Neisseria gonorrhoeae

Neisseria gonorrhoeae

Humans are the only natural host for N. gonorrhoeae, the agent of gonorrhea. Gonorrhea is an acute pyogenic

infection of nonciliated columnar and transitional epithelium; infection may be established at any site where these cells are found. Gonococcal infections occur primarily in the urethra, endocervix, anal canal, pharynx, and conjunctiva. Disseminated infections from the primary site may also occur, as seen in the Case in Point.

The first use of the term gonorrhea was in the second century, when it implied a “flow of seed.” For centuries thereafter, the diseases syphilis and gonorrhea were confused because the two were often present together in the same infected individual; this still occurs. In 1530, it was thought that gonorrhea was an early symptom of syphilis. The issue was further confused in 1767 by a classic blunder by a physician who inoculated himself intentionally with pus from a patient with symptoms of gonorrhea. The pus gave the physician syphilis instead. Gonorrhea was also called the “clap” from the French word clapoir meaning Brothel.

Virulence Factors

The pathogenic Neisseria spp. have several characteristics that contribute to their virulence. These virul factors include the following: capsule,pili,cell membrane proteins,receptors for human transferring Lipooligosaccharide (endotoxin); lipid A moiety and core lipooligosaccharide (LOS) that differentiates it from the lipopolysaccharide found in most gramnegative bacilli and is loosely attached to the

underlying peptidoglycan.

IgA protease that cleaves IgA on mucosal surfaces N. ganorrhoeae is divided into five morphologically

distinct colony types. Types T1 through T5 are based on the presence or absence of pili, the fine. Hairlike projections that are important in the initial attachment of the organism to host tissues. Pili also inhibit

phagocytosis of the organism by neutrophils and aid in the exchange of genetic material between cells. Types

T1 and T2, which possess pili, are virulent forms, whereas T3 through T5, devoid of pili, are avirulent strains. Piliated organisms usually predominate when first isolated from uncomplicated genitourinary infections, but on subculture, pili are lost and colony types T3 through T5 appear. Antigenic phase switching allows the gonococcus to regain its pili, and antigenic variation allows the organism to vary the antigenic structure contributing to the organism’s ability to evade the immune system. The capsule, cell outer membrane proteins I, II, and III, and the LOS not only serve as protective devices of the organism but are also important in antigenic variation.

Moreover, the LOS endotoxin is a major in vivo virulence factor that mediates damage to body tissues and elicits the inflammatory response. The organism releases outer membrane fragments called “blebs.” During periods of rapid growth, these blebs contain LOS.

The major outer membrane porin proteins (por, formerly Por I) form channels for nutrients to pass into and waste products to exit the cell. They are coded for by two genes: porA and parB. Both genes are expressed in N. meningitidis, but only porB is expressed in N. ganorrhoeae. PorB is also protective against the host’s inflammatory response and serum complement mediated killing. Protein II (Opa, for opacity) is a group of

proteins that facilitate the adherence to phagocytic and epithelial cells. Protein III (reduction modified protein

[RMP]) blocks host serum bactericidal OgG) action against the organism. A schematic diagram of the cellular structure of N. gonorrhoeae is shown in Figure 18-2.

 

Epidemiology

N. gonorrhoeae infections are transmitted most commonly by sexual contact. The primary reservoir is the

asymptomatic carrier. Gonorrhea is a national reportable disease; therefore all culture confirmed cases

must be reported to state health laboratories. Gonorrhea is second to Chlamydia trachoma tis in the number

of sexually transmitted bacterial infections in the United States. There were 335,104 cases of gonorrhea

reported in 2003. Actual numbers of infected individuals are probably much higher than reported, due to a

large reservoir of asymptomatic carriers and other unreported cases. Reported cases increased steadily

through the 1960s but declined almost 75% from 1975 to 1997 and another 10% from 1999 to 2003. In women,

most cases are seen in ages 15 to 19 years; in men, ages 20 to 24 years. The highest rates are seen in the

southeastern United States and in high-density, urban areas where individuals are more likely to have

multiple partners and unprotected sexual intercourse. The fear of acquired immunodeficiency syndrome

(AIDS) and a subsequent reduction in high-risk sexual behavior are thought to contribute to the decline in

reported cases of gonorrhea.

 

Clinical Infections

Gonorrhea has a short incubation period of approximately 2 to 7 days. In men, acute urethritis, usually resulting

in purulent discharge and dysuria (painful urination), is the common manifestation. Asymptomatic

gonococcal infection in men is uncommon; only 3’X, to 5% of cases may be asymptomatic, whereas 95‘X.

show acute infections. N. gonorrllOeae strains with a nutritional requirement for arginine, hypoxanthine, and

uracil (AHU strains) are often isolated from asymptomatic men. Complications in males include ascending

infections such as prostatitis and epididymitis. The endocervix is the most common site of

infection in women, resulting in vaginal discharge and dysuria; however, up to 50% of cases in women may be

asymptomatic. Symptoms of infection, when present, include dysuria, vaginal discharge, and lower abdominal

pain. Untreated gonococcal cervicitis is a major cause of pelvic inflammatory disease (PID) in women,

which may cause sterility, ectopic pregnancy, or perihepatitis (Fitz-Hugh-Curtis syndrome).

Blood-borne dissemination of N. gonorrhoeae occurs in less than 1% of all infections, resulting in purulent arthritis, as seen in the Case in Point, and rarely septicemia. The organism will not be recovered from routine

blood cultures, however, because growth of N. gonorrhoeae is inhibited by sodium polyanethol sulfonate, the

anticoagulant in blood culture media. Fever and a rash on the extremities may also be present. The majority

of disseminated gonococcal infections is attributed to the AHU strains and occurs in women, acquired from

infected but asymptomatic males.

Other conditions associated with N. gonorrhoeae include anorectal and oropharyngeal infections. Infections

in these sites are more common in homosexual males but can also occur in women. Most infections are

asymptomatic or have nonspecific symptoms. Pharyngitis is the chief complaint in symptomatic oropharyngeal

infections, whereas discharge, rectal pain, or bloody stools may be seen in rectal gonorrhea. Approximately

30% to 60% of females with genital gonorrhea have concurrent rectal infection.

Newborns can acquire ophthalmia neonatorum, a gonococcal eye infection, during vaginal delivery

through an infected birth canal. This condition, which can result in blindness if not treated immediately, is rare in the United States because application of antimicrobial eyedrops, generally erythromycin, is legally required

at the birth of every infant. Ocular infections can occur in adults due to inoculation of the eye with

infected genital secretions or, rarely, as a result of a laboratory accident.

Laboratory Diagnosis

Specimen Collection and Transport Specimens collected for the recovery of N. gonorrhoeae,

as noted previously, may come from genital sources or from other sites such as the rectum, pharynx, and joint

fluid. The laboratory should be notified when cultures for N. gonorrhoeae from sites such as the rectum or

throat are requested, because normal laboratory protocols for such specimens would not recover the

organism.

The specimen of choice for genital infections in males is the urethra, and in females, the endocervix.

In males, purulent discharge can be collected directly onto a swab for culture. When no apparent discharge

is present, the swab is inserted 2 to 3 cm into the anterior urethra and slowly rotated to collect material.

Swabs for rectal culture should be inserted 4 to 5 em into the anal canal. Disinfectants should be avoided in

preparing the patient for collection of the specimen. Because N. gonorrhoeae is extremely susceptible to

drying and temperature changes, direct plating of the specimen to gonococcal-selective media gives optimal

results (Figure 18-3). Calcium alginate and some cotton swabs are inhibitory to N. gonorrhoeae, so Dacron or rayon swabs are preferred. Inoculated swabs should be placed in a transport system such as Amies medium with charcoal, transported to the laboratory immediately, and plated within 6 hours. Several commercial transport systems, such as JEMBEC plates (James E. Martin Biological Environmental Chamber; Figure 18-4), Bio-Bag, Gono-Pak, and Transgrow, contain selective media and a carbon dioxide atmosphere to provide optimal conditions until the specimen reaches the laboratory. These systems are especially useful when the clinic or physician’s office is some distance from the laboratory.

Direct Microscopic Examination Smears for direct Gram stains should be prepared from

urogenital specimens when the culture is collected. A Gram stain is not recommended for pharyngeal

specimens because commensal Neisseria spp. can be present. Demonstration of gram-negative intracellular

diplococci, appearing as kidney or coffee bean shaped, from a symptomatic male with discharge correlates at a rate of 95% with culture and is evidence of gonococcal infection (Figure 18-5, A). Many times avirulent forms (lacking pili) of the organism are seen as extracellular, gram-negative diplococci in the direct smear of the clinical specimen. Because women have vaginal commensal flora that resemble gonococci, direct Gram stain correlates in only 50% to 70% of cases with culture.

The direct Gram stain may be helpful in a symptomatic woman with discharge, but culture is necessary

for confirmation. A Gram stain with more than five polymorphonuclear neutrophils (PMNs) per field but

no bacteria (Figure 18-5, B) may suggest nongonococcal urethritis with organisms such as C. trachomatis or Ureaplasma urealyticum.

Culture

Cultivation of N. gonorrhoeae requires the use of chocolate agar (CHOC), but this enriched medium also supports the growth of many other organisms found as commensals in specimens collected for recovery of

gonococci. To prevent overgrowth of the normal flora and to enhance the recovery of the pathogenic species,

a selective medium containing inhibitors for gramnegative and gram-positive organisms and yeast is

used. Commonly used selective media are described in Table 18-2.

New York City (NYC) medium, a transparent agar, has the added advantage of supporting the growth of

possible urogenital pathogens Mycoplasma hominis and Ureaplasma urealyticum. Over the past several years, up to 10% of N. gonorrhoeae strains have been reported as sensitive to vancomycin, an antimicrobial agent used in most selective media for N gonorrhoeae. To recover these vancomycin-sensitive strains, including a nonselective CHOC as a primary plating medium is good

practice. Several other bacteria will grow on selective gonococcal media. Some of these are  Acinetobacter spp., Capnocytophaga spp., and Kingella denitrificans. These

are differentiated from N gonorrhoeae by the oxidase and catalase tests.

All specimens received in the laboratory for recovery of Neisseria spp. should be held at room

temperature and plated as soon as possible. Because Neisseria spp. are susceptible to cold, media should be warmed to room temperature before inoculation. Specimens on swabs should be rolled in a Z pattern on the media and cross-streaked with a loop to facilitate growth of isolated colonies.

Incubation

Inoculated plates should be incubated at 35° C in a 3% to 5% CO2 atmosphere. This is easily accomplished by

use of a candle extinction jar (Figure 18-6) or a commercially available CO2 incubator. Sufficient humidity is

provided by the moisture evaporating from the media in a closed jar; most commercially available CO2 incubators are automatically humidified, or a pan of water can be placed in the bottom. Scented or colored candles may be inhibitory to the gonococci, so only white wax candles are used in the candle extinction jar. Presumptive Identification Microscopic Morphology The Gram stain must be performed on all suspected Neisseria isolates to verify the appearance of gram-negative diplococci. Some gram-negative rods, such as Kingella and Acinetobacterspp., are occasionally able to grow on gonococcal selective media. To differentiate these from the gramnegative diplococci, the organism can be streaked to a plate with a 10-unit penicillin disk added (Figure 18-7).

After growth, the edge of the zone of inhibition is stained to visualize the morphology.

Colonial Morphology Cultures are examined daily for growth and held for 72 hours. Colonies of N gonorrhoeae

on CHOC or selective agars are small, tan, translucent, and raised after 24 to 48 hours of incubation. As noted previously, five colony types of N gonorrhoeae have been described: T1 and T2 have pili and are

considered virulent types; these colonies are smaller and raised and they appear bright in reflected light.

Types T3 through T5 do not have pili and usually grow as larger, flatter colonies. The AHU strains produce

smaller colonies and grow more slowly; they are often more difficult to identify with biochemical methods.

The gonococci can produce autolytic enzymes that may make the isolate nonviable, so primary plates should

not be incubated once sufficient growth is obtained. A fresh subculture should be used for identification tests.

Oxidase Test The filter paper or direct plate oxidase test must be done on all isolates. In the filter paper method, oxidase reagent (1% dimethyl-p-phenylenediamine- dihydrochloride or tetramethyl-p-phenylenediamine- dihydrochloride) is placed on filter paper, and a colony from the plate is rubbed onto the reagent with

an applicator stick or a non-nichrome needle. In a positive reaction on a fresh isolate, purple color should

develop in 10 seconds (Figure 18-8, A). Alternatively, the oxidase reagent may be dropped directly onto a

colony. In a positive reaction, the colony turns pink then black (Figure 18-8, B). If subculture of the positive

colony is needed, it must be done while the colony is still pink; when black, the organism is no longer viable.

Definitive Identification

A presumptive diagnosis of gonorrhea was once acceptable if an oxidase-positive, gram-negative diplococcus

was recovered on gonococcal-selective media; this presumptive diagnosis is no longer acceptable. Oxidasepositive, gram-negative diplococci, such as Neisseria cinerea and N meningitidis, and M. catarrhalis can grow on selective media from sites where gonorrhea is expected.

These organisms would be incorrectly reported as N gonorrhoeae if no further identification were performed.

Many different methods are currently used for the speciation of Neisseria and Moraxella species or for

confirmation only of N gonorrhoeae isolates. Both culture and nonculture tests are available for the detection

of N gonorrhoeae and are listed by type in Tables 18-3 and 18-4. All have advantages and disadvantages, and

the selection of a particular method depends on the demographic profile of the patients, sensitivity and

specificity of the method with low- or high-prevalence groups, cost of materials and technical time, and

number of tests performed.

Carbohydrate Utilization The traditional method

for the identification of Neisseria spp. has been by carbohydrate utilization in cystine trypticase agar

(CTA) , containing 1% of the individual carbohydrate and phenol red as a pH indicator (Procedure 18-1).

If the organism uses the particular carbohydrate, then acid, characterized by a yellow color, is produced

in 24 to 72 hours as shown in Figure 18-9. Many problems are associated with this method, however, and

it has been replaced by new, rapid, more accurate methods.

The rapid carbohydrate degradation tests require pure cultures but can be read in 2 to 4 hours rather than

the 24 to 72 hours needed for the CTA carbohydrate test. These rapid tests also detect acid production from

various carbohydrates, but they are based on the presence of preformed enzymes for carbohydrate utilization

rather than on bacterial growth. Problems noted with these methods, however, include the following: · Weak acid production from glucose by certain strains of N. gonorrhoeae · Misidentification of sucrose-negative strains of Neisseria subflava as N. meningitidis · Strains of N. cinerea that give positive glucose reactions

Additional tests such as superoxol or colistin susceptibility are needed when acid production is equivocal.

The superoxyl test uses 30% HPz and is performed in the same way as the catalase test. Colonies of N. gonorrhoeae produce immediate, vigorous bubbling. N. meningitidis and Neisseria lactamica produce weak delayed bubbling in this reagent. Other Neisseria spp. produce weak delayed bubbling or none at all.

Chromogenic substrate tests detect enzymes that hydrolyze the substrates and produce colored end

products. Only strains that are isolated on selective media should be tested. The advantage of these tests

is the identification of Neisseria spp. strains with aberrant carbohydrate utilizations. Problems noted with

these tests include misidentification of nonpathogenic species, such as N. cinerea, N. sicca, N. subflava, and

N. mucosa, as N. gonorrhoeae or N. meningitides

The multi test conventional-chromogenic enzyme methods combine enzyme substrate tests with other

biochemical tests and allow identification of strains isolated on selective or nonselective media. These tests

can also speciate other genera, such as Haemophilus (see Chapter 19). Characteristics of clinically significant

species of Neisseria, Moraxella, and Kingella are listed in Table 18-5. Key differentiating reactions of the major

pathogens include use of glucose only by N. gonorrhoeae, whereas N. meningitidis uses glucose and maltose. M. catarrhalis is asaccharolytic but, unlike the Neisseria spp., is DNase and butyrate esterase positive.

 CfA CARBOHYDRATE METHOD

1. Prepare a heavy inoculum in saline of a pure isolate from a fresh subculture of the organism. The subculture must not be from a selective agar.

2. Inoculate the top portions only of the following eTA media: glucose, maltose, lactose, and sucrose, plus a carbohydrate-free eTA control.

3. Incubate tightly capped tubes at 35° to 37° e in a non-carbon dioxide atmosphere. Incubation in an

acid CO2atmosphere can produce the appearance of carbohydrate degradation in all tubes and,

hence, false-positive reactions.

4. Yellow at the top of them media in 24 to 72 hours is considered positive. Bright yellow throughout the

media usually indicates contamination with other organisms.

Immunologic Assays Immunologic methods employ monoclonal antibodies for the identification of N gonorrhoeae. These methods do not require pure or viable

organisms and can be done from the primary plates. Immunologic methods include coagglutination and fluorescent

antibody testing. The coagglutination tests use monoclonal antibodies attached to killed Staphylococcus

aureus cells; N gonorrhoeae produces agglutination. No reported cross-reaction with N cinerea exists, but

rare isolates of N lactamica have been reported as N. gonorrhoeae. The fluorescent antibody (FA) method uses monoclonal antibodies that recognize epitopes on the principal outer membrane protein (Por) of N. gonorrhoeae.

The method is extremely sensitive and specific; no demonstrated cross-reactivity with N. cinerea or other Neisseria spp. has been reported. The FAmethod also microscopically confirms the diplococci’s morphologic appearance.

Nucleic Acid Assays Alternatives to culture for N. gonorrhoeae are available. These methods detect gonococcal antigen or nucleic acid directly in cervical, urine, and/or urethral exudates. Nucleic acid detection methods include both nonamplified and amplified probe technologies (see Table 184). One nucleic acid probe test is a nonisotopic chemiluminescent DNAprobe that hybridizes specifically with ribosomal RNA (rRNA) of N. gonorrhoeae. Nucleic acid detection methods are

rapid and sensitive but they do have some drawbacks. Some disadvantages of the nucleic acid probes include the following: · Lesser sensitivity than cervical culture in females; used in high-risk populations only · Not approved for pharyngeal or rectal specimens; not used in children or sexual abuse cases · Cannot identify an N. gonorrhoeae infection produced by a f)-lactamase-producing strain · Do not allow for recovery of an organism to be used for susceptibility testing · False-positive results reported with some strains of N. lactamica and N. cinerea Nucleic acid amplification tests (NMTs) amplify a specific nucleic acid sequence, by polymerase chain reaction (PCR) , for example, before detecting the target

sequence with a probe. They are extremely sensitive and do not require viable organisms. Because urine can

be used in these procedures, self-collection of the specimen is possible and pelvic examination or intraurethral

swabs are not required. These methods have the additional advantage of being less sensitive to transport and

storage conditions than culture. NMTs can also detect both N. gonorrhoeae and C trachomatis in the same specimen. Disadvantages of NMTs include the following: · False-negative results may occur if the specimen contains inhibitors of amplification. · Because amplification is highly sensitive, strict attention to procedural factors and quality control is mandatory, including prescribed work areas, specimen handling to avoid cross-contamination, and ··· SNoMmTepsoostmihtieavrye bNaeenidslesesnrsieagsaetnsivpseipt.ivcemoanoytrnoclusro.rsins e.react. Probe technology and amplification methods can be expensive. These methods are probably not costeffective if they are the only probe or amplification test used in the laboratory or if low volumes of N. gonorrhoeae tests are requested. Added to the

expense is the need to repeat the test after equivocal results are obtained.

Additional Methods Some strains of N. gonorrhoeae have a specific need for one or more nutritional factors

to be included in artificial media for growth to occur. These strains are called auxotypes; of the approximately

30 known auxotypes, the most common is AHU, which requires arginine, hypoxanthine, and uracil. The

AHU strains are usually highly susceptible to penicillin, commonly recovered from patients with disseminated

gonococcal infection, and can cause asymptomatic urethral infection in males. The auxotyping procedure is

labor intensive; for this reason, it is not recommended for the routine clinical microbiology laboratory.

In low-prevalence populations, such as children, or in cases of suspected sexual assault/abuse, nonculture

detection methods must not be used. The isolation of the gonococcus from children beyond the neonate

period is indicative of sexual abuse. The specimens should be cultured onto selective gonococcal media;

isolates must exhibit typical colonial morphology, microscopic morphology of gram-negative diplococci,

and be oxidase and catalase positive. Isolates must be identified to species level by the use of two biochemical,

enzymatic, or serologic procedures with different principles. The organism should also be frozen in case

further testing is needed. With various methods, isolates such as N. cinerea, M. catarrhalis, and K. denitrificans,

all of which closely resemble N. gonorrhoeae biochemically, have been incorrectly reported as N. gonorrhoeae. Potentially serious medical, social, and legal ramifications could obviously result.

Antimicrobial Resistance

Until 1976, almost all strains of N. gonorrhoeae in the United States were susceptible to penicillin. The first plasmid-mediated penicillinase-producing N. gonorrhoeae (pPNG) strains were isolated in that year, largely

imported from Southeast Asia or Africa. By 1980, more than half of the reported PPNG cases were of domestic

origin. The Gonococcal Isolate Surveillance Project (GISP) reported that PPNG has decreased in the United

States from 11% in 1991 to 1% in 2003. In addition to plasmid-mediated penicillin resistance, in which the

organism acquires a plasmid with genes for f)-Iactamase production, N. gonorrhoeae can exhibit chromosomemediated penicillin resistance (penR), which was initially noted in the United States in 1983. These isolates are f)-Iactamase negative. Resistance in these strains is due to a combination of mutations at several chromosomal loci resulting in altered penicillin-binding proteins. The incidence of PenR increased from 0.5% in 1988 to 5.7% in 1999 and then decreased to 1.3% in 2003. In addition to PenR in N. gonorrhoeae, there is also

plasmid-mediated resistance to tetracycline as well as chromosomally mediated resistance to tetracycline, spectinomycin, and fluoroquinolones. Chromosomally mediated resistance to penicillin and tetracycline

increased from 3’1.’,in 1989 to a peak of 8.7% in 1997 and then decreased to 3.8% in 2003.

Spectinomycin resistance, first reported in the United States in 1981, is due to a chromosomal mutation

resulting in high-level resistance to the antimicrobial agent. Only five spectinomycin-resistant isolates

of N. gonorrhoeae were reported by GISP in 2003.

Quinolone-resistant N. gonorrhoeae (QRNG) was first identified in the United States in 1991. In 2003, GISP

reported that 4.3% of the isolates were resistant to fluoroquinolone. The mechanisms of resistance of

N. gonorrhoeae are summarized in Table 18-6. In 2005, the Clinical and laboratory Standards Institute (ClSD, formerly the National Committee for Clinical laboratory Standards (NCClS), issued performance standards

for antimicrobial disk susceptibility testing (Document M1OO-S15) of organisms including N. gonorrhoeae.

Treatment

Several antimicrobials are currently recommended for uncomplicated gonococcal therapy by the U.S. Public

Health Service. These primary therapies are two broadspectrum cephalosporins (ceftriaxone or cefixime) and

three fluoroquinolones (ciprofloxacin, ofloxacin, or levofloxacin).

These agents are generally active against organisms with plasmid or chromosomal resistance and cure uncomplicated gonorrhea of the pharynx, rectum, and urogenital sites. Since the 1990s, QRNG has been

reported in many areas of the world, especially Asia, Hawaii, and California; therefore the Centers for Disease

Control and Prevention (CDC) recommends that fluoroquinolones not be used to treat gonorrhea acquired in

areas of high QRNG prevalence. Because there is also an increase in QRNG among men who have sex with men (MSM), the CDC recommended in 2004 that fluoroquinolones not be used to treat gonorrhea in MSM patients. Because co-infection with C. trachoma tis is fairly common in patients with gonorrhea, dual therapy is

frequently prescribed. One of the primary therapies is used, plus azithromycin or doxycycline for C.

trachoma tis. Routine use of dual therapy can be cost effective, can decrease the prevalence of chlamydial

infection, and may reduce the development of resistant strains of N. gonorrhoeae..

table 18-3 table 18-4 table 18-5 figure 18-1 figure 18-2 figure 18-3 figure 18-4 figure 18-5 figure 18-6 figure 18-7 figure 18-8 figure 18-9 table 18-1 table 18-2

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