Laboratory Diagnosis of Neisseria meningitides infections

Laboratory Diagnosis

Laboratory diagnosis depends on demonstration of meningo-cocci in clinical specimens by microscopy and culture.

◗ Specimens

Cerebrospinal fluid and blood are the specimens of choice for demonstration of meningococci in the early stage of men-ingitis. Nasopharyngeal swabs are useful to detect carriers. The CSF is collected by lumbar puncture and blood by veni-puncture in strict aseptic conditions. CSF is never refrigerated as Haemophilus influenzae, another agent of meningitis, may die at the cold temperature. CSF specimens are transported immediately to laboratory for processing. Similarly, blood is collected in blood culture media containing either glucose broth or sodium taurocholate broth. Nasopharyngeal speci-mens are collected using sterile swabs and are transported in Stuart’s transport medium to the laboratory.

CSF

Meningococcal meningitis produces various inflammatory changes in the CSF:

·           The CSF in bacterial meningitis is more turbid.

·           It contains more than 1000 WBC/mL, and the cells are pre-dominantly PMN cells.

·           The total protein content is increased. The total glucose level, which is normally 60% of simultaneous blood glucose level, is lowered (hypoglycorrhachia).

·           The intracranial pressure may be elevated.

CSF received in the laboratory is processed in three parts:

1.        First part is centrifuged and smear is prepared from the deposit for Gram staining. The supernatant is tested for meningococcal antigens.

2.        The second part of CSF is used for direct culture.

3.        The third part is incubated overnight with an equal vol-ume of glucose broth and then subcultured onto the blood agar and chocolate agar.

◗ Microscopy

Gram staining of the CSF is a very useful method for detec-tion of meningococci. Meningococci are seen as Gram-negative diplococci present mainly inside the leukocytes and some may even be present extracellularly. These cocci can be demon-strated in the CSF in approximately 50% of the patients with meningococcal meningitis. In fulminant meningococcemia, Gram staining of the peripheral blood buffy coat may reveal Gram-negative diplococci.

◗ Culture

Isolation of N. meningitidis from the CSF, blood, and other clinical specimens by culture confirms the diagnosis of menin-gococcal infection. The CSF is inoculated immediately on a nonselective medium, such as blood agar or chocolate agar, and incubated at 35–36°C under 5% CO2 for 18–24 hours. The colo-nies of meningococci are small, round, translucent, and convex with a smooth glistening surface.

Blood is inoculated immediately into blood culture bottles containing either glucose broth or sodium taurocholate broth and incubated at 35–36°C. Subcultures are made on blood agar or chocolate agar from these broths and are reincubated overnight at 35–36°C in the presence of 5% CO2. The cultures should be incubated for 4–7 days with daily subculture. Blood culture is often positive during early stage of meningitis and in meningococcemia.

◗ Other specimens

Other specimens, such as nasopharyngeal swabs and petechial exudates are processed in a similar way as described earlier for CSF.

◗ Identification of bacteria

Serogrouping of the bacterial isolates grown on culture is car-ried out by slide agglutination with specific hyperimmune serum.

◗ Antigen detection

Detection of soluble polysaccharide antigen in the CSF is a useful method for diagnosis of meningococcal meningitis. Counter-current immunoelectrophoresis, latex agglutination test, and bacterial coagglutination test using specific antibod-ies are the rapid tests frequently used to detect the soluble antigen in the CSF. Antigen detection is useful when bacteria are scanty in the CSF. However, antigen detection is not use-ful in the meningitis caused by Group B meningococci because N. meningitidis serogroup B is relatively nonimmunogenic anddoes not react with specific antibodies.

◗ Serodiagnosis

Indirect hemagglutination test and ELISA are useful for the demonstration of antibodies against specific polysaccharide antigen in the serum. Serodiagnosis is useful in the cases of chronic meningococcal infection where cultures have proved negative for meningococci.