Neisseria gonorrhoeae grows well only on chocolate agar and on specialized medium enriched to ensure its growth. It requires carbon dioxide supplementation. Small, smooth, nonpigmented colonies appear after 18 to 24 hours and are well developed (2-4 mm) after 48 hours. Gonococci possess numerous pili which are structurally similar to those of meningococci and extend beyond the outer membrane (Figure 30–6), (Table 30–1). The gonococcal outer membrane is composed of phospholipids, LPS, LOS, and several distinct OMPs. The OMPs include porins (Por1BA and Por1BB) and adherence proteins known as Opa. Opa proteins are a set of at least 12 proteins that get their name from the opaque appearance they give to colonies as a result of adhesion between gonococcal cells. A variable number of the Opa proteins may be expressed at any one time.
Neisseria gonorrhoeae pili. This view is a cross-section of the microcolony of gonococci on the surface of an epithelial cell originally shown in Figure 22–2 (inset). Pili are actively attaching to the epithelial cell surface and using a contractile force (twitching motility) to move and modify the surface. (Photomicrographs kindly provided by Dustin L. Higashi and Magdalene So.)
Chocolate agar and CO2 are required
Fresh isolates have pili
LOS and OMPs are in outer membrane
Opa proteins are adherence OMPs
Neisseria gonorrhoeae and N meningitidis are among several microorganisms whose surface structures are known to change antigenically from generation to generation during growth of a single strain. The mechanisms involved have been more extensively studied in gonococci but appear to be similar in both species. The major gonococcal structures known to undergo antigenic variation are pili, Opa proteins, and LOS. The genetic mechanisms are discussed below and illustrated in Figure 22–5.
Pili, OMPs, and LOS vary in gonococci and meningococci
Gonococcal pili are antigenically variable to an extraordinary extent. There are multiple genetic mechanisms, but the most important one appears to be recombinational exchange between the multiple pilin genes present in the chromosome of every strain. Some of these genes are complete and able to express pilin (pilE). Others are not, due to lack of an effective promoter and are thus silent (pilS). When recombination between expression and silent loci results in the donation of new sequences to an expression locus, the result can be expression of a pilin with changes in its amino acid composition and thus its antigenicity. The recombination could also involve exogenous DNA from another cell or strain, because gonococci naturally take up species-specific DNA by transformation. The process is complex, involving other genes that play a role in the assembly of pili and their functional characteristics, such as cellular adhesion. The numerous possible outcomes include no pilin subunits, pilin subunits unable to assemble, mature pili with altered functional characteristics, and fully functional pili with a new antigenic makeup.
Genes for pilin subunits may be expressed or silent
Recombination between multiple genes occurs
Outcome may be nonfunctional or antigenically altered pili
The multiple gonococcal Opa proteins are each encoded by separate genes scattered around the genome. Various combinations of these genes may be either “on” or “off” at any one time. The switch is set during the transcription of each Opa gene for the next cell generation. As a result of a process called replicative slippage, the number of repeats of particular gene sequence can vary. When the time comes for translation, the number of repeats determines whether the gene will be in or out of frame to translate its Opa protein. If it is in frame, the gene is “on”; if not, the switch is “off.” Variation in gonococcal LOS has been observed in volunteer subjects challenged with intraurethral N gonorrhoeae, but the genetic mechanism is unknown.
Multiple Opa genes may be “on” or “off”
Translational frame shift controls the switch
LOS also varies antigenically
These changes in the gonococcal surface are random events which may or may not have survival value depending on the circumstances. During the early stages of infection there could be positive selection for the expression of pili and Opas that mediate adherence. If the host has antibodies against one or more of these proteins they would be removed and the infecting population would shift to cells expressing pili or Opas to which there is no immunologic experience. An example of how these antigenic variants could be selected is shown in Figure 30–7. Taken together, these multifactorial, antigenic variations of the gonococcal surface may serve the dual purposes of escape from immune surveillance and timely provision of the ligands required to bind to human cell receptors.
Gonococcal antigenic variation. A. A population of gonococci is shown with surface pili. There are two antigenic types of pili in the population, one of which is dominant. B. IgG against the dominant blue pilin type is introduced and binds all the cells with that pilin type on the surface. C. Later, the bound gonococci with their pili are clumped at the bottom. The minor (red) pilin type present in A now predominates, and a new one (green) has appeared but is still a minority member of the population. Antibody directed against the now dominant member would allow the new green one to take over. The same kind of population change occurs based on antigenic variation of outer membrane proteins. The genetic mechanisms involved in generating multiple antigenic types are illustrated in Figure 22–5.
In contrast to meningococcal disease, gonorrhea is primarily localized to mucosal surfaces with relatively infrequent spread to the bloodstream or deep tissues. Infection is sexually acquired by direct genital contact, and the primary manifestation is pain and purulent discharge at the infected site. In men, this is typically the urethra, and in women, the uterine cervix. Direct extension of the infection up the fallopian tubes produces fever and lower abdominal pain, a syndrome called pelvic inflammatory disease (PID). For women, sterility or ectopic pregnancy can be long-term consequences of gonorrhea.
Gonorrhea is one of our greatest public health problems. The more than 300 000 cases reported in the United States each year are felt to represent less than 50% of the true number and the rates for adolescents are alarmingly high and increasing by 10% a year. The highest rates are in women between the ages of 15 and 19 years and in men between the ages of 20 and 24 years. No truly effective means of control is yet in sight. Our ability to stem the tide of changed sexual mores continues to be hampered by lack of an effective means to detect asymptomatic cases, resistance of N gonorrhoeae to antibiotics (see Treatment), and, to some extent, lack of appreciation of the importance of this disease. The latter is evidenced by failure of patients to seek medical care and reluctance to report cases to public health authorities due to privacy concerns. In the minds of too many, syphilis is dreaded and “unclean,” whereas gonorrhea is only “the clap” (“clap” is from the archaic French clapoir, “a rabbit warren”; later, “a brothel”).
Rates among adolescents are high and increasing
Inability to detect asymptomatic cases hampers control
Gonorrhea is acquired by genital contact with an infected person. The major reservoir for continued spread is the asymptomatic patient. Screening programs and case contact studies have shown that almost 50% of infected women are asymptomatic or at least do not have symptoms usually associated with venereal infection. Most men (95%) have acute symptoms with infection. Many who are not treated become asymptomatic but remain infectious. Asymptomatic male and female patients can remain infectious for months. The attack rates for those engaging in sexual intercourse with an infected person are estimated to be 20% to 50%. The organism may also be transmitted by oral–genital contact or by rectal intercourse. When all these factors operate in a sexually active population, it is easy to explain the high prevalence of gonorrhea. Although gonococci can survive for brief periods on toilet seats, nonsexual transmission is extremely rare. Virtually all gonococci isolated from children can be traced to sexual abuse by an infected adult.
Intercourse risk is up to 50%
Asymptomatic cases are highest in women
Nonsexual transmission is rare
Gonococci are not normal inhabitants of the respiratory or genital flora. When introduced onto a mucosal surface by sexual contact with an infected individual, adherence ligands such as pili and Opa proteins allow initial attachment of the bacteria to receptors (CD46, CD66, integrins) on nonciliated epithelial cells (Figure 30–2). Initial attachment is mediated by the pili, which have been shown to generate an active force with movement in microcolonies across the cell surface (Figure 30–6). This is followed by a tighter attachment owing to Opa proteins. This close binding provides an opportunity for other OMPs (Por1BA) to trigger signaling cascades activating multiple enzymatic systems within the host cell. These reactions lead to induction of phagocytosis of the gonococci in a process involving microfilaments and microtubules of the invaded cell. The microvilli surround the bacteria and appear to draw them into the host cell in the same manner as meningococci. Thus, after initial attachment the gonococcus appears to induce the host cell to actively take it inside (Figure 30–2). Once inside, the bacteria transcytose the cell and exit through the basal membrane to enter the submucosa.
Pili and Opa proteins mediate attachment to nonciliated epithelium
Gonococci induce their own phagocytosis
Bacteria quickly pass to submucosa
Survival in the Submucosa
Once in the submucosa, the bacteria must survive and resist innate host defenses as well as adaptive immune responses acquired from a previous infection. As with meningococci, siderophores on the gonococcal surface enable the organisms to scavenge iron needed for growth from human iron transport proteins. Although gonococci lack the polysaccharide capsule of the meningococcus, they still have multiple mechanisms that protect them against serum complement and antibody. One of these is LOS sialyation in which the gonococcus is able to incorporate host sialic acid onto its own surface. This provides a mechanism for blocking surface C3b deposition by direct LOS/sialic acid binding of factor H or by facilitating its binding to surface porins.
Receptors scavenge iron
Sialated LOS binds factor H
Even when phagocytes do encounter gonococci, surface factors such as pili and Opa proteins interfere with effective phagocytosis. The organisms are also able to defend against oxidative killing inside the phagocyte by upregulation of catalase production and an efficient antioxidant defense system. Taken together, these factors provide ample evidence that killing by neutrophils is sufficiently retarded to allow prolonged survival of gonococci in mucosal and submucosal locations.
Phagocytosed gonococci resist killing
In contrast to meningococci, N gonorrhoeae bacteria tend to remain localized to genital structures, causing inflammation and local injury, which no doubt facilitate their continued venereal transmission. Purulent exudates containing “sticky” clusters of gonococci held together by Opa proteins could be the primary infectious unit. Infection may spread to deeper structures by progressive extension to adjacent mucosal and glandular epithelial cells. These include the prostate and epididymis in men and the paracervical glands and fallopian tubes in women (Figure 30–8). Spread to the fallopian tubes is facilitated by pilus-mediated attachment to sperm and then to the microvilli of nonciliated fallopian tube cells. Injury to the fallopian epithelium is mediated by the local effect of outer membrane LPS. Gonococci are also known to turn over their peptidoglycan rapidly during growth, releasing peptidoglycan fragments which are also toxic to the ciliated epithelium of the fallopian tube.
Local spread is to epididymis and fallopian tubes
LOS and peptidoglycan shedding cause local injury
In a small proportion of infections, organisms reach the bloodstream to produce disseminated gonococcal infection (DGI). When this happens, the systemic findings have their own pattern (see Manifestations) and seldom take on the endotoxic shock picture of meningococcemia. Although differences have been noted between N gonorrhoeae strains that remain localized and those that produce DGI, their connection to pathogenesis is unknown. Both DGI and salpingitis tend to begin during or shortly after completion of menses. This may relate to changes in the cervical mucus and reflux into the fallopian tubes during menses.
DGI differs from meningococcal endotoxic shock
Reflux during menses may facilitate spread
Genetic Regulation of Virulence
Through all the stages of gonorrhea, gonococci are able to use a particularly rich variety of genetic mechanisms in deployment of the virulence factors previously described at the right time. Some are regulatory responses to environmental cues, such as iron in relation to iron-binding proteins, whereas others involve changes in the genome. Antigenic changes in both pili and Opa proteins have been demonstrated in human infection, including the isolation of antigenic variants from different sites in the same patient. These presumably take place by the recombinational and translational mechanisms described above (see Antigenic Variation) as the organisms replicate in the patient.
Regulation, recombination, and translational changes deploy virulence factors
The apparent lack of immunity to gonococcal infection has long been a mystery. Among sexually active persons with multiple partners, repeated infections are the rule rather than the exception. Both serum and secretory antibodies are generated during natural infection, but the levels are generally low, even after repeated infections. Another aspect is that even when antibodies are formed, antigenic variation defeats their effectiveness and allows the gonococcus to escape immune surveillance. Antigenic variation of pili, Opa proteins, and LOS is particularly likely to be important. Outbreaks have been traced to a single strain that demonstrated multiple pilin variations and Opa types in repeated isolates from the same individual or from sexual partners. In experimental models, passive administration of antibody directed against one pilin type has been followed by emergence of new pilin variants presumably through the sequence illustrated in Figure 30–7. It appears that although some immunity to gonococcal infection is present, its effectiveness is compromised by the ability of the organism to change key structures during the course of infection.
Antibody response is weak
Gonococcus varies multiple structures to avoid immune surveillance
GONORRHEA: CLINICAL ASPECTS
The clinical spectrum of gonorrhea differs substantially in men and women (Figure 30–8). In men, the primary site of infection is the urethra. Symptoms begin 2 to 7 days after infection and consist primarily of purulent urethral discharge and dysuria. Although uncommon, local extension can lead to epididymitis or prostatitis. The endocervix is the primary site in women, in whom symptoms include increased vaginal discharge, urinary frequency, dysuria, abdominal pain, and menstrual abnormalities. As mentioned previously, symptoms may be mild or absent in either sex, particularly women.
Gonorrhea in men and women. The majority of cases in women are asymptomatic. Local extension up the fallopian tubes causes salpingitis. The majority of men have acute urethritis, and only a small percentage have local extension to the epididymis. A very small part of either spectrum results in bacteremia and disseminated gonococcal infection.
Urethritis and endocervicitis are primary infections
Rectal gonorrhea occurs after rectal intercourse or, in women, after contamination with infected vaginal secretions. This condition is generally asymptomatic, but may cause tenesmus, discharge, and rectal bleeding. Pharyngeal gonorrhea is transmitted by oral–genital sex and, again, is usually asymptomatic. Sore throat and cervical adenitis may occur. Infection of other structures near primary infection sites, such as Bartholin glands in women, may lead to abscess formation.
Rectal and pharyngeal infections relate to sexual practices
Inoculation of gonococci into the conjunctiva produces a severe, acute, purulent conjunctivitis. Although this infection may occur at any age, the most serious form is gonococcal ophthalmia neonatorum, a disease acquired during childbirth by a newborn from an infected mother. The disease was formerly a common cause of blindness, which is now prevented by the administration of prophylactic topical eye drops or ointment (silver nitrate, erythromycin, or tetracycline) at birth.
Transmission at birth causes ophthalmia neonatorum
Pelvic Inflammatory Disease
The clinical syndrome of pelvic inflammatory disease (PID) develops in 10% to 20% of women with gonorrhea. The findings include fever, lower abdominal pain (usually bilateral), adnexal tenderness, and leukocytosis with or without signs of local infection. These features are caused by spread of organisms along the fallopian tubes to produce salpingitis and into the pelvic cavity to produce pelvic peritonitis and abscesses (Figure 30–9). PID is also known to develop when other genital pathogens ascend by the same route. These organisms include anaerobes and Chlamydia trachomatis, which may appear alone or mixed with gonococci. The most serious complications of PID are infertility and ectopic pregnancy secondary to scarring of the fallopian tubes.
Tubo-ovarian abscess. This large abscess in the fallopian tube is part of the spectrum of pelvic inflammatory disease (PID) of which Neisseria gonorrhoeae is a major cause. (Reproduced with permission from Connor DH, Chandler FW, Schwartz DQ, et al: Pathology of Infectious Diseases. Stamford CT: Appleton & Lange, 1997.)
Salpingitis and pelvic peritonitis cause scarring and infertility
Disseminated Gonococcal Infection
Any of the local forms of gonorrhea or their extensions such as PID may lead to bacteremia. In the bacteremic DGI phase, the primary features are fever, migratory polyarthralgia, and a petechial, maculopapular, or pustular rash. Some of these features may be immunologically mediated. Gonococci are infrequently isolated from the skin or joints at this stage despite their presence in the blood. The bacteremia may lead to metastatic infections such as endocarditis and meningitis, but the most common is purulent arthritis. The arthritis typically follows the bacteremia and involves large joints such as elbows and knees. Gonococci are readily cultured from the pus.
Skin rash, arthralgia, and arthritis are associated with bacteremia
Purulent arthritis involves large joints
The presence of multiple pairs of bean-shaped, Gram-negative diplococci within a neutrophil is highly characteristic of gonorrhea when the smear is from a genital site (Figure 30–1). The direct Gram smear is more than 95% sensitive and specific in symptomatic men. Unfortunately, it is only 50% to 70% sensitive in women, and its specificity is complicated by the presence of other bacteria in the female genital flora that have similar morphology. Experience is required in reading smears, particularly in women. A positive Gram smear is generally accepted as diagnostic in men. It should not be used as the sole source for diagnosis in women or when the findings have social (divorce) or legal (rape, child abuse) implications.
Direct smear is useful in men
Interfering flora complicates interpretation in women
Attention to detail is necessary for isolation of the gonococcus because it is a fragile organism that is often mixed with hardier members of the genital flora. Success requires proper selection of culture sites, protection of specimens from environmental exposure, culture on appropriate media, and definitive laboratory identification. In men, the best specimen is urethral exudate or urethral scrapings (obtained with a loop or special swab). In women, cervical swabs are preferred over urethral or vaginal specimens. The highest diagnostic yield in women is with the combination of a cervical and an anal canal culture; this is because some patients with rectal gonorrhea have negative cervical cultures. Rectal cultures in men and throat cultures are needed only when indicated by sexual practices.
Urethra and cervix are preferred culture sites
Swabs may be streaked directly onto culture medium or promptly transmitted (in <4 hours) to the laboratory in a suitable transport medium. Laboratory requests must specify the suspicion of gonorrhea so that media that satisfy the nutritional requirements of the gonococcus and inhibit competing normal flora can be seeded. The selective medium (eg, Martin-Lewis agar) is an enriched selective chocolate agar with antibiotics. The exact formulation has changed over the years, but includes agents active against Gram-positive bacteria (vancomycin), Gram-negative bacteria (colistin, trimethoprim), and fungi (nystatin, anisomycin) at concentrations that do not inhibit N gonorrhoeae.
Transport media required unless plating is immediate
Selective medium inhibits competing flora
Colonies appear after 1 to 2 days of incubation in carbon dioxide at 35°C. They may be identified as Neisseria by demonstration of typical Gram stain morphology and a positive oxidase test. Classically, speciation is by carbohydrate degradation pattern, but this approach has been replaced by immunologic procedures (immunofluorescence, coagglutination, enzyme immunoassay) using monoclonal antibodies to unique antigens. Neisseria species other than N gonorrhoeae are unusual in genital specimens, but speciation is the only way to be certain of the diagnosis.
Isolates are identified by fermentation or immunoassay
Much effort has been directed at developing immunoassay and nucleic acid amplification (NAA) methods that detect gonococci in genital and urine specimens without culture. Such methods have particular importance for screening populations in which culture is impractical. After a series of improvements NAA methods are now considered at least as sensitive as culture for diagnosis of gonorrhea. They are also highly specific when performed on genital specimens but less so for samples from nongenital sites. Throat specimens are most likely to generate false positives due to the regular presence of commensal Neisseria and the roughly 10% probability of closely related N meningitidis colonization. Thus, NAA results are considered diagnostic from genital sites (including urine) but must be confirmed by culture from other sites. The cost/benefit ratio of NAA tests has been improved by combining them with Chlamydia detection (Chapter 39), which targets the same clinical population.
NAA methods are sensitive and specific from genital sites
Gonococci and Chlamydia are combined in testing
Attempts to develop a serologic test for gonorrhea have not yet achieved the needed sensitivity and specificity. A test that would detect the disease in asymptomatic patients would be very useful in control of this disease.
The treatment of gonorrhea, as with other sexually transmitted diseases, includes individual patient issues as well as public health concerns. Patients who do not complete a course of treatment once they begin to feel better present a risk of continued transmission and selection of resistant strains. For this reason, definitive treatment at the time of the initial visit has been the favored approach. For decades, this was easily accomplished with a single intramuscular injection of penicillin G.
Compliance dictates treatment on first encounter
Penicillin, which once was active against all known gonococci at extremely low concentrations (<0.1 μg/mL), is no longer used. This is due to the development of multiple mechanisms of resistance. Mutations that altered the affinity of penicillin for its transpeptidase (or penicillin-binding protein [PBP]) target were the first to be recognized. Other mutations in porins—either restricting penicillin transport into the cell or efflux systems pumping it out—have also been discovered either alone or in combination with the transpeptidase alterations. Over decades, a subpopulation of gonococci slowly emerged that required even higher minimum inhibitory concentrations (up to 8.0 μg/mL). For a time this was managed by increasing the penicillin dose, which for the single injection treatment approached the maximum volume that could be humanely administered (even injecting both buttocks). Finally, the most powerful resistance mechanism, penicillinase production, appeared during the Vietnam War and spread throughout the world. These strains produce a plasmid-encoded β-lactamase identical with that of members of Enterobacteriaceae and are resistant at a level that far exceeds achievable therapeutic levels. This was the end for penicillin and gonorrhea.
PBP alterations cause incremental resistance
-Lactamase–producing strains are highly resistant
This situation has caused a shift in treatment of genital gonorrhea to third-generation cephalosporins because of their resistance to the β-lactamases prevalent in gonococci. In addition, it is now recommended that all patients treated for gonorrhea also be treated for Chlamydia infection. For gonorrhea, ceftriaxone is given in a single large intramuscular injection. Oral cephalosporins are no longer considered preferred treatment. For Chlamydia, either azithromycin or doxycycline (both oral) is added. Resistance rates up to 25% have taken fluoroquinolones out of the picture. Other options are available for those who cannot take cephalosporins or for the disseminated forms of gonococcal infection. Patients are treated for both gonorrhea and Chlamydia infection.
Ceftriaxone is combined with azithromycin or doxycycline
Condoms provide a high degree of protection against both infection with N gonorrhoeae and transmission to a sexual partner. Spermicides and other vaginal foams and douches are not reliable protection. The classic public health methods of case–contact tracing and treatment are important, but difficult because of the size of the infected population. The availability of a good serologic test would greatly aid control, as it has for syphilis. The development of a vaccine is a high but distant goal. Achieving it awaits further understanding of immunity and its relationship to the shifting target provided by the gonococcus.
Condoms block transmission
Vaccine strategies await better understanding of immunity