Neisseria

TAXONOMY Back to top

According to the second edition of Bergey’s Manual of Systematic Bacteriology the

genus Neisseria belongs to the family Neisseriaceae of the order Neisseriales (124), which is

placed into the class Betaproteobacteria. Since the 1980s, several alterations have been

made within the taxonomy and classification of the familyNeisseriaceae due to knowledge

gained from molecular analyses. The exclusion and subsequent reassignment of the

genera Moraxella, Acinetobacter, and Psychrobacter to the Gammaproteobacteria were first

proposed by the use of DNA-rRNA and DNA-DNA hybridization techniques (110) and later

confirmed by 16S rRNA gene sequencing (45, 61). Today, the family Neisseriaceae is the

only family within the order Neisseriales, which in addition to the

genus Neisseria contains Eikenella, Kingella, and 27 other genera.

DESCRIPTION OF THE GENUS NEISSERIA Back to top

Most members of the genus Neisseria are cocci with a diameter of up to 2 μm, presenting as

single bacteria or in pairs. The species N. elongata, N. weaveri, and the proposed new

species N. bacilliformis sp. nov. are exceptions and consist of short rods, frequently arranged

as diplobacilli or in chains. While Neisseria species are gram negative, occasionally a

tendency to withstand decolorization is noted. Capsules (N. meningitidis)and pili (N.

meningitidis and N. gonorrhoeae) may be present, yet flagella are not formed. N.

meningitidis is the only species expressing a polysaccharide capsule, of which 12 different

serogroups are distinguishable (53). Strains of several species like N. flavescens, N.

sicca, and N. subflava may produce a yellowish pigment.Neisseria species grow optimally

under aerobic conditions and a temperature of 35 to 37°C. Nevertheless, isolation of N.

gonorrhoeae from body sites with reduced oxygen tensions suggests ability of anaerobic

growth, which, given the inability to generate energy from fermentation, was suggested to

be due to nitrite respiration (74). Microaerobic growth by denitrification of nitrite via NO has

also been shown for N. meningitidis(3). While many species are not nutritionally demanding,

the human-pathogenic species N. gonorrhoeae and N. meningitidis are fastidious, showing

particular susceptibility to unfavorable environmental factors such as extreme temperatures,

desiccation, and alkaline or acidic conditions. All species are oxidase positive and, with the

exception of N. elongata subsp. elongata and N. elongata subsp. nitroreducens, catalase

positive. Neisseriaspecies produce acid from carbohydrates by oxidation, not fermentation.

Some species, like N. elongata and N. cinerea, are asaccharolytic. Most members of the

genus are able to reduce nitrite. The natural habitat of the members of this genus is the

mucous membranes of mammals including humans. The species N. gonorrhoeaeand N.

meningitidis are human pathogens. Exotoxins are typically not produced (124). All species

classified in the genus Neisseria are naturally competent for DNA uptake and display a high

frequency of horizontal gene transfer (118). As a consequence, phylogenetic analyses within

this genus based on different genes may yield incongruent results (117). Of note, this

distortion also applies to 16S rRNA gene sequencing, which has been used to define interand

intrageneric relationships within the Neisseriaceae and Moraxellaceae (61). Multiplelocus

instead of single-locus approaches might therefore be more suitable for the resolution

of species identification within the genus Neisseria (10). According to Euzeby ’s “List of

Prokaryotic names with Standing in Nomenclature”

(http://www.bacterio.cict.fr/n/neisseria.html), the genus Neisseria consists of 25 species.

EPIDEMIOLOGY AND TRANSMISSION Back to top

N. gonorrhoeae causes gonorrhea, which is the second most commonly reported notifiable

disease in the United States (http://www.cdc.gov/std/stats07/gonorrhea.htm). N.

gonorrhoeae is always considered pathogenic, and humans are the only hosts of this

bacterium. It is mainly transmitted through sexual practices and infects the mucosal surfaces

of urethra, cervix, rectum, and pharynx and the eye. The risk of infection is greatly

influenced by sexual behavior yet can be reduced, although not eliminated, by the use of

condoms (134). Furthermore, the eye can be infected intrapartally during passage of the

fetus through the birth canal. The rate of gonorrhea has decreased by 74% from 1975 to

1997 in the United States following the implementation of the gonorrhea control program in

the mid-1970s (http://www.cdc.gov/std/stats07/gonorrhea.htm). From 1997 onwards,

however, the overall incidence has remained largely unchanged. In 2007, 355,991 cases

were reported in the United States, which translates to an overall incidence of 119 cases per

100,000 population. Rates vary considerably among states, with values below 19/100,000 in

the Northeast and West to over 250/100,000 in the South. Prior to 1996, disease rates were

consistently higher among men, but incidences have been similar or slightly higher among

women in recent years. The age groups with the highest burden of disease are adolescents

and young adults between 15 and 24 years. In 2007, gonorrhea rates remained highest

among African Americans (663 per 100,000), with African American women between 15 and

19 years particularly affected (2,956 per 100,000). Overall rates in the United Kingdom were

comparable to those of the United States in 2007 (130 per 100,000), yet a significant

downward trend since 2002 has been observed (http://www.hpa.org.uk). While the age

distribution is very similar to that of the United States, men have consistently higher rates

than women in the United Kingdom. Moreover, Great Britain has observed a threefold

increase in the number of individuals diagnosed with gonorrhea among men who have sex

with men (MSM).

N. meningitidis also occurs exclusively in humans and plays a dual role of commensal and

potential invasive pathogen. On average, the mucosal surfaces of oro- and nasopharynx of

10% of the population is colonized by this bacterium (34). Carriage is strongly age

dependent, with adolescents and young adults attaining rates of over 30% in contrast to

infants with carriage rates of a few percent (34). As a consequence of repeated episodes of

carriage, the percentage of sera with bactericidal activity against pathogenic strain increases

with age. Transmission occurs through large droplet secretions from the oropharynx and is

favored by repeated or close contact, given the low yield of growth from saliva compared to

nasopharyngeal swabs (98). Nevertheless, outbreaks and clusters are rare in developed

countries (44). Disease occurs in only a minute proportion of individuals acquiring N.

meningitidis and follows a typical age distribution with infants and adolescents having the

highest incidences. Apart from genetic host polymorphisms (19), individuals with underlying

conditions like properdin deficiencies (47), late complement deficiencies (47, 103), and

splenic impairment including asplenia (59) are at increased risk for invasive meningococcal

disease (IMD). Also, behavioral risk factors, including exposure to smokers (39) and kissing

(127), have been described to contribute to acquisition of disease.

IMD is rare in developed countries. In the United States the incidence for the year 2007 was

estimated to be 0.34 per 100,000 (26) with serogroups B, C, and Y constituting 91% of all

cases. The rate in European countries is rather variable, with the United Kingdom reaching

incidences of 2.5/100,000 with an 80% dominance of serogroup B (2007 and 2008) and

Germany reporting an incidence of 0.55/100,000 with a 70% proportion of serogroup B in

2008. In contrast, African countries in the so-called meningitis belt regularly report epidemic

waves, mainly caused by serogroup A, with rates soaring to over 300 per 100,000 (21).

Several predominant clones have successively caused the majority of IMD in Africa (24). A

number of vaccines have been developed for the prevention of IMD. In 2005, the Advisory

Committee on Immunization Practices recommended vaccination of young adolescents (11 to

12 years of age) with a quadrivalent polysaccharide-protein conjugate vaccine (26) covering

serogroups A, C, W135, and Y. Due to high rates of serogroup C disease in the 1990s and

early 2000s, several European countries implemented vaccination campaigns with conjugate

vaccine against serogroup C, which led to dramatic reduction of disease with this capsule

type (126). Until recent times, vaccination against serogroup B had been deemed impossible

due to poor immunogenicity of the serogroup B capsule. Nevertheless, outer membrane

vesicle vaccines were used to combat local epidemics, e.g., in New Zealand (99). Also,

recent advances in the development of vaccines based on outer membrane proteins

(55, 104) promise to provide broad coverage against a wide array of disease-causing strains.

CLINICAL SIGNIFICANCE Back to top

Members of the genus Neisseria have a high affinity to mucosal membranes of mammals and

humans. A wide variety of species can be isolated from humans including N. gonorrhoeae, N.

cinerea, N. elongata, N. flavescens, N. lactamica, N. meningitidis, N. mucosa, N.

polysaccharea, N. sicca, and N. subflava. Several species are predominantly recovered from

animals, like N. animalis, N. animaloris, and N. zoodegmatis (throat of cats and dogs)

(129a), N. denitrificans (throat of guinea pigs), N. dentiae (dental plaques of domestic

cows), N. macacae(oropharynges of rhesus monkeys), and N. weaveri (oral flora of dogs).

Similar to N. elongata, the new speciesN. bacilliformis likely colonizes the oral cavity and

respiratory tract of humans (58). Most human Neisseriaspecies are considered normal

inhabitants of the upper respiratory tract, which cause disease in an opportunistic fashion.

Rarely, species of animal origin can cause wound infections in humans after bites. N.

meningitidis mostly appears as a mere commensal of the human oropharynx yet can cause

life-threatening, acute disease in previously healthy individuals. N. gonorrhoeae, however, is

always considered a pathogen, even if obvious signs of disease are absent.

Uncomplicated infection by N. gonorrhoeae (gonorrhea) manifests most commonly as acute

urethritis in men. The major symptoms are urethral discharge, sometimes associated with

dysuria, typically without frequency or urgency. Coinfection of the preputial (Tyson’s),

urethral (Littre’s), and bulbo-urethral (Cowper’s) glands is possible. Also, completely

asymptomatic infections occur in up to 10% of cases. Most cases of untreated urethritis

resolve spontaneously after several weeks. Further localized complications after gonococcal

urethritis include acute epididymitis, penile edema, and abscesses of the above-mentioned

glands. In women, the endocervix is the primary site of genital infection. Additionally, N.

gonorrhoeae may infect the urethra, the rectum, the periurethral (Skene’s) glands, and the

ducts of the greater vestibular (Bartholin’s) glands. The squamous epithelium of the vagina is

typically not infected in sexually mature women. In contrast to infection in men,

asymptomatic infection in women is common (29). Also, if symptoms appear, they often

cannot clearly be attributed to infection by N. gonorrhoeae, given that concurrent infection

by Chlamydia trachomatis andMycoplasma genitalium is common. The main complaints

include increased vaginal discharge, dysuria, and intermenstrual bleeding. Ascension of the

infection may result in pelvic inflammatory disease, which manifests by various combinations

of endometritis, salpingitis, tubo-ovarian abscess, and peritonitis. Acute perihepatitis (Fitz-

Hugh-Curtis syndrome) can develop following direct extension of N. gonorrhoeae from the

fallopian tube to the liver capsule and the surrounding peritoneum. While over 80% of rectal

infections remain asymptomatic, some patients complain of acute proctitis. Pharyngeal

infection is acquired by oral sexual exposure and is mostly asymptomatic (96) yet can also

cause overt pharyngitis or tonsillitis (6). While probably less transmissible than rectal or

urethral gonorrhea, its silent nature and considerable prevalence among MSM render

pharyngeal infection a common reservoir for gonorrhea in sexually active MSM (96).

Gonococcal conjunctivitis in adults usually results from autoinoculation, oculogenital, or

orogenital exposure. If not treated promptly, corneal ulceration may rapidly develop.

Conjunctivitis of the newborn (ophthalmia neonatorum) is transmitted during birth and is

favored by premature rupture of the membranes and preterm delivery. Historically a

common cause for blindness, it can be prevented by administering a 1% aqueous solution of

silver nitrate or an antibiotic ointment (usually containing erythromycin) into the

conjunctivae after delivery. Disseminated gonococcal infection (DGI) reflects bacteremic

dissemination, possibly generation of immune complexes, and indirect immunological

mechanisms. It complicates less than 1% of mucosal infections (63). DGI usually manifests

as septic arthritis and a characteristic syndrome of polyarthritis and dermatitis and should be

suspected in patients presenting with tenosynovitis, arthritis, and vasculitic skin lesions (69).

IMD commonly presents as meningitis, acute sepsis, or a combination of both. In addition,

unusual presentations include transient mild bacteremia, chronic meningococcal sepsis,

pneumonia (mainly by serogroup Y), septic arthritis, and endocarditis (23). Symptoms of

meningitis vary widely and can include a stiff neck, headache, confusion, and photophobia.

Lethality of meningococcal meningitis without sepsis can be as low as 3% (108). Sequelae

such as sensorineural hearing loss, developmental delay, and speech defects afflict a

substantial part of survivors, yet in a lower proportion than in other forms of acute bacterial

meningitis (68). Petechial lesions are telltale signs of meningococcal sepsis, which can

coalesce and become ecchymotic. Nevertheless, nonpurpuric maculopapular rashes that can

be confused with viral exanthems have also been associated with meningococcemia.

Meningococcal septic shock can take a fulminant course with a lethality of 30% (131), and

concentrations in plasma can reach up to 108 meningococci/ml (18), which in turn lead to

massive activation of cytokines and vasoactive anaphylatoxins. Meningococcal shock

syndrome is characterized by myocardial depression, vasoplegia, capillary leakage, and

disseminated intravascular coagulation (131). Complications of IMD include arthritis,

pericarditis, cranial nerve dysfunction, meningococcal pericarditis, and rarely, cerebral or

spinal infarction. In addition, adolescent survivors of IMD have been described as suffering

from a series of long-term consequences including poorer physical and mental health, quality

of life, and educational achievement (13). Bacteremia can also manifest without signs of

sepsis in the form of chronic meningococcemia, a condition associated with low-grade

relapsing fever, arthritis, and rash (101). Meningococcal pneumonia has been recognized as

an infrequent clinical syndrome for more than 100 years (67). Most meningococcal

pneumonias are caused by serogroup Y; they are responsible for 45% of IMD in the United

States (25) and affect adults disproportionately (138). Preceding viral illness, notably

pandemic influenza (49), has been reported to promote its development. N. meningitidis is

an uncommon cause of acute bacterial conjunctivitis and can also be the etiologic agent of

urethritis in men.

The clinical significance of Neisseria species other than N. gonorrhoeae and N. meningitidis is

covered under “Evaluation, Interpretation, and Reporting of Results” below.

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Neisseria gonorrhoeae

The selection of specimens for culture-based diagnosis of gonorrhea depends on the sex of

the individual, the level of sexual maturity, and anatomical sites exposed. The anterior

portion of the male urethra is sampled by introducing a swab up to 2 cm in a rotatory

fashion. Samples from the endocervical canal are obtained by introducing a swab after

removal of mucus plugging the cervical orifice. In MSM and women practicing anal

intercourse, rectal samples should be taken. Swabs heavily contaminated with feces have to

be discarded. In symptomatic patients, direct swabbing of lesions under rectoscopic guidance

improves culture yield. A pharyngeal swab should be obtained from individuals who

performed fellatio on a person with genital gonorrhea. Vaginal swabs are inadequate for

culture-based diagnosis in sexually mature women but can be used in prepubescent females.

If the hymen is intact, however, the specimen is collected from the vaginal orifice.

Dacron (polyethylene terephthalate)- or rayon (viscose)-tipped swabs, e.g., Transwab

(Medical Wire, Corsham, United Kingdom), Bactiswab (Remel, Lenexa, KS), or Minitip Amies

(Copan Innovation, Brescia, Italy), are preferable for culture-based diagnosis of gonorrhea.

Calcium alginate swabs should be avoided due to reported toxicity (80). Also, cotton buds

and oil-based lubricants can contain unsaturated fatty acids, which inhibit N. gonorrhoeae.

Although direct plating maximizes the yield of gonococci in culture, this approach is not

always practical or possible. Here, Amies-based semisolid transport media can be used to

transport swabs to the processing laboratory. There are, however, considerable performance

differences of commercial Amies-based transport systems after 24 and 48 hours, which are

not uniformly rectifiable by the addition of charcoal (57). Therefore, it is advisable to

inoculate swab specimens transported in these media within 6 hours after collection. During

the time of transport, media should be kept at room temperature and not refrigerated.

Survival and transport of gonococci for over 24 hours can be achieved by culture medium

transport systems, which allow direct plating of specimens in a clinical environment. They

usually consist of a solid medium onto which swabs are inoculated directly after collection

and a CO2-generating system within a resealable container. A CO2-rich atmosphere is

generated by tablets containing citric acid and sodium bicarbonate that are activated after

contact with water. Commercially available systems include Biocult-GC (Orion Diagnostica,

Espoo, Finland) and John E. Martin Biological Environmental Chamber GC-Lect Agar (Becton

Dickinson and Company, Franklin Lakes, NJ).

Similar to culture samples, specimens for molecular detection of N. gonorrhoeae are best

sampled by using rayon- or Dacron-tipped swabs, since calcium alginate was reported to

inhibit PCR (38). The inhibitory influence of aluminum shafts is rather contentious, and

preliminary testing in conjunction with the employed molecular kit is advisable. Transport

and collection systems specifically designed for molecular detection include the Digene

Female Swab Specimen Collection Kit (Qiagen Inc., Valencia, CA) and the STD Swab

Specimen Collection and Transport Kit (F. Hoffmann-La Roche Ltd., Basel, Switzerland).

Some molecular kits can also be used for urine and vaginal swabs (see “Nucleic Acid

Amplification Tests” below). For these sample types, recommendations by the producer of

the molecular detection kits employed have to be followed.

Neisseria meningitidis

The types of specimens that can be used for the detection of N. meningitidis include blood,

cerebrospinal fluid (CSF), nasopharyngeal and oropharyngeal swabs, bronchoalveolar lavage

fluids, joint aspirates, urethral and endocervical swabs, petechial aspirates, and biopsy

specimens. Genital and rectal specimens may be obtained by using the collection and

inoculation procedures described above. Pharyngeal swabs used for determination of

meningococcal carriage are best taken from the posterior pharyngeal wall through the mouth

and plated directly after sampling (109). Alternatively, swabs may be put into Amies-based

transport media and plated preferably within 5 hours after collection. Growth of N.

meningitidis and N. gonorrhoeae in commercial blood culture media is adversely affected by

the anticoagulant sodium polyanetholesulfonate (107), for which currently no suitable

substitute is available. Its inhibitory action is reduced by the addition of gelatin at a

concentration of 1 g/liter to most commercially available blood culture media.

Laboratory Safety Issues for Handling of Meningococcal

Cultures

Rare cases of fatal meningococcal disease in laboratory staff have been described (114). A

risk factor for laboratory- acquired infection is exposure to droplets or aerosols containing N.

meningitidis (114). Laboratories working with live N. meningitidis isolates should comply with

biological safety containment level 2 standards, including the use of class II biological safety

cabinets whenever infectious splashes or aerosols may be created, e.g., during mobilization

of organisms from culture plates, handling of liquid cultures, performing of carbohydrate

utilization tests, oxidase testing, and slide agglutination.

DIRECT EXAMINATION Back to top

Microscopy

A direct smear for Gram staining should be prepared with a different swab than that used for

the collection of specimen for culture. The swab should be rolled softly onto the glass slide to

conserve cellular morphology. A presumptive diagnosis of gonococcal urethritis in men is

made by visualization of gram-negative diplococci associated with or within

polymorphonuclear leukocytes. The sensitivity of microscopy depends on the anatomical site

investigated and is highest in urethral slides of men, where it reaches 89% (86). For

endocervical and rectal smears of MSM, however, it drops to 51% and 54%, respectively

(86). The specificity of microscopic diagnosis for these sites has been reported to be over

90%. Microscopy is not useful for the diagnosis of pharyngeal gonorrhea. Nevertheless,

microscopic diagnosis is mandatory from normally sterile material.

A Gram stain of CSF is required for all cases of suspected bacterial meningitis sent to the

laboratory. Visualization of gram-negative diplococci is sufficient for the presumptive

diagnosis of meningococcal meningitis (Fig. 1). If more than 1 ml of CSF is available, the

specimen should be centrifuged at 1,000 × g for 10 minutes and the pellet used for

microscopic examination and culture. Cytocentrifugation also increases the sensitivity of

microscopic investigation. On Gram-stained smears from CSF, meningococci appear as gramnegative

diplococci both inside and outside polymorphonuclear leukocytes, which will

typically be abundant in samples from bacterial meningitis. Organisms may tend to resist

decolorization.

Antigen Detection

Diagnosis of meningococcal meningitis can be made through the use of commercially

available antigen detection kits. These methods are useful, if no or only limited access to

microscopes is available. They are of questionable clinical usefulness when compared with

Gram stain (102) and should therefore not be used as a substitution for microscopy.

Commercially available latex agglutination tests, which consist of latex particles coated with

monoclonal antibodies targeting the capsular polysaccharide of common serogroups, include

the Pastorex Meningitis kit (Inverness Medical UK Ltd., Stockport, United Kingdom) and

Wellcogen N. meningitidis A, C, Y, and W135 (Oxoid Ltd., Basingstoke, United Kingdom).

These assays have a reasonable sensitivity and specificity (42) yet are useless for the

detection of uncommon serogroups (130). In laboratories handling only a small number of

cases the cost for purchase and storage of antigen detection kits outweighs any potential

benefits for patient management.

Nucleic Acid Detection

Neisseria gonorrhoeae

Nucleic acid tests permit the rapid and sensitive detection of N. gonorrhoeae from clinical

samples without the requirement of bacterial viability. They have been in use since the early

1990s and can be categorized in nucleic hybridization assays and nucleic acid amplification

tests (NAATs).

Hybridization Assays

The two commercially available hybridization assays include Digene CT/GC Dual ID HC2

(HC2; Qiagen) and Gen-Probe Pace 2 (P2; Gen-Probe Inc., San Diego, CA), which use RNA

probes targeting genomic DNA and DNA probes targeting rRNA, respectively. The detection

method of the RNA-DNA hybrids in the HC2 assay involves antibody-mediated recognition of

the hybrids and subsequent binding of alkaline phosphatase-conjugated antibodies, which act

on a chemiluminescent substrate. Signal amplification results from multiple alkaline

phosphatase molecules being attached to a conjugated antibody, of which several bind to a

single captured hybrid. In the P2 assay the DNA probes are labeled with a chemoluminescent

substance, which is quantified after separation of the stable DNA-RNA hybrids from

nonhybridized probe. The sensitivity of hybridization tests is probably higher than that of

culture (41, 66).

Nucleic Acid Amplification Tests

All main commercial NAATs developed to date use multiplex NAATs, targeting both N.

gonorrhoeae andChlamydia trachomatis (see chapter 60). Of the first-generation tests, which

include Roche Amplicor CT/NG (F. Hoffmann-La Roche Ltd., Basel, Switzerland) and Abbott

Ligase Chain Reaction (LCx, Abbott Molecular, Maidenhead, Berkshire, United Kingdom), only

Roche Amplicor CT/NG continues to be available. The Amplicor assay uses PCR for

amplification of DNA and targets the DNA-cytosine methyltransferase gene. It has shown

cross-reactivity with strains of several commensal Neisseria species, contributing to low

positive predictive values (PPV) on urogenital specimens in several studies (136). The CDC

issued guidelines suggesting additional testing for N. gonorrhoeae NAATs in cases where the

PPV is expected to be lower than 90% (72), which apart from cross-reactivity (i.e., low

specificity) can be due to low disease prevalence. With the Amplicor assay, additional testing

has been carried out with real-time PCR assays targeting the porA pseudogene (135) and

the gyrA gene (33). A further real-time-based confirmatory test was attempted using gene

melt curve analysis with labeled probes hybridizing with variable stretches of the 16S rRNA

genes (88), thus enabling distinction of N. gonorrhoeae from other Neisseria species.

Nevertheless, confirmatory tests themselves have differing sensitivities and specificities (88),

which can limit their usefulness, as shown for poorly specific assays targeting

the cppB (cryptic plasmid protein B) gene (20, 88). The Becton Dickinson ProbeTec SDA

assay (SDA) (Becton Dickinson) is a second-generation test that uses strand displacement

amplification, a technique not requiring thermal cycling, for the multiplication of DNA. It

targets a region within the multicopy pilin gene-inverting protein homologue (82). This test

was also shown to have a PPV of less than 90% in certain populations (30). Furthermore,

cross-reactivity with N. flavescens, N. subflava, N. lactamica, and N. cinerea was reported

(100). In analogy to Amplicor PCR, porA pseudogene and cppB were used as a confirmatory

test for SDA. The rate of confirmation with porA, however, has been reported to be only 74%

for anorectal and 30% for oropharyngeal specimens in MSM from Australia (94). Similarly,

concordance with cppB in urogenital specimens has been only 77% (77), although this figure

could represent a shortcoming of the confirmatory assay itself. Additional testing by another

NAAT, Aptima Combo 2 and Aptima GC (see below), showed high concordance for cervical

and urethral swabs (60) and for male urethral swabs and first-catch urine (31). Aptima

Combo 2 (AC2) (Gen-Probe) is a further second-generation test, which uses transcriptionmediated

amplification for the replication of gonococcal 16S rRNA and a chemoluminescent

single-stranded DNA probe for product detection. Aptima GC (AG) represents a confirmatory

assay based on the same technology and even uses the same capture probe as AC2 but

targets a slightly different region of the rRNA subunit. Evaluations regarding the performance

of this assay have largely been favorable. Specificity and sensitivity were shown to be higher

than for the Amplicor assay in Australia (83). A study using AC2 with AG as a confirmatory

assay demonstrated a PPV of 97% among 60,000 female urine and cervical swabs despite

low prevalence (56). Nevertheless, PPV varied between 75% and 100% for urogenital

specimens in a multicenter study (31). The specificities of AC2 and those of SDA were very

similar and always over 94% in several studies investigating first-void urine (81), vaginal

(92), rectal (95, 112), and pharyngeal (5, 112) specimens. Finally, the Abbott RealTime

CT/NG (Abbott Molecular) assay is a new real-time PCR test, which like its predecessor,

Abbott LCx, targets a region in the N. gonorrhoeae opacity (Opa) gene (89). To date, not too

many comparative analyses have been published, yet one study analyzing 500 first-void

urine specimens (81) reported performance identical to that of AC2. No confirmatory tests

have been developed for Abbott RealTime.

In summary, NAATs provide several advantages over culture-based diagnosis yet also have a

series of important limitations. The main advantages are their superior sensitivity over

culture, evidenced in numerous clinical studies (5, 95, 112, 132), and the less stringent

collection and transport conditions. The current list of NAATs with FDA approval includes

Amplicor, Abbott RealTime, AC2, and ProbeTec for urine and urethral swab specimens. Some

NAATs are licensed for further sample types including self-collected and clinician-collected

vaginal swabs (Abbott RealTime and AC2) and endocervical swabs (Amplicor, AC2, and

ProbeTec). Importantly, no NAAT is currently cleared for oropharyngeal, rectal, ocular, or

pediatric specimens, which to date have to be investigated by culture-based means. Major

limitations of NAATs include high cost, carryover contamination, high quality control

requirements, and the absence of antibiotic resistance data (136). Furthermore, the assays

are susceptible to inhibition by substances present in patient samples, e.g., those commonly

found in urine (84), and also to inhibition by competing amplification in the case of

coinfection with C. trachomatis (136). The complexity of the assay, involving steps such as

nucleic acid extraction, amplification, and detection, requires stringent quality control and

staff training. Nevertheless, the latest commercial assays such as AC2 and Abbott RealTime

can be integrated into fully automated molecular testing systems, such as Tigris-DTS (Gen-

Probe, San Diego, CA) and m2000 (Abbott Molecular, Maidenhead, Berkshire, United

Kingdom), respectively, thus reducing hands-on processing of samples.

Neisseria meningitidis

Several in-house methods have been developed to enable culture-independent diagnosis of

IMD, which are especially useful when previous antibiotic treatment or unfavorable transport

conditions lead to a negative culture. The DNA targets used for molecular diagnosis

include ctrA (52), IS1106 (16), siaD (14, 15) (or mynA for serogroup A

meningococci), porA (111), porB (128), fetA (123), and housekeeping genes used for

multilocus sequence typing (MLST). Specifically, ctrA was evaluated as a target in real-time

detection of meningococcal DNA (40). Apart from facilitating laboratory confirmation of

meningococcal disease, the polysialyltransferase genes (siaD or mynA in the case of

serogroup A) can be used for serogrouping, while porA, porB, fetA, and housekeeping genes

allow culture-independent typing (44). False-positive results have been reported for

IS1106, which should therefore not be used as a single assay for routine screening (16).

Moreover, ctrA is negative in rare cases of IMD caused by N. meningitidis harboring the

capsule-null locus (48).

ISOLATION PROCEDURES Back to top

Cultivation of N. gonorrhoeae requires the use of chocolate agar, which supports the growth

of many other commensal bacteria. To isolate N. gonorrhoeae from mucosal and other

nonsterile body sites, several selective media containing a mixture of inhibitory agents have

been developed. All of them contain the antibiotics vancomycin and colistin for the

suppression of gram-positive and gram-negative bacteria, respectively. The prototype

medium, developed by Thayer and Martin (122), consists of a chocolate agar base, which in

addition to the above antibiotics contains nystatin for the inhibition of yeasts. The addition of

trimethoprim to the modified Thayer-Martin medium and following formulations prevents

swarming of Proteus species. The Martin-Lewis medium contains anisomycin instead of

nystatin, which has increased activity against Candida albicans. Further modifications include

the GC-Lect Agar (Becton Dickinson), which provides additional control

againstCapnocytophaga species and against vancomycin-resistant gram-positive

contaminants by the addition of lincomycin. Moreover, the reduced vancomycin

concentration in GC-Lect Agar enhances the recovery of uncommon vancomycin-sensitive N.

gonorrhoeae. The media are available in petri-style or John E. Martin Biological

Environmental Chamber-style plates. In contrast to above media, the New York City medium

is a clear peptone-corn starch agar containing yeast dialysate, citrated horse plasma, and

lysed horse erythrocytes. It contains the antibiotics vancomycin, colistin, amphotericin B,

and trimethoprim.

Specimens are to be inoculated on warmed or room temperature media. Plates should be

incubated at 35 to 37°C with 3 to 7% CO2 in a moist atmosphere after inoculation. This is

accomplished in a commercially available CO2 incubator equipped with a humidifier. A moist,

CO2-rich atmosphere can also be generated with a candle extinction jar using white,

nonscented candles. Cultures should be examined daily for growth and held for a minimum

of 72 hours.

For culture-based detection of N. meningitidis from primarily sterile materials, such as CSF or

joint fluid, specimens should be inoculated onto sheep blood agar and chocolate agar.

Specimens from mucosal surfaces (e.g., respiratory material) have to be inoculated

additionally on selective media (see above) that exclude growth of most

commensal Neisseria species. Incubation conditions are identical to those for N.

gonorrhoeae,at 35 to 37°C under 5% CO2 tension (109). Nevertheless, in contrast to N.

gonorrhoeae, N. meningitidis tends to grow more readily on solid media and almost

invariably grows on blood agar plates. In addition, vancomycin susceptibility, impeding

recovery of some gonococcal strains from selective media, has not been described. Media

must be examined for suspicious growth at 24, 48, and 72 h. After 72 h a negative culture

result can be issued.

IDENTIFICATION Back to top

Presumptive Identification

Colonial Morphology

After 48 hours of growth on chocolate agar, colonies of N. gonorrhoeae are up to 1 mm in

diameter, opaque, grayish white, glistening, and convex. Morphology can vary subject to the

presence of pili and opacity proteins. Colonies of N. gonorrhoeae expressing pili and opacity

proteins are wrinkled and well defined with a clear edge, while nonpiliated colonies have

more diffuse edges and are more glistening. Due to rapid pilus phase variation, colonial

morphology can appear heterogeneous after primary inoculation.

Colonies of N. meningitidis have smooth, entire edges and are about 1 mm in diameter after

18 h of growth on blood agar. They are gray, convex, glistening, and occasionally mucoid.

Blood agar beneath the colonies may display a gray-green color.

Microscopic Morphology

A Gram stain must be performed on suspected N. gonorrhoeae and N. meningitidis colonies

to confirm the presence of uniform gram-negative diplococci. Consistent results are obtained

with <24-h-old colonies, before autolytic processes appear. Microscopic examination of

suspicious colonies growing on selective plates is essential, since gram-negative rods

belonging to the genera Moraxella (e.g., M. osloensis), Acinetobacter, andKingella can

occasionally grow on them. Nevertheless, the microscopic appearance of gram-negative rods

grown on solid media, particularly Acinetobacter, can be identical to that of Neisseria spp.

Oxidase Test

Performance of the oxidase test is mandatory for colonies suspected to belong to Neisseria.

Both N. gonorrhoeae and N. meningitidis give a positive reaction. In the filter paper method,

oxidase reagent (1% dimethyl-p-phenylene-diamine-dihydrochloride or tetramethyl-pphenylene-

diamine-dihydrochloride) is placed on filter paper, onto which a colony is rubbed

with a wooden stick (nickel-chrome loops may give a false-positive reaction). A fresh isolate

should produce a deep purple color within 10 s. Commercial strips (Microbact Oxidase Strips,

Oxoid, United Kingdom) are a useful alternative.

Definitive Identification

Carbohydrate Utilization Assays

Neisseria species produce acid from carbohydrates by oxidation, not fermentation. The only

carbohydrate used by N. gonorrhoeae is glucose, while N. meningitidis additionally

catabolizes maltose (Table 1). Rarely, however,N. gonorrhoeae (142) and N.

meningitidis (129) fail to acidify carbohydrate-containing media. Also, several asaccharolytic

species including N. cinerea, N. flavescens, and N. elongata never produce acid at all from

sugars. The traditional cystine tryptic agar sugar method has been virtually replaced by rapid

carbohydrate utilization tests in most routine laboratories. These tests give results within 4

hours and are integrated into commercial kits like ApiNH (bioMerieux, Marcy-l’Etoile, France)

and RapID NH (Remel).

Chromogenic Enzyme Substrate Tests

Identification of N. gonorrhoeae can be confirmed by direct detection of enzyme activities

using chromogenic substrates. The tested enzymes usually include β-galactosidase, γ-

glutamyl-aminopeptidase, and proline-iminopeptidase (Pip), which are specific for N.

lactamica, N. meningitidis, and N. gonorrhoeae, respectively. The substrates used for the

above enzymes, bromo-chloro-indolyl-β-galactoside (or -galactopyranoside), γ-glutamylnitroanalide,

and proline-methoxynapthylamide, change their colors after a positive reaction

to blue, yellow, and red, respectively. The three enzymes can be assayed in a one-tube

format in the two commercially available kits, GonoCheck II (EY Laboratories Inc., San

Mateo, CA) and Neisseria PET (BioConnections, Wetherby, United Kingdom). In both tests, β-

galactosidase and γ-glutamyl-aminopeptidase are assayed first. This requires an incubation

step at 37°C for 30 minutes. In a second step, which takes up to 2 minutes, Pip is tested by

the addition of the appropriate reagent (Neisseria PET) or replacement of the screwcap and

subsequent inversion of the tube (GonoCheck II).

A study reported poor sensitivity of these tests in the confirmation of N. gonorrhoeae (2).

False-negative results were obtained with Pip-negative N. gonorrhoeae isolates, which were

shown to constitute 4% of all N. gonorrhoeae isolates in a recent survey in England and

Wales (1). Moreover, N. meningitidis may be γ-glutamyl-aminopeptidase negative (142).

Immunologic Methods for Culture Confirmation

All commercially available tests for the culture confirmation of N. gonorrhoeae rely on the

recognition of gonococcal protein I (with its variants IA and IB) by a pool of monoclonal

antibodies. Phadebact Monoclonal GC Test (Bactus AB, Huddinge, Sweden) is a

coagglutination assay employing inactivated Staphylococcus aureuscells coated with

antibodies bound via their Fc portions to staphylococcal protein A. Cross-reactions with M.

catarrhalis, N. cinerea, and N. lactamica were reported. Nevertheless, more recent studies

found the test to be highly sensitive and specific for culture confirmation of N.

gonorrhoeae (2, 12). The BD GonoGen II (Becton, Dickinson) is a colorimetric test employing

antibodies adsorbed to metal sol particles, which give the reagent its raspberry red color.

False-positive reactions with N. lactamica and N. meningitidis were observed (70).

Furthermore, the solubilizing buffer of GonoGen II was described to only insufficiently extract

protein I (2), resulting in false-negative reactions for some isolates. However, repeat testing

with an extended extraction method led to high specificity and sensitivity of the test (2). The

MicroTrak Culture Confirmation Test (Trinity Biotech, Bray, Ireland) uses fluorescein

isothiocyanate-labeled antibodies for confirmation of N. gonorrhoeae. Positive specimens are

identified by apple-green fluorescent diplococci under a fluorescence microscope. Among the

immunologic methods, MicroTrak was appraised as the most labor-intensive (2). While

earlier evaluations pointed to high specificity yet limited sensitivity (70), false negatives were

not observed in a recent study (2).

Multitest Identification Systems

Several kits combine carbohydrate utilization tests and direct enzyme detection assays for

rapid confirmation of isolates belonging to Neisseria. The Api NH system can be used for the

identification of Neisseria, Haemophilus, and Moraxella catarrhalis and uses 13 miniaturized

tests. In total, the test comprises four sugar utilization tests (assessing glucose, fructose,

maltose, and sucrose), eight enzyme substrate tests, and an acidimetric penicillinase test. In

contrast, the RapID NH (Remel) system contains only two carbohydrate utilization tests (for

glucose and sucrose), 10 enzyme substrate tests, and a resazurin reduction test. The Api NH

and RapID NH kits are inoculated with dense bacterial suspensions adjusted to McFarland

standards of 4 and 3, respectively. Results are obtained after incubation at 37°C for 2 and 4

hours, respectively. The denser bacterial inoculum used in ApiNH might explain the slightly

higher sensitivity compared to RapID NH (2). The automated bacterial identification platform

Vitek 2 (bioMerieux) can also be used for identification of Neisseriaspecies. Its NHI card

contains 30 biochemical tests. Valenza and colleagues reported misidentification of N.

gonorrhoeae as N. cinerea in one isolate owing to the lack of glucose utilization (129). In

another study, all N. gonorrhoeae strains were identified correctly, yet 6% of them received

a low-discrimination result (106).

MALDI-TOF MS

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF

MS) has generated a lot of interest as an emerging technique in the identification of bacterial

pathogens. A species may be determined within a few minutes from whole cells, cell lysates,

or crude bacterial extracts. A recent study analyzing 29, 13, and 15 strains of N.

gonorrhoeae, N. meningitidis, and other Neisseria species, respectively, reports that direct

bacterial profiles are sufficiently different to allow species identification of

pathogenicNeisseria organisms (65). Nevertheless, further evaluations on extended strain

collections are needed.

Hybridization Test

The Accuprobe culture identification test (Gen-Probe) is a DNA probe assay for N.

gonorrhoeae isolated from culture. Similar to AC2 and AG, Accuprobe targets gonococcal

rRNA. After lysis of bacteria, released rRNA is bound by single-stranded-DNA probes labeled

with chemiluminescence. Labeled DNA-RNA hybrids are detected in a luminometer. While the

test has not been evaluated of late, a study confirmed high sensitivity and specificity (141).

DNA Sequencing

Interpretive criteria for identification of bacteria and fungi by DNA target sequences have

been published by CLSI (36). Harmsen et al. have established a reference database for 16S

rRNA sequences including a representative set of Neisseria spp. obtained from reference

strain collections (http://rdna.ridom.de/). The database in most cases allows the

identification to species level of an organism belonging to the genusNeisseria (61). Due to

the possibility of horizontal gene transfer, however, results obtained from a single locus must

be interpreted in light of additional parameters, e.g., growth on selective media, biochemical

tests, slide agglutination, and further PCR assays. Differences in 16S rRNA sequences

between N. meningitidis, N. cinerea, N. gonorrhoeae, and N. lactamica may be as low as 1 to

4% over 700 bp. Other targets such as gyrB and recAhave not been evaluated sufficiently.

The pan-Neisseria MLST has the capacity to provide sufficient information for accurate

species assignment (10).

TYPING SYSTEMS Back to top

Neisseria gonorrhoeae

Methods used for typing of N. gonorrhoeae include Opa typing, pulsed-field gel

electrophoresis, multiantigen sequence typing, and MLST. While both Opa typing and pulsedfield

gel electrophoresis are highly discriminatory, they are cumbersome and poorly portable.

Multiantigen sequence typing represents a portable, sequence-based typing method of N.

gonorrhoeae based on the sequencing of coding regions of the highly polymorphic antigens

Por and TbpB (β subunit of transferring-binding protein) (91). MLST (85), based on the

sequence-based typing of seven household genes, was also highly discriminatory for a

sample of 149 N. gonorrhoeae isolates (10).

Neisseria meningitidis

N. meningitidis is a highly variable organism, and a vast array of techniques have been

developed to describe isolated variants. The simplest method of typing is based on the

nature of the polysaccharide capsule. In total, 12 different serogroups can be distinguished,

which include A, B, C, H, I, K, L, X, Y, Z, W135, and 29E (53). Serogrouping is usually

performed by slide agglutination with a set of commercially available sera (supplied by Remel

or Becton Dickinson) (Fig. 2). A further level of differentiation can be achieved by serotyping

and serosubtyping, which designate the serological characterization of the outer membrane

proteins PorB and PorA, respectively. Today, DNA sequence-based typing schemes of

hypervariable outer membrane proteins have replaced sero(sub)typing. Protocols are

available at http://neisseria.org. Sequence-based typing methods have increasingly gained

acceptance, and a European consensus recommends serogrouping and MLST in conjunction

with the typing of two variable regions of PorA, and the variable region of FetA (50) (Table

2). Protocols for multiple-locus variable-number tandem repeat analysis have also been

developed for meningococci (113).

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

Neisseria gonorrhoeae

The Clinical and Laboratory Standards Institute (CLSI) recommends the use of GC agar

containing 1% growth supplement for disk diffusion testing of N. gonorrhoeae (37). Colony

suspensions of isolates have to be adjusted to a 0.5 McFarland standard before inoculation to

media. CLSI further recommends agar dilution for the measurement of MICs, yet due to their

ease of use, gradient test systems (e.g., Etests) represent an acceptable and frequently used

surrogate.

The difficulties in treatment and control of gonorrhea are aggravated by the ability of N.

gonorrhoeae to mount resistance against a wide range of antibiotics. Although penicillin was

the treatment of choice up to the 1970s, the emergence and increase of penicillinaseproducing

N. gonorrhoeae (PPNG) (139) and chromosomally mediated penicillin resistance

(Penr) led to the abandonment of penicillin as a treatment option. Similarly, plasmidmediated

(TRNG) and chromosomally mediated (Tetr) resistance against tetracycline resulted

in the replacement of this drug by broad-spectrum cephalosporins in the 1980s and later by

the fluoroquinolones. Nevertheless, resistance against fluoroquinolones emerged in the

1990s in Southeast Asia (121) and spread widely to many countries, including the United

States (51). Since April 2007, quinolones are no longer recommended to treat gonococcal

infections in the United States (28). The Gonococcal Isolate Surveillance Project (GISP),

which was established in 1986 to monitor trends in antimicrobial resistance in the United

States, uses six mutually exclusive categories for the description of chromosomally and

plasmid-mediated resistance to penicillin and tetracycline (51): PPNG (β-lactamase positive),

TRNG (MIC, ≥16 μg/ml), PPNG-TRNG, Penr, Tetr (MIC, 2 to 8 μg/ml), and Penr combined

with Tetr (CMRNG). Quinolone resistance (QRNG) represents an additional nonexclusive

category. According to the GISP Annual Report 2007 (27), PPNG, TRNG, and PPNG-TRNG

accounted for 0.4%, 5.6%, and 0.5%, respectively, of all sampled strains. Penr, Tetr, and

CMRNG increased to 2.2%, 5.1%, and 9.3%, respectively (27). In total, 15% of GISP

isolates were resistant to ciprofloxacin in 2007 in the United States (27). In contrast, the

rate of QRNG was earlier reported to be 31% in Europe (90) and close to 100% in many

Asian settings (137). A total of 27 strains (0.4%) were categorized as azithromycin

“nonsusceptible” in GISP isolates in 2007 (37). The CLSI does not define a threshold for

resistance of azithromycin and categorizes isolates with MICs of >2 μg/ml as

“nonsusceptible.” However, the Gonococcal Resistance to Antimicrobials Surveillance

Programme (GRASP), which monitors England and Wales, reported 6 “highly” resistant

isolates in 2007 with MICs of >256 μg/ml by Etest (32). Nevertheless, in 2008, no highly

resistant isolates were collected by GRASP (http://www.hpa.org.uk/GRASP2008). The rise in

QRNG and PPNG led to the replacement of quinolones by broad-spectrum cephalosporins as

the treatment of choice for gonorrhea. Ceftriaxone is the most active cephalosporin

against N. gonorrhoeae but has to be given as an intramuscular (i.m.) injection in a

preparation containing a local anesthetic. The most widely recommended oral broadspectrum

ceph alosporin is cefixime, yet other oral agents, including ceftibuten, cefozopran,

cefdinir, and cefpodoxime, are used as well. Susceptibility testing for cefixime was

discontinued in 2007 by GISP, although strains revealing decreased susceptibility with MICs

of >0.5 μg/ml were occasionally isolated (27). In addition, treatment failures following

therapy with the oral broad-spectrum cephalosporins cef ixime and ceftibuten have been

reported, but not with the injectable ceftriaxone (121). Alterations in genes

includingpenA, encoding penicillin-binding protein 2 (PBP2); mtrA, leading to derepression of

an efflux pump; penB1b,encoding a porin; ponA, encoding PBP1; and others have been

made responsible for cephalosporin resistance (121). Current treatment guidelines (28)

recommend a single dose of ceftriaxone i.m. or cefixime orally for treatment of

uncomplicated gonococcal infections of the cervix, urethra, and rectum. Pharyngeal

gonorrhea should be treated with ceftriaxone i.m., while a cephalosporin-based intravenous

treatment is recommended for the initial treatment of DGI. Fluoroquinolones may be used for

treatment only if antimicrobial susceptibility can be documented by culture. Molecular tools

for rapid detection of resistance (78, 115) have been developed.

Neisseria meningitidis

According to CLSI, testing is performed by disk diffusion on Mueller-Hinton agar or broth

microdilution using cation-adjusted Mueller-Hinton broth (37). Alternatively, gradient test

systems (e.g., Etest) are frequently used. In contrast to N. gonorrhoeae, N. meningitidis is

usually penicillin susceptible, and β-lactamase production is rare (133). In many countries

penicillin is still regarded as a treatment of choice for IMD. Nevertheless, reduced

susceptibility, resulting from modification of PBP2, has been increasingly recorded for several

years. Its molecular basis lies in a combination of five amino acid polymorphisms on

positions 504, 510, 515, 541, and 566 of the PenA protein’s transpeptidase region (119). In

a manner analogous to cefixime resistance in N. gonorrhoeae (121), penA genes of

intermediate-resistant strains were found to have a mosaic structure, suggesting multiple

events of interspecies horizontal DNA transfer originating from commensal Neisseriaspecies

(119). Moreover, MICs of cefotaxime were reported to be higher in strains with intermediate

resistance to penicillin (4). CLSI defines cef otaxime nonsusceptibility at MICs above 0.12

μg/ml but does not provide a threshold for resistance (37). While cefotaxime

nonsusceptibility is rare globally, disquietingly high MICs of up to 8 μg/ml were recently

reported from a sample of eight nonsusceptible strains in India (87). Nevertheless, spread to

other countries has not taken place and the mechanism of nonsusceptibility remains to be

elucidated. Rifampin and ciprofloxacin are used for chemoprophylaxis in close contacts of

patients. Rifampin-resistant strains have a MIC of >2 μg/ml and result from point mutations

in the RNA polymerase β subunit (rpoB) gene (105). Despite this one-step mechanism, the

rate of resistance is very low (120). Resistance against ciprofloxacin has recently emerged in

the United States (140) and is associated with a point mutation at position 91 of the gene

encoding subunit A of DNA gyrase (gyrA). While the rate of resistant isolates is low in the

United States (140) and Europe, an alarmingly high proportion of 65% was reported from a

recent outbreak of IMD in India (97). The CLSI defines ciprofloxacin resistance as a MIC of at

least 0.12 μg/ml or a zone diameter of less than 32 mm when using the 5-μg ciprofloxacin

disk diffusion method (37). Enriquez and colleagues suggested that a 30-μg nalidixic acid

disk is more reliable for screening of ciprofloxacin resistance (46). Azithromycin is used for

mucosal eradication of contacts in areas of high rates of ciprofloxacin resistance (140). Due

to the lack of resistant strains (73) CLSI only defines nonsusceptibility for strains with a MIC

of over 2 μg/ml (37).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

Neisseria gonorrhoeae

Due to the imperfect specificity of many diagnostic methods used for identification of N.

gonorrhoeae, the PPV of each procedure highly depends on the prevalence of disease. If

medicolegal ramifications are likely to result from a positive test, as is the case, for example,

in victims of sexual assault, special scrutiny has to be applied to all laboratory procedures

involved in the issuing of a positive result.

In cases where probability of a positive test is low, such as in the detection of N.

gonorrhoeae from pharyngeal samples in a laboratory not serving a specialized clinic for

genitourinary medicine, special protocols should be in place to ensure confirmation of results.

This is especially important for specimens from children and adolescents and the

documentation of sexual abuse. Here, suspect N. gonorrhoeae should be confirmed by at

least two different methods, including (i) multitest identification systems, (ii) immunologic

methods, (iii) DNA probe culture confirmation, (iv) sequencing of the 16S rRNA gene, and

(v) MALDI-TOF MS. Additionally, strains and DNA need to be conserved.

In settings of high prevalence, such as in laboratories serving genitourinary medicine clinics,

tests giving a “yes-no answer” may be preferable over systems identifying the exact species.

Here two levels of confidence may be attached to the laboratory report. A presumptive

diagnosis of gonorrhea may be issued if one of the following criteria is met: (i) microscopic

visualization of typical gram-negative intra cellular diplococci on examination of a smear of

urethral exudate (male) or endocervical secretions (female), (ii) growth of oxidase-positive

bacteria from the male urethra or female endocervix on selective media with colonial

morphology and microscopic appearance (gram-negative diplococcic) suggestive of N.

gonorrhoeae. A definitive diagnosis requires (i) isolation of oxidase-positive gram-negative

diplococci from sites of exposure (e.g., urethra, endocervix, throat, and rectum) by culture

on selective media; and (ii) confirmation by biochemical or molecular methods. Due to the

different performances of available NAATs, the significance of a positive result will differ

between settings.

The choice of approach is often determined by workload and prevalence of gonorrhea in the

service area of the laboratory. Laboratories that rarely encounter N. gonorrhoeae should

prefer kits that give a full species identification.

Neisseria meningitidis

N. meningitidis is always considered a pathogen when isolated from usually sterile body

fluids such as blood or CSF. Also, when isolated from the urethra, cervix, or the conjunctiva,

a pathogenic role is likely. In the above cases, N. meningitidis should always be reported and

the strain be forwarded to a reference laboratory. As a notable difference from pneumonia

and the detection of meningococci from mucosal surfaces, many national guidelines consider

meningococcal conjunctivitis to be an indication for chemoprophylaxis of the patient and

close contacts, due to high immediate risk of invasive disease (8, 11). Detection of N.

meningitidis from bronchoalveolar lavage fluid or sputum has to be interpreted in liaison with

the clinician. Growth from oropharyngeal or nasopharyngeal specimens usually reflects

asymptomatic carriage and may be omitted from laboratory reports, since it can lead to

confusion regarding the pathogenic significance. Eradication of the organism in asymptomatic

carriers should not be recommended. Similarly, typing of meningococcal carriage isolates

should not routinely be performed. Furthermore, obtaining nasopharyngeal swabs to detect

meningococci from close contacts of a patient with a case of invasive disease should

exclusively be restricted to scientific projects.

Commensal Neisseria Species

Neisseria bacilliformis

Like N. elongata and N. weaveri, N. bacilliformis are rods, not cocci. On blood agar, colonies

have sizes up to 1 mm after 24 hours and are smooth and glistening (58). The color of

colonies ranges from light grey to buff (Fig. 3). Catalase reaction and reduction of nitrate are

variable, and strains are asaccharolytic (58). Strains are associated with the human

respiratory tract and were occasionally recovered as causative agents of endocarditis

(58, 93).

As the species name suggests, colonies of N. cinerea have an ash-gray color and are up to

1.5 mm in diameter. Isolates are asaccharolytic; i.e., they do not acidify carbohydratecontaining

media. Due to this carbohydrate utilization profile, N. cinerea can be confused

with glucose-negative N. gonorrhoeae (129). Furthermore, growth on selective media is

occasionally possible despite colistin sensitivity (76). N. cinereacolonizes the oropharynx of

over 24% of adults (75). Furthermore, it has been attributed a role in ocular infections in

infants (43).

Neisseria elongata

N. elongata forms grayish white, semiopaque colonies, which have a diameter of up to 3 mm

after 48 h of incubation. In contrast to the majority of species within Neisseria, N.

elongata cells are short rods of ca. 0.5 μm in diameter. The species consists of three

subspecies: N. elongata subsp. elongata, N. elongata subsp.glycolytica, and N.

elongata subsp. nitroreducens. As an exception within the genus Neisseria, N.

elongata subsp.elongata is catalase negative and does not produce acid from glucose or

other carbohydrates. In contrast, subsp. glycolytica is catalase positive and weakly acidifies

glucose media. Finally, subsp. nitroreducens is catalase negative and reduces nitrate. As

other Neisseria species, N. elongata mainly appears as a colonizer of the human oropharynx.

Nevertheless, several cases of endocarditis caused by N. elongata were published (64).

Neisseria flavescens

N. flavescens produces smooth and opaque yellow colonies. N. flavescens does not generate

acid from sugars but produces polysaccharide from sucrose, which can be detected by

pouring an iodine-containing solution (e.g., Lugol’s) over colonies growing on brain heart

infusion agar with sucrose. The iodine test is positive if colonies develop a deep-blue color,

indicating the presence of a starch-like polysaccharide. N. flavescenscolonizes the pharynx of

humans and only rarely causes disease, such as endocarditis (116).

Neisseria mucosa

N. mucosa typically grows in large, adherent, and mucoid colonies, which are mostly

nonpigmented. In carbohydrate utilization tests strains of this species are glucose, maltose,

fructose, and sucrose positive. N. mucosa is found in the nasopharynx of humans, where it

represents an apathogenic commensal. It has been associated with infective endocarditis in

rare cases (125), and due to variable susceptibility to penicillin, the choice of antibiotic

treatment has to be supported by susceptibility testing.

Neisseria lactamica

N. lactamica is readily confused with N. meningitidis, since it is morphologically similar and

may grow on selective media. Nevertheless, it acidifies lactose in addition to glucose and

maltose and is γ-glutamyl-aminopeptidase negative. N. lactamica is a commensal of the

upper respiratory tract of infants and children. In contrast to N. meningitidis, colonization of

the oropharynx with N. lactamica begins as soon as 2 weeks after birth (9). It is rarely

pathogenic, although exceptional cases of meningitis and septicemia have been described. N.

lactamica usually displays reduced susceptibility against penicillin.

Neisseria polysaccharea

Strains of N. polysaccharea present as small, yellow-grayish, translucent colonies. Like N.

meningitidis, they acidify glucose and maltose but not fructose or lactose. In addition, they

may grow on selective media and are γ-glutamyl-aminopeptidase positive. In contrast to N.

meningitidis, however, the iodine test is positive, indicating the production of polysaccharide

from sucrose (similar to N. flavescens). It colonizes the nasopharynx of children and has so

far not been associated with disease.

Neisseria sicca

The colonies formed by N. sicca are large (≤3 mm), dry, wrinkled, and grayish white,

although some strains may produce a yellowish pigment. Its carbohydrate utilization profile

is indistinguishable from that of N. mucosa, but it does not reduce nitrate. This bacterium is

a common oropharyngeal commensal in humans. Nevertheless, it can appear as an

opportunistic pathogen. N. sicca was recently implicated, for example, as the causative agent

of endocarditis (71).

Neisseria subflava

N. subflava appears as smooth, variably transparent colonies with a yellowish pigment. This

species contains the previous species N. subflava, N. perflava, and N. flava (124). Strains

of N. subflava acidify glucose and maltose. In addition, N. subflava bv. subflava and

bv. flava produce acid from fructose, while bv. perflavaacidifies sucrose and produces

polysaccharide from sucrose. N. subflava is a common commensal of the human oropharynx,

yet has occasionally been associated with invasive diseases such as meningitis, endocarditis,

and bacteremia (7). Similar to N. lactamica, reduced susceptibilities to penicillin and also to

cefixime and ciprofloxacin were reported recently (56).

Neisseria weaveri

Colonies of N. weaveri are of variable size (1 to 2 mm), smooth, flat, and slightly glistening

(124). They have entire edges and are grayish in color. As most species of the

genus Neisseria, they are strongly catalase and oxidase positive. Like N. bacilliformis and N.

elongata, N. weaveri cells are rods, not cocci. N. weaveri does not use carbohydrates and

does not reduce nitrate (62). Strains are infrequently recovered from human dog bite

wounds and oral cavities of dogs (62). While septicemia in an immunosuppressed individual

has been described (22), invasive disease is very rare.