity, offering protection against gonococcal bacteremia caused by serum-sensitive strains. Generally, inflammation does not spread systemically. If treated with Yaodui—appropriately and adequately—local inflammation will gradually subside. After inflammation resolves, necrotic mucosa is repaired, replaced by squamous epithelium or connective tissue. Severe or recurrent infections can lead to connective tissue fibrosis, causing urethral strictures. If treatment is inadequate, gonococci may spread to the posterior urethra or cervix, ascending to cause inflammation in the urogenital tract and adjacent organs, such as paraurethral glanditis, Cowper’s glanditis, prostatitis, seminal vesiculitis, epididymitis, endometritis, etc. In severe cases, hematogenous dissemination may occur, spreading the infection systemically. Gonococci can also remain latent deep within glandular tissues for extended periods, becoming a cause of recurrent chronic gonorrhea. After inflammation in these infected organs subsides, connective tissue fibrosis may lead to strictures or obstruction of the vas deferens or fallopian tubes, resulting in ectopic pregnancy and sterility.

bubble_chart Clinical Manifestations

The clinical manifestations of gonococcal infection depend on the extent of the infection, the body's sensitivity, the virulence of the bacteria, the site of infection, and the duration of the infection. It is also related to the overall health status, excessive sexual activity, and alcohol abuse. Based on clinical manifestations, gonorrhea can be classified into uncomplicated gonorrhea and complicated gonorrhea; asymptomatic and symptomatic gonorrhea; disseminated gonorrhea; and acute versus chronic gonorrhea.

I. Uncomplicated Gonorrhea

(1) Uncomplicated Gonorrhea in Males

Acute gonococcal urethritis (acute gonorrhea): The incubation period ranges from 1 to 14 days, typically 2 to 5 days. Initially, it presents as acute anterior urethritis, with redness, swelling, and itching of the urethral orifice, accompanied by mild stabbing pain. This is followed by the discharge of thin mucus, causing discomfort during urination. After about 2 days, the secretions become thicker, and pus discharges from the urethral orifice, appearing deep yellow or yellow-green. Concurrently, urethral discomfort worsens, with redness and swelling spreading to the entire glans penis and part of the urethra. Symptoms include frequent urination, urgency, dysuria, difficulty urinating, restricted movement, and often painful nocturnal erections. There may also be swelling, redness, and tenderness of the inguinal lymph nodes, which may suppurate. Acute symptoms are most severe in the first week. Without treatment, symptoms gradually lessen or disappear after about a month. Two weeks after the onset of acute anterior urethritis, about 50–70% of patients experience gonococcal invasion of the posterior urethra, manifesting as urinary urgency, frequency, and acute urinary retention. The characteristic dysuria is pain or intensified pain at the end of urination, described as needle-like, sometimes accompanied by perineal heaviness. Terminal hematuria may occur. Symptoms typically resolve within 1–2 weeks. Systemic symptoms are generally mild, with a few patients experiencing fever up to around 38°C, malaise, and loss of appetite.

Chronic gonococcal urethritis (chronic gonorrhea): Symptoms persisting for more than 2 months are termed chronic gonococcal urethritis. Due to inadequate treatment, gonococci may hide in the urethral glands, paraurethral glands, or urethral crypts, leading to a chronic course. If the patient has a weak constitution, anemia, or subcutaneous node disease, the condition may become chronic from the outset, often involving combined infection of the anterior and posterior urethra, particularly affecting the bulbar urethra, membranous urethra, and prostatic urethra. Clinically, patients often experience urethral itching, burning during urination, or grade I stabbing pain, thin urine stream, weak urination, and dribbling. Many patients have a small amount of serous crust sealing the urethral orifice in the morning, and pressing the perineum or penile root may yield thin mucus discharge. The urine is generally clear but may contain gonorrheal threads.

(2) Uncomplicated Gonorrhea in Females

The primary site of gonococcal infection in females is the uterine cervix. Gonococci can attach to stratified squamous epithelium, and electron microscopy shows infection at the squamous-columnar junction of the cervix. Patients with gonococcal cervicitis often have no early subjective symptoms, making the incubation period difficult to determine. Cervical congestion, tenderness, and increased purulent discharge are common, often accompanied by vulvar itching and burning, occasionally with lower abdominal pain and lumbago. These atypical symptoms often lead patients to delay seeking treatment, making them a major source of contagion. Gonococcal urethritis typically occurs 2–5 days after intercourse, with urethral orifice congestion, tenderness, and purulent discharge, along with grade I urinary frequency, urgency, dysuria, and burning during urination. Pressing the urethra yields purulent discharge. Gonococcal bartholinitis is usually unilateral, with redness, swelling, and severe pain at the gland’s opening, potentially forming an abscess in severe cases. Systemic symptoms such as fever may occur. Gonococcal vaginitis is rare, with milder symptoms in prolonged cases. Some patients experience abdominal heaviness, lumbago, and increased leucorrhea, while others may have lower abdominal pain and hypermenorrhea. Gonococcal vulvovaginitis presents as inflammation of the vulva and vagina, with abundant vaginal purulent discharge, sometimes yellow-green discharge from the vagina and urethra, painful urination, and vulvar redness and swelling. Discharge may spread to the anus, causing irritation. In severe cases, it may infect the rectum, leading to gonococcal proctitis.

II. Complicated Gonorrhea

(1) Complicated Gonorrhea in Males:

淋 nature of disease Urethritis has various complications, mainly including prostatitis, seminal vesiculitis, and epididymitis.

1. Prostatitis: Acute prostatitis is caused by gonococci entering the excretory ducts and glands of the prostate, presenting with fever,

chills, perineal pain, and urinary tract infection symptoms such as dysuria. Upon examination, the prostate is swollen and tender. However, gonococci are not a common cause of acute prostatitis. Gonococcal prostatitis mainly manifests as a chronic sexually transmitted disease, with mild symptoms such as perineal discomfort, penile pain, and a "sticky" urethral orifice in the morning. Gonorrheal threads may be seen in the urine, and prostatic massage fluid may contain pus cells and reduced lecithin. Smears or cultures may reveal gonococci. Digital rectal examination may detect small nodules on the prostate, accompanied by discomfort or pain. Scarring and contraction near the excretory ducts due to pus expulsion can affect ejaculation, leading to infertility.

2. Epididymitis: This usually occurs after acute urethritis and is mostly unilateral. Symptoms include low-grade fever, swollen and painful epididymis, and referred pain in the ipsilateral groin and lower abdomen. Initially, the boundary between the epididymis and testis is clear but gradually becomes indistinct. The testis becomes tender, swollen, and extremely painful. Urine is often turbid. Concurrent prostatitis and seminal vesiculitis may also occur.

3. Seminal Vesiculitis: In the acute phase, symptoms include fever, frequent urination, urgency, dysuria, and turbid terminal urine with blood. Rectal examination may reveal swollen seminal vesicles with severe tenderness. Chronic seminal vesiculitis is usually asymptomatic, but rectal examination may show hardened and fibrotic seminal vesicles.

4. Cowperitis: This occurs in the perineum or its vicinity, presenting with finger-sized nodules and pain. Acute cases may suppurate and rupture, compressing the urethra and causing dysuria. Systemic symptoms such as fever may also occur, with slow progression.

5. Urethral Stricture: Recurrent episodes can lead to urethral stricture, and a few cases may develop vas deferens stricture or obstruction, resulting in dysuria, thinning of the urinary stream, and, in severe cases, urinary retention. Secondary vas deferens stricture, seminal vesicle cysts, and infertility may also occur.

(II) Complicated Gonorrhea in Women

The main complications of gonorrhea in women include gonococcal pelvic inflammatory disease, such as acute salpingitis, endometritis, secondary tubo-ovarian abscess and its rupture leading to pelvic abscess, peritonitis, etc. Symptoms often appear suddenly after menstruation, including high fever, chills, headache, nausea, vomiting, lower abdominal pain, and increased purulent leucorrhea. Bilateral adnexa may be thickened and tender.

III. Gonorrhea in Other Sites:

1. Gonococcal Conjunctivitis: Newborns typically develop symptoms 2–3 days after birth, mostly bilaterally, with swollen and red eyelids and purulent discharge. In adults, it is often self-inoculated and unilateral, with similar manifestations as in newborns. Due to pus overflow, it is colloquially called "purulent eye." Delayed treatment can lead to corneal clouding, perforation, and blindness.

2. Gonococcal Pharyngitis: This is mainly seen in those who practice oral sex, often referred to as "oral淫" and is more common in Western homosexuals or heterosexuals. It presents as acute pharyngitis or tonsillitis, occasionally accompanied by fever and cervical lymphadenopathy. Symptoms include dry throat discomfort, sore throat, and pain on swallowing.

3. Gonococcal Anorectalitis: This is mainly seen in male homosexuals. In women, it is often caused by self-infection from the vagina. Symptoms include tenesmus, bloody and purulent stools, congested anal canal mucosa, and purulent discharge. Gonococcal cultures are positive.

IV. Disseminated Gonococcal Infection

Disseminated gonococcal infection occurs when gonococci spread systemically via the bloodstream, leading to severe systemic infection. The incidence is about 1% of gonorrhea cases. The strains causing disseminated gonococcal infection are often AHU-auxotype, which are stably resistant to normal human serum. Additionally, normal human serum contains IgM antibodies against gonococcal lipopolysaccharide, which, with the help of complement, are bactericidal to most gonococci. Patients deficient in complement components such as C5, C6, C7, and C8 are highly susceptible to gonococcal sepsis or gonococcal meningitis.

(1) Gonococcal septicemia: The patient initially develops fever, with body temperature potentially reaching 40°C, but typically ranging between 38°C-40°C. Shivering is uncommon, though some patients may experience skin papules, ecchymosis, pustular, hemorrhagic, or necrotic skin lesions, with some lesions exhibiting pain symptoms. At the lesion sites, gonococci can be detected via fluorescent immunoassay staining or cultured for gonococcal growth, and PCR tests show positive results for gonococcal DNA. The pathological tissue of the lesions presents as superficial ulcers with pus formation, diffuse inflammation in the dermis and subcutaneous tissue, infiltration of polymorphonuclear leukocytes, involvement of small blood vessels, thrombosis, and localized necrosis.

(II) Gonococcal arthritis: Joint swelling and pain, affecting one or several suppurative arthritis. Generally asymmetrical, rarely involving the hip, shoulder, or spinal joints. Gonococci are found in joint fluid tests, which can lead to bone destruction, fibrosis, and bony ankylosis.

(III) Gonococcal keratosis: Possibly caused by gonococci or their toxins, though gonococci are not found in the lesions. Often occurs concurrently with gonococcal arthritis. Lesions commonly appear on the hands, feet, ankles, heels, and waist. Typically flat, keratotic, slightly raised patches or plaques, conical in shape, yellow, copper-red, or gray-white. Lesions on the metatarsus show hyperkeratosis and large areas of keratinization.

(IV) Gonococcal endocarditis: In the decades before antibiotics, gonococci were a major pathogen of endocarditis. Currently, gonococcal endocarditis is almost never seen. It shares the same clinical manifestations as other types of endocarditis. When endocarditis occurs, it often affects the aortic or mitral valves, leading to subacute or acute endocarditis due to rapid valve destruction, which can be fatal.

(V) Gonococcal meningitis: Uncommon, may be accompanied by arthritis and typical rashes, distinguishing it from meningococcal meningitis.

V. Impact of Gonorrhea on Pregnancy and Newborns

When female gonorrhea is complicated by salpingitis, it can lead to infertility. The incidence of infertility due to female gonorrhea is about 20%, increasing with the number of infections. For women infected more than three times, the infertility rate can reach 70%. Cervical gonococcal inflammation can cause premature rupture of membranes, intra-amniotic infection, fetal intrauterine infection, intrauterine growth retardation, and premature labor. Newborns affected by premature labor, low birth weight, and sepsis have high morbidity and mortality rates. Postpartum ascending gonococcal infection can cause endometritis, puerperal fever, and, in severe cases, postpartum sepsis, neonatal gonococcal conjunctivitis, and gonococcal vulvovaginitis.

VI. Laboratory Tests

Gonococcal laboratory tests include smear examination, culture for gonococci, antigen detection, drug sensitivity testing, PPNG determination, and genetic diagnosis.

(I) Smear Examination

Collect urethral or cervical secretions from the patient and perform Gram staining. Gram-negative diplococci are identified within polymorphonuclear leukocytes.

For patients with simple gonococcal anterior urethritis and copious purulent discharge, the smear method has a positivity rate of about 90%, allowing for a preliminary diagnosis. Female cervical secretions contain many contaminants, resulting in lower sensitivity and specificity, with a positivity rate of only 50–60% and potential false positives. Therefore, the World Health Organization recommends culture methods for female patients. In chronic gonorrhea, gonococci are scarce in secretions, leading to low positivity rates. Prostatic massage fluid may be collected to improve detection rates.

The presence of Gram-negative diplococci in pharyngeal smears does not confirm gonorrhea, as other Neisseria species are normal flora in the throat. Additionally, atypical smear-positive cases should undergo further testing.

(II) Culture Examination

Gonococcal culture is an important supportive evidence for diagnosis. The culture method is a relatively sensitive approach for both males and females with mild or asymptomatic sexually transmitted diseases. A positive culture result can confirm the diagnosis. Before the advent of genetic diagnosis, culture was the only method recommended by the World Health Organization for screening gonorrhea. Currently, internationally recommended selective culture media include modified Thayer-Martin (TM) medium and New York City (NYC) medium. Domestically, chocolate agar or blood agar media are used, both containing antibiotics to selectively inhibit the growth of many other bacteria. Cultures are incubated at 36°C, 70% humidity, and in an environment containing 5-10% CO₂ (candle jar), with results observed after 24-48 hours. Post-culture identification includes colony morphology, Gram staining, oxidase testing, and sugar fermentation tests. The positive culture rates are 80-95% for males and 80-90% for females.

（3）Antigen Detection

1. Solid-phase enzyme immunoassay (EIA): Can be used to detect gonococcal antigens in clinical specimens. It is employed in areas with very high prevalence where culture is not feasible or when specimens require long-distance transportation. It can also be used to diagnose gonococcal infections in female populations.

2. Direct Immunofluorescence Assay: This test uses monoclonal antibodies against the outer membrane protein I of Neisseria gonorrhoeae for direct immunofluorescence. However, due to its currently low sensitivity in both male and female specimens, poor specificity, and the variability in judgment by laboratory personnel, this assay is not yet recommended for diagnosing gonococcal infections.

（4）Genetic Diagnosis

1. Gene Probe Diagnosis for Neisseria gonorrhoeae

Gene probe diagnosis for Neisseria gonorrhoeae utilizes the following probes: plasmid DNA probes, chromosomal gene probes, and rRNA gene probes.

（1）Plasmid DNA Probes

① Cryptic Plasmid DNA Probe: Gonococcal plasmids are classified into three types: conjugative plasmids, which are the largest (36 kb DNA); medicinal property-resistant plasmids, which include two plasmids with DNA lengths of 5.6 kb and 7.1 kb; and cryptic plasmids (4.2 kb). The cryptic plasmid is present in 96% of clinical gonococcal isolates and is absent in other Neisseria species, making its sequence suitable as a specific DNA probe for detecting Neisseria gonorrhoeae. Torres used nucleic acid hybridization with a cryptic plasmid probe to test 134 gonococcal strains and 131 related strains. The results showed 124 positive gonococcal hybridizations (93%), with some cross-reactivity observed with other Neisseria species. Sensitivity tests indicated the probe could detect 10² CFU of Neisseria gonorrhoeae. Studies confirmed that the CPPB gene sequence from the cryptic plasmid is present in all gonococcal chromosomes (including strains lacking the plasmid). Thus, the CPPB gene probe exhibits excellent specificity and sensitivity. Using a non-radioactive digoxigenin labeling system, Torres et al. tested 201 clinical specimens with the CPPB gene probe, achieving 95% sensitivity and 98% specificity.

② Medicinal Property-Resistant Plasmid DNA Probe

Gonococcal drug-resistant plasmids can be categorized into: ① Penicillinase-producing Neisseria gonorrhoeae (PPNG), which is β-lactamase-positive; and ② Plasmid-mediated high-level tetracycline-resistant Neisseria gonorrhoeae (TRNG).

PPNG strains were first isolated in the laboratory in 1976. These strains contain genes encoding penicillinase, which can integrate into either the chromosome or plasmid DNA (with the latter being more common). These plasmids are termed penicillinase-producing plasmids, with sizes of 7.4 kb and 5.3 kb. In 1998, Pescador designed a specific probe targeting the β-lactamase gene in Neisseria gonorrhoeae, using enzyme chemiluminescence labeling and liquid-phase hybridization. A luminometer was used to measure the light emitted by specific hybrids, enabling the detection of 10⁴–10⁵ CFU of PPNG strains within 4 hours. Although TRNG strains are resistant to tetracycline, they are typically sensitive to β-lactams and quinolone antibiotics. Therefore, in laboratory susceptibility testing, they may be classified as sensitive bacteria. Pescador used an oligonucleotide probe targeting the tetM gene of TRNG, which mediates tetracycline resistance. Using enzyme chemiluminescence labeling and liquid-phase hybridization, the probe could directly detect 1.5 × 10⁴ CFU of gonococci carrying the tetM gene in clinical specimens within 4 hours.

（2）Chromosomal Probe

Chromosomal probes include gene probes with known functions, such as the pilus DNA probe and the paI gene probe, which play important roles in the process of Neisseria gonorrhoeae infecting human cells; and gene probes with unknown functions, which have sequences complementary to specific chromosomal sequences but whose functions are currently unknown. Due to the low copy number of complementary sequences in Neisseria gonorrhoeae, these two types of chromosomal probes generally have low detection sensitivity and are rarely used unless for specific research purposes.

(3) rRNA Gene Probe

The rRNA gene probe uses DNA complementary to rRNA as the probe, with the target sequence being the rRNA sequence. The characteristics of the rRNA gene probe are: ① It can enhance the sensitivity of probe detection, as the rRNA gene probe can simultaneously detect rRNA molecules and DNA molecules; ② rRNA exhibits evolutionary conservation; ③ The hybridization method is simple and rapid; ④ Due to the high abundance of rRNA, the specimen does not require enrichment. The gonococcal detection probe PACE C, produced by Gen-Probe in the United States, targets rRNA and its gene sequences, employing radioactive labeling, and can complete detection within 2 hours. Peter used this probe to test 395 clinical specimens, achieving sensitivity and specificity of 92.9% and 99.4%, respectively. He concluded that the PACE C system is a reliable method for screening gonococci in clinical specimens. This probe can also detect asymptomatic gonococcal infections, which is currently difficult to achieve with culture methods.

2. Gene Amplification Detection of Gonococci

Although the probe technology described above for detecting gonococci has significantly improved sensitivity, specificity, and convenience compared to culture methods, it still has certain limitations, such as often requiring high concentrations of gonococci in the specimen. The emergence of PCR technology and ligase chain reaction has further enhanced the sensitivity of gonococcal detection, offering advantages of speed, sensitivity, specificity, and simplicity, enabling direct detection of extremely low levels of pathogens in clinical specimens.

(1) Extraction of Gonococcal DNA

① DNA Extraction from Cultured Bacteria

The cultured gonococci are lysed in an alkaline lysis solution at a concentration of 10² cfu/ml. The lysis solution consists of 1M NaCl, 1M NaOH, and 1% sodium dodecyl sulfate. After mixing, the solution is boiled for 1 minute, then neutralized with 100μl of 1M Tris pH 7.0. The DNA is extracted once with Tris-balanced phenol, once with phenol-chloroform, and then precipitated with anhydrous ethanol or isopropanol. The extracted DNA is dissolved in 30μl of distilled water or TE buffer.

② Extraction from Clinical Swab Specimens

The swab with secretions is squeezed and washed for 1 minute in 2ml of sterile saline or PBS buffer to dissolve the specimen as much as possible. The swab is discarded, and the suspension is centrifuged at 2,000–3,000 rpm for 5 minutes. The supernatant is removed, and the cells are resuspended in 100μl of 1×PCR buffer containing 0.45% Tween 20 and 200μg/ml proteinase K. The cell suspension is incubated at 50–60°C for 1 hour, then heated at 95°C for 10 minutes to inactivate proteinase K. After centrifugation at 12,000 rpm for 10 minutes, the supernatant contains the DNA template.

(2) Design of PCR Primers

Since the CPPB gene of the gonococcal cryptic plasmid exists in both the gonococcal chromosome and the 4.2 kb cryptic plasmid, and this cryptic plasmid is present in 96% of gonococci, many PCR primers are designed for 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 2μl of gonococcal DNA extract and add it to 28μl of reaction solution. The final PCR reaction solution contains 100μmol/L of each dNTP, 0.5μmol/L of each primer, 1U of Taq DNA polymerase, and Mg²⁺ 1.5mmol/L. Add 30μl of sterile mineral oil and centrifuge at 1000r/min for 30s. Perform PCR amplification cycles under the following conditions: denaturation at 94℃ for 1min, then 94℃ for 30s, 57℃ for 1min, and 72℃ for 1min. A total of 30 cycles are performed, followed by a final extension at 72℃ for 5min.

The amplification products are electrophoresed on a 2% agarose gel for 30min, stained with ethidium bromide, and visualized under UV light. The amplified DNA fluorescent bands should match the expected size of the target sequence amplified by the primers.

（4）Sensitivity and Specificity of PCR

Since the CPPB gene is also present on the chromosome of gonococci without cryptic plasmids, and 96% of gonococci carry cryptic plasmids, primers targeting the CPPB sequence exhibit extremely high sensitivity. Experiments have shown that the conventional one-step PCR (GC1-GC2) method can detect as few as 3 gonococci, while the single-tube nested PCR (GC2-GC4) can detect ≤0.3 gonococci (9 CPPB genes). Specificity tests confirm that these primers only amplify gonococcal DNA and do not produce specific products for non-gonococcal Neisseria.

（5）Single-Tube Nested PCR Method

This method is based on traditional nested PCR but involves special design of two pairs of PCR primers. The outer primers (GC1, GC2) are 25bp with a higher annealing temperature (68℃), while the inner primers (GC3, GC4) are 17bp with a lower annealing temperature (46℃). Other components of the PCR reaction solution are the same as in conventional PCR. By controlling the annealing temperature (68℃), the outer primers are amplified first. After 20–30 cycles (first PCR), the annealing temperature is lowered (46℃) to allow the inner primers to perform nested amplification using the first PCR products as templates. This PCR method achieves a sensitivity sufficient to detect 0.3 gonococci.

(6) Neisseria gonorrhoeae ligase chain reaction (LCP) detection method

Currently, PCR testing for Neisseria gonorrhoeae is widely used, with its specificity and sensitivity continuously improving. At the same time, another genetic diagnostic technique—the Ligase Chain Reaction (LCP)—has also been applied to the detection of Neisseria gonorrhoeae due to its high specificity and sensitivity. The difference between LCP and PCR lies in the fact that LCP uses four pairs of primers, and the enzyme employed is ligase. Ligase can connect two adjacent primers. The connected pair of primers can serve as a template for another pair of primers, which are then connected under the action of ligase and can further serve as a template. This process is repeated for 30–40 cycles. The template preparation method used in LCP is equivalent to that of PCR. The probes used in LCP can be designed not only on the CPPB gene but also on chromosomal gene sequences, such as the opa-1 gene. The Abbott Laboratory in the United States designed four LCP probes within a 48bp region of the opa-1 gene. Since the opa-1 gene is repeated 11 times in the chromosome of Neisseria gonorrhoeae, this set of LCP probes exhibits high sensitivity and specificity. The LCP reaction process:

Add the template to the LCP reaction solution. The LCP reaction solution consists of: 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; 40 fmol/L each of two adjacent labeled probes; 40 fmol/L each of two unlabeled probes; and 15U of heat-stable ligase. The reaction conditions are: 97°C for 1s, 55°C for 1s, and 62°C for 50s, for 40 cycles. Add 100 μL of the reaction product to the microplate wells for color development, and finally read the optical values using a microplate reader. According to Buimer's experiments, the sensitivity of LCP in detecting male urethral swab samples was 100%, urine samples 88.9%, and female cervical swab samples 95.4%. The specificity of the LCP method was as high as 100%, significantly higher than that of PCR, thereby avoiding false positives.

3. Considerations for Clinical Genetic Diagnosis of Neisseria gonorrhoeae

Currently, the PCR method is primarily used for the genetic diagnosis of Neisseria gonorrhoeae in clinical testing. However, several issues should be noted when applying this method in clinical detection.

(1) Primer Design In addition to the Neisseria gonorrhoeae PCR primers listed above, primers can also be designed from other genes.

However, the primer sequences must be specific. Due to the large size of bacterial chromosomes, many gene sequences remain unclear. Additionally, bacteria share varying degrees of homology with one another, and plasmid sequences among bacteria also exhibit homology. Therefore, primer design must involve comparative analysis of gene databases, along with specificity and sensitivity experiments, to select primers suitable for clinical testing.

(2) Clinical Specimen Processing For clinical specimens, the PCR template should be as pure as possible. This requires collecting samples from accurate locations. For asymptomatic patients, an adequate number of samples should be taken to ensure the collection of pathogenic bacteria. Furthermore, since clinical specimens are often complex in composition, simply processed specimens may not yield ideal PCR amplification results, possibly due to excessive impurities. Further purification, such as phenol-chloroform extraction, can improve outcomes. Although this purification method is cumbersome, commercially available DNA purification kits now allow for the relatively simple extraction of high-purity DNA from clinical specimens.

(3) Detection methods for PCR products Not long ago, clinical PCR detection of Neisseria gonorrhoeae almost exclusively used electrophoresis for PCR product identification. This method had many issues, such as false-positive and false-negative results due to the subjectivity of visual observation. Currently, hybridization colorimetry has replaced electrophoresis, improving the specificity and sensitivity of result interpretation.

In summary, both the PCR method and the LCP method have significantly improved sensitivity and specificity compared to traditional culture methods, while also greatly reducing the required time. With the continuous advancement of genetic diagnostic techniques, the PCR and LCP methods will become routine detection methods for Neisseria gonorrhoeae.

(5) Drug Susceptibility Testing: After a positive culture, further drug susceptibility testing is conducted. Sensitivity tests are performed using the disk diffusion method, or the minimum inhibitory concentration (MIC) is determined using the agar plate dilution method to guide antibiotic selection.

(6) PPNG Detection: For β-lactamase, the quantitative acidometric disk method is used, employing Whatman No. 1 filter paper. PPNG

strains can change the color from blue to yellow, with a positive result indicating PPNG and a negative result indicating N-PPNG.

bubble_chart Diagnosis

The diagnosis must be confirmed through comprehensive analysis based on exposure history, clinical manifestations, and laboratory tests.

(1) Exposure history: The patient has a history of extramarital sexual behavior or visiting prostitutes, a spouse with an infection history, shared items with gonorrhea patients (especially those at home), or a newborn whose mother has a history of gonorrhea.

(2) Clinical manifestations: The main symptoms of gonorrhea include frequent urination, urgency, dysuria, purulent discharge from the urethral orifice or cervical/vaginal orifice, or manifestations such as gonococcal conjunctivitis, enteritis, pharyngitis, or symptoms of disseminated gonorrhea.

(3) Laboratory tests: Microscopic examination of urethral discharge in males with acute gonococcal urethritis has preliminary diagnostic significance, while for females, it serves only as a reference. Culture should be performed to confirm gonococcal infection. In well-equipped facilities, genetic diagnostic methods can be used for definitive diagnosis.

bubble_chart Treatment Measures

Gonorrhea is an extremely contagious and easily reinfected sexually transmitted disease. It is often complicated by infections such as chlamydia. The gonococcus is prone to developing drug resistance and can lead to complications and sequelae, necessitating sufficient attention in treatment. Since the use of sulfonamides in 1935 and penicillin in 1944 to treat gonorrhea, relatively good therapeutic outcomes have been achieved. However, the emergence of penicillin-resistant, tetracycline-resistant, and other drug-resistant strains has posed challenges in the treatment of gonorrhea.

I. Treatment Principles:

1. Early diagnosis and early treatment. 2. Timely, adequate, and standardized medication. 3. Tailored treatment methods based on different conditions. 4. Partner tracing and simultaneous treatment. 5. Follow-up and re-examination after treatment. 6. Attention to concurrent infections such as chlamydia, mycoplasma, and other STDs.

II. Treatment Plans:

1. Gonococcal urethritis and cervicitis: - Procaine penicillin G, 4.8 million units mixed with 100 mL saline for intravenous drip. - Ampicillin 4.0 g taken orally once or administered via intravenous drip. - Amoxicillin 4.0 g administered at once. Any one of the above three drugs may be selected. For those allergic to penicillin, tetracycline 0.5 g every 6 hours for 7 days may be used. Erythromycin-class drugs such as Lijunsha, azithromycin, or roxithromycin may be taken as directed for 7 days.

For penicillinase-producing Neisseria gonorrhoeae (PPNG), or penicillin-resistant gonococci, when the prevalence of penicillin-resistant gonococci exceeds 5%, penicillin should be combined with sulbactam sodium. Alternatively, other drugs may be selected:

1. Cephalosporins: Ceftriaxone 3.0 g intravenous drip, or cefotaxime sodium 4.0 g intravenous drip.

2. Spectinomycin, also known as Trobicin, 2 g intramuscular injection once. Some recommend 4 g intramuscular injection once for females.

3. Quinolones: Ofloxacin (Tarivid) 600 mg taken orally once, or ofloxacin 200 mg intravenous drip.

Note: Quinolones are contraindicated in pregnant women and children.

4. β-lactamase inhibitors combined with penicillin-class drugs: - Unasyn (ampicillin-sulbactam) 1.5 g intramuscular injection once. - Timentin (piperacillin sodium-sulbactam sodium) 3.0 g intramuscular injection or intravenous drip once.

Since some gonorrhea patients also have concurrent chlamydia infections, ceftriaxone sodium 3.0 g intravenous drip is often used in treatment, along with oral azithromycin or roxithromycin 250 mg twice daily.

2. Gonococcal pharyngitis: - Ceftriaxone sodium 3.0 g intravenous drip. - Ofloxacin 250 mg orally three times daily. - Compound sulfamethoxazole (Co-trimoxazole) 1 g twice daily for 7 days.

3. Gonococcal proctitis: - Ceftriaxone sodium 3.0 g intravenous drip. - Cefixime 3.0 g intravenous drip. - Ceftazidime 3.0 g intravenous drip.

4. Gonococcal ophthalmia (adults): - Aqueous penicillin G 10 million units intravenous drip once daily for 5 days.

5. Pediatric gonorrhea: - For children weighing ≥45 kg, administer adult doses. <45kg的兒童按以下方法給藥：頭孢曲松鈉125mg，一次肌肉注射；或樂施福定25mg/kg，一次肌肉注射；或壯觀黴素40mg/kg，一次肌肉注射。

6. Complicated gonorrhea:

- Gonorrhea complicated with salpingitis and epididymitis: - Aqueous procaine penicillin G 4.8 million units intravenous drip twice daily for 7 days. - For PPNG-induced cases: Timentin 3.0 g once daily for 7 days, or spectinomycin 2 g intramuscular injection once daily for 10 days, or ceftriaxone sodium, ceftazidime, or cefixime 3.0 g intravenous drip once daily for 7 days.

② Disseminated gonorrhea: Aqueous penicillin G 10 million units intravenously once daily for 7 days, or ceftriaxone sodium 3.0g intravenously once daily for 7 days.

bubble_chart Prognosis

Patients with gonorrhea can be completely cured if treated promptly and correctly during the acute phase. Uncomplicated gonorrhea has a cure rate of 95% with a single high-dose medication. Inadequate treatment may lead to complications, including infertility, ectopic pregnancy, pelvic inflammation, urethral stricture, blindness, or disseminated gonorrhea. Therefore, it is crucial to seize the opportunity to thoroughly treat gonorrhea during the acute phase.

bubble_chart Prevention

1. Promote knowledge about sexually transmitted diseases, advocate noble moral values, and strictly prohibit prostitution and solicitation.

2. Using condoms can reduce the incidence of gonococcal infection.

3. Prophylactic use of antibiotics can lower the risk of infection. Taking norfloxacin or amoxicillin before and after sexual intercourse can effectively prevent sexually transmitted disease infections.

4. Simultaneous treatment of sexual partners is necessary.

5. Patients should pay attention to personal hygiene and isolation, avoiding sharing beds or baths with family members, especially children and young girls.

6. Implement the system of applying silver nitrate solution or other antibiotic eye drops to newborns to prevent gonococcal ophthalmia.

bubble_chart Differentiation

(1) Nongonococcal urethritis: The incubation period is relatively long, ranging from 7 to 21 days. Urethral discharge is minimal or absent, appearing as serous or mucoid, thin in consistency, with mild symptoms and no systemic manifestations. The primary pathogens are Chlamydia trachomatis and Ureaplasma urealyticum.

(2) Candidal vaginitis: The main clinical symptoms include vulvar and vaginal cutaneous pruritus. Leucorrhea increases, appearing white and watery or gel-like. The vaginal mucosa shows congestion, edema, and adherent white membranes, with grade I erosions where the white membranes detach. Microscopic examination of the white membranes reveals spores and hyphae.

(3) Trichomonal vaginitis: The main clinical symptoms include vaginal cutaneous pruritus, with discharge often appearing frothy. The vaginal mucosa and cervix exhibit marked congestion and characteristic strawberry-like hemorrhagic spots. The vaginal mucosa frequently bleeds, resulting in bloody discharge. Trichomonads can be detected in the discharge.

(4) Bacterial vaginosis: Leucorrhea increases, appearing gray and homogeneous, with an elevated pH and a fishy odor. Microscopic examination shows a reduction in lactobacilli and an increase in Gram-negative bacteria.