Laboratory Diagnostics of Gonococcal and Meningococcal infections
N. gonorrhoeae is a fastidious organism, requiring complex media for growth and adversely affected by drying and fatty acids. Soluble starch is added to the media to neutralize the toxic effect of the fatty acids. It is highly susceptible to enviromental, physical, and chemical agents. The optimum growth temperature is 35ºC to 37ºC, with poor survival of the organism at cooler temperature. A humid atmosphere supplemented with CO2 is required or enhances growth of N. gonorrhoeae.
Antigenic structure as it relates to virulence
The outer surface is not covered with a true carbohydrate capsule, as is found in Neisseria meningitidis. Pili mediate attachment of the organism to epithelial and mucosal cell surfaces and are antiphagocytic. Three outer membrane proteins (OMPs) have been studied extensively:
1. Por proteins are porin proteins that form pores channels in the outer membrane.
2. Opa (opacity) proteins are a family of membrane proteins that mediate binding to epithelial cells.
3. Rmp (reduction-modifiable) proteins. Prevents complement-mediated bactericidal antibody function and thus may contribute to dissemination of the disease.
Another major antigen in the cell wall is lipooligosaccharide (LOS). This antigen is composed of lipid A and a core oligosaccharide, similar to gram-negative lipopolysaccharide (LPS), and possesses endotoxin activity. Other important gonococcal proteins are an immunoglobulin (Ig) A1 protease; the enzyme inactivates local secretory Ig A and thus may play a role in facilitating the adherence of gonococci to mucosal surfaces; ß-lactamase, which degrades penicillin.
Diseases caused by
Gonorrhoea is one of the most commonly reported sexually transmitted disease in the United States. A higher proportion of females than males are generally asymptomatic; these individuals act as the reservoir for maintaining and tranmitting gonococcal infections. More than one sexually transmitted disease may be acquired at the same time, for example gonorrhea in combination with syphilis, chlamydia, human immunodeficiency virus (HIV), and hepatitis B virus. Patients with gonorrhea may therefore have to be treated for more than one pathogen.
Genital infection in men is primarily restricted to the urethra. Purulent urethral discharge and dysuria are developing after a 2-7 day incubation period. Although complications are rare, epididymitis, prostatitis, and periurethral abscesses can occur. The primary site of infection in women is the cervix, although gonococci can be isolated in the vagina, urethra and rectum. Vaginal discharge, dysuria, and abdominal pain are commonly reported in symptomatic patients. Other diseases associated with N. gonorrhoeae include purulent conjunctivitis particularly in newborns infected during vaginal delivery (ophthalmia), anorectal gonorrhea in homosexual males, and pharyngitis.
Specimens obtained depend on the disease process and include urethral, cervical, rectal, pharyngeal, and/or conjunctival exudates.
The direct demonstration of gram-negative intracellular diplococci within PMNs is diagnostic only when observed in the urethral exudates of males with characteristic clinical manifestations.
Gram stains of smears from female urethral and cervical exudates, from rectal, pharyngeal, and conjunctival exudates of male and females, are unreliable due to the potential presence of nonpathogens resembling gonococcal morphology, and presence of meningococcuses. All such specimens must be cultured and the isolated organism identified. These techniques require initial cultivation on Thayer-Martin medium. Incubation for 48 h at 35-37ºC under aerobic conditions in the presence of 3-10% carbon dioxide is optimal for isolation of the organism, which may be identified as a gram-negative, oxidase positive, diplococcus that ferments glucose, but not maltose, sucrose, or lactose.
Ceftriaxone, cefixime, ciprofloxacin, or ofloxacin can be administered in uncomplicated cases. In vitro susceptibility should be determined in cases unresponsive to therapy, because antibiotic resistance is increasing. Penicillin should be avoided, because resistance is common. Doxycycline or azithromycin should be added for infections complicated by Chlamydia.
N. meningitidis causes endemic or epidemic disease of worldwide prevalence. The most commonly recognized from of this disease is meningitis.
N. meningitidis is an obligate parasite of humans, harbored in the nasopharynx, and transmitted by droplet nuclei from or direct intimate contact with a case or carrier. Factors contributing to sesceptibility include fatigue and exposure to inclement weather. Meningococci are destroyed rapidly in the enviroment and are highly susceptible to physical and chemical agents.
This organism is a gram-negative, nonmotile, encapsulated, piliated diplococcus flattened on one site to give the appearance of a “kidney bean” or “coffee bean”. The antigenic diversity of the capsule forms the basis for classification of the meningococci into 13 serogroups. The great majority of meningicoccal disease is caused by serogroups A, B, C, W, and Y. The capsules are antiphagocytic and may facilitate meningeal invasion. Outer membrane proteins (OMPs) of the organism are also antigenically diverse, which enables serotyping meningococci within each serogroup. In addition, OMPs acts as porins. The organisms are strict aerobes that grow best on chocolate agar at 35-37ºC in the presence of 3-10% carbon dioxide.
Pathogenesis and Clinical Manifestations
The basic pathogenesis process of primary meningococcal disease is initiated in the nasopharynx from a case or carrier. Adherence to the mucosal surface with resultant colonization is mediated by pili and possibly facilitated by secretory Ig A cleavage by Ig A1 protease. Phagocytosis is inhibited by the capsule and the organisms continue to multiply, producing nasopharyngitis. Most patients produce complement-dependent bactericidal antibody, predominantly of the Ig M type, and opsonic antibody, both of which restrict the organisms to the mucosal surface of the nasopharynx and eventually cause their riddance. Disseminated meningococcal disease manifests most often as septicemia and meningitis. Onset may correlated with the absence of complement-dependent bactericidal antibody and opsonic antibody, the presence of serum Ig A antibody that blocks the initiation of immune lysis, or complement component deficiencies. The clinical manifestations of septicemia are the result of Lipooligosaccharide (LOS), which is an abundant component of the organism, is released upon multiplication and autolysis. LOS differs from LPS in that the former has shorter, nonrepeat, O-antigenic side chains and thus has a lower molecular weight. Meningitis is the most common complication of meningococcal septicemia. Clinical manifestations are fever, stiff neck, vomiting, severe headache, convulsions, bulging of the fontanels, and progression to a coma within a few hours.
Specimens obtained depend on the disease process and mainly include nasopharynx swabs, blood culture, and cerebrospinal fluid. Gram stains of specimens may show gram-negative, intracellular and extracellular diplococci in association with PMNs.
Primary isolations require initial blood cultures and culture on chocolate agar plates or, if a mixed flora is anticipated, Thyer-Martin medium, which is an enriched chocolate agar medium, containing antibiotics to inhibit gram-positive organisms and gram-negative rods. Incubation for 48 h at 35-37ºC under aerobic conditions in the presence of 3-10% carbon dioxide is optimal for isolation of the organisms, which may be identified as gram-negative, oxidase-positive, diplococcus that ferments glucose and maltose, and agglutinates in the presence of serogroup-specific anticapsular antibody.
Early treatment with penicillin has reduced the case fatality rate in disseminated disease from 40-90% to 10-15%, but the antibiotic is ineffective in eradicating the carrier state. Chloramphenicol and ceftriaxone are effective in penicillin-allergic individuals. Polysaccharide vaccines conjugated with protein carriers offer protection for infants younger than 2 years.
Microbiological research of the material
in meningococcal infections and bacteria carriers
Mucus from pharynx and nose, blood, pus, spinal fluid, exudates
Inoculation part colony in the slanted serum agar
(to accumulate pure culture)