Laboratory Diagnosis of meningitis

Laboratory diagnosis of AM is essential to differentiate between aseptic and septic meningitis. Purulent or septic meningitis is life-threatening but potentially treatable disease whereas viral or AM usually subsides spontaneously (Lee & Davies ,2007).

The identification of specific causes of AM help in prognosis. The EVs are responsible for the majority of cases of AM which need no treatment and have a self limited course, so it is essential to exclude other causes of meningitis such as HSV, VZV, bacterial meningitis to avoid unnecessary hospitalization and the use of antibiotics and therapeutic medications (Rotbart; 1995).

The most important laboratory technique for diagnosis of meningitis is CSF examination which can be considered as a corner stone for differentiation between viral and bacterial meningitis (Lee & Davies ,2007).

Examination of CSF :

(a)-Cerebrospinal fluid:

Cerebrospinal fluid (CSF) : is a clear body fluid that occupies the subarachnoid space and the ventricular system around and inside the brain. Essentially, the brain "floats" in it.More specifically,CSF occupies between the arachnoid mater (the middle layer of the brain cover, meninges) and the pia mater (the layer of the meninges closest to the brain). Moreover , it constitutes the content of all intra-cerebral (inside
the brain, cerebrum) ventricles, cisterns and sulci (singular sulcus) as well as the central canal of the spinal cord. It is an

approximately isotonic solution and acts as a "cushion" or buffer for the cortex. Also providing a basic mechanical and immunological protection to the brain inside the skull )Zakharov et al.,2003).

Amount and constitution:
CSF is produced at a rate of 500 ml/day. Since the brain can only contain from 135-150 ml, large amounts are drained primarily into the blood through arachnoid granulations in the superior sagittal sinus. The CSF contains approximately 0.3% plasma proteins or 15 to 40 mg/dL depending on sampling site. CSF pressure ranges from 60 - 100 mmH2O or 4.4 - 7.3 mmHg with most variations due to coughing or internal compression of jugular veins in the neck ( Dixon et al.,2002).
Function:

CSF has many putative roles including mechanical protection of the brain, distribution of neuroendocrine factors and prevention of brain ischemia. The prevention of brain ischemia is made by decreasing the amount of CSF in the limited space inside the skull. This decreases total intracranial pressure and facilitates blood perfusion (Saunders et al.,1999).

(b) - CSF collection :

CSF is usually collected by an experienced medical officer or health worker by lumbar puncture through aseptically inserting a needle into the subarachinoid space usually at the level of lumber spine .The procedure may be dangerous if the intracranial pressure ( ICP) is raised , so the clinician should be check that there is no papilloedema before the proceeding (Rotbart , 2000 ).

About 6 ml is collected in fresh sterile screw-capped container and divided into 2 parts. A part for culture and should be incubated at 37◦c.The other part should be transported on ice and examined for other tests as cell count, microscopically examination and biochemical analysis including measurement of glucose , protein and for antigen detection (Negrini et al ., 2000).

Lumbar puncture should be avoided in patients with depressed levels of consciousness and shock. Contraindications to lumbar puncture include the following: prolonged or focal seizures , focal neurological signs,widespread purpuric or petechial rash ,pupillary dilatation or asymmetry , impaired oculocephalic reflexes , abnormal posture or movement - decerebrate or decorticate movement or cycling , signs of impending brain herniation ( inappropriate low pulse, raised blood pressure, irregular respiration) , coagulation disorder , papilledema and hypertension (American Academy of Neurology ,2005).

(c) - Storage and transport :

According to Wong et al .,( 2000) the specimens which will be examined for viral detection may be stored by freezing up to 5 days at 4 c and for 6 days or more at -20◦ c after proper mixing in viral transport media. Viral transport media are used to transport small volume of fluid specimen, small tissue, scrapings and swab specimen.These media contain serum , albumin and gelatin to stabilize virus and antimicrobial agents as penicillin,streptomycine and a more potent mixture include vancomycine,gentamycine and amphotricine.CSF is potentially highly infectious and must be handled and transported with great caution .

(I) – Nonspecific discriminatory tests:

A-Macroscopical examination:

Normal CSF is clear and colorless like water. A yellow color "xanthochromic" may result from subarachnoid hemorrhage and traumatic lumbar puncture .



The normal number of WBCs in the CSF in adults vary from 0 to 3 cells/cmm, a maximum of 5 cells/cmm and they are mainly lymphocytes. In children less than one year old , they vary from 0 to 30 cells/cmm and they are mainly polymorphs. Normal CSF contains a small number of lymphocytes and monocytes. The lymphocytes present in the CSF are similar to those in the peripheral blood. Small lymphocytes predominate and 75 to 95% are T lymphocytes (Puccioni-Sohler ,2002 ).


Cell counts over 1000/cmm usually are caused by bacterial infections while counts of 500-1000 /cmm may be bacterial or viral and need further evaluation. In AM the total WBCs count is usually <500/cmm with lymphocytes predominance. However, several studies proved that 20-75% of AM especially those caused by EVs , PMN's were predominant in initial CSF samples (Glimaker et al; 1992). Blood samples should be collected before starting antibiotic therapy for culture and serodiagnosis of viral agents as mumps ,herpes simplex or as Mycoplasma Pneumonia which may be difficult to be cultivated and require serological confirmation of infection (Cinque et al ; 1996) . C-Direct Gram Stained of CSF : Gram staining is a common procedure in the traditional bacteriological laboratory. The technique is used as a tool for the differentiation of Gram-positive and Gram-negative bacteria as a first step to determine the identity of a particular bacterial sample (Beveridge , 1990) . As a general rule Gram-negative bacteria are more pathogenic than Gram-positive bacteria due to their outer membrane structure (Søgaard et al ., 2007). ***Gram-negative bacteria : The proteobacteria are a major group of Gram-negative bacteria. These include many medically relevant Gram-negative cocci, bacilli and many bacteria associated with nosocomial infections (Beveridge ,2001). ***Gram-positive bacteria : They include Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms (Beveridge ,2001). A part of CSF should be centrifuged to get a deposit which stained by Gram stain . The stain may help in diagnosis but the bacterial pathogen may be missed in up to 30% of culture proven cases of bacterial meningitis and is negative in all cases of A.M ( Waler and Rathore ; 1995). According to Saitoh et al. (2005), if a tuberculuse (TB) meningitis is suspected , Zheil-Neelsen smear is done for detection of acid fast bacilli (AFB) and culture on one or two slope of Lowenstein-Jensen medium and incubated for 2-6 weeks at 37◦C .The growing organisms are identified by colonial morphology, staining characters and biochemical reactions . The direct smear examination for acid-fast Mycobacterium is a rapid method but requires a minimum of 104 cells / ml which makes detection extremely difficult for samples such as CSF, pleural effusion and peritoneal fluid ( Kulkarni, et al. 2005). If Yeast cells are seen when performing a cell count or detected in a Gram stain , a small drop of CSF sediment is transferred to a slide with addition of small drop of India ink for the detection of Cryptococcus neoformans if present ( Johansen , et al.2004). D- Culture of CSF: Immediately, part of the CSF deposit seeded heavily on culture media as blood agar, chocolate agar as ( Thayer Martin media ) and incubated in humid air at 37◦C for 24-48 hours plus 5-10% CO2 . Tube of cooked-meat broth and another blood agar plate as Columbia , Brucella or Scheadler should be seeded by deposit and incubated for 2-5 days in an anaerobic atmosphere when there is a suspicious of anaerobic infection. The culture inspected after overnight incubation for identifying any growth , if no growth appears after overnight incubation , the plates are reincubated for another day and reinspected for growth (Nigrovic et al ., 2004 ) . The culture method in tuberculous meningitis is a definitive diagnosis but it is limited by the slow growth rate over several weeks. The sensitivity of the culture method decreases in certain samples with a low number of M. tuberculosis. It could be as low as 40 % for CSF, pleural effusion and peritoneal samples (Schluger et al., 2001). The radiometric method (BACTEC) is based on the measurement of the radioactive CO2 released by metabolism of the radioactive or carbon labeled palmitic acid present in the liquid culture media from mycobacterial growth. This method reduces the culture time, but still requires 7 – 10 days for positive cultures (Bonington et al, 2000). Mycobacteria Growth Indicator Tube (MGIT) system is a non-radiometric method, it has an oxygen sensitive fluorescent sensor embedded in silicone base to serve as an indicator of mycobacterial growth. As the actively growing and respiring mycobacteria consume the dissolved O2, the sensor glows indicating mycobacterial growth. This is observed by using an ultra violet lamp with a wave length of 365 nm (Chitra and Prasad , 2001). Though the use of culture method in leptospirosis confirms diagnosis and also aids in identifying the prevalent serovar, it is rarely used, as it is very tedious, complicated, expensive technically demanding time consuming, requiring prolonged incubation minimum 1 month before declaring a sample negative and may not be successful (low sensitivity). Additionally they are highly infectious organisms requiring strict biosafety facilities (Dutta & Christopher , 2005). Blood or CSF culture for diagnosis of brucellosis in patients with primary infections gives excellent sensitivity results but in individuals with previous contact with the microorganism or occupational exposure and symptoms of acute or persistent infection (very frequent in endemic areas) culture gives poor results( Jordi & Miquel , 2004). The risk of spread of infection, difficulty in culture, time period of more than 6 weeks and positivity of blood or CSF culture in only 50 to 70% patients do not make culture method the investigation of choice for the diagnosis of brucellosis (Kochar et al ., 2000). Diagnosis of Lyme disease by culture method can be done but it is difficult as it requires specialized medium not generally available in most microbiological laboratories and prolonged incubation at relatively low temperatures. Even in specialized labs, the number of spirochetes present in the samples are so low that the yield of culture and even PCR testing are low. The best sensitivity reported in CSF culture in Lyme meningitis is only 10 % as spirochetes are so few in number or even none in the aliquot tested. As a result , diagnosis depends heavily on demonstration of specific anti-B.burgdorferi antibody in serum or CSF (Pachner & Steiner , 2007). Although T. pallidum cannot be grown in culture, there are many tests for the direct and indirect diagnosis of syphilis.Still, there is no single optimal test. Direct diagnostic methods include the detection of T. pallidum by Dark-field microscopy examination of fluid or smears from lesions, histological examination of tissues or nucleic acid amplification methods such as PCR. Indirect diagnosis is based on serological tests for the detection of antibodies. Serological tests are fall into two categories: nontreponemal tests for screening and treponemal tests for confirmation . All nontreponemal tests measure both immunoglobulin (Ig) G and IgM antiphospholipid antibodies formed by the host in response to lipoidal material released by damaged host cells early in infection and to the lipid from the cell surfaces of the treponeme itself (Sam , 2005). In diagnosis of CNS candidiasis ,the significance of a positive culture from the CSF may be unclear. Contamination of the CSF sample may occur because of colonization of the skin or when cultures have been taken from external reservoirs that contain CSF. Several non-culture-based methods have been developed for diagnosing invasive fungal infections of the CNS, such as cryptococcal meningitis and CNS aspergillosis. Similarly, a Candida cell wall component, mannan, has been used as a target for serological tests. Although the detection of circulating mannan was found to be of limited value in the diagnosis of invasive candidiasis, detection of mannan in CSF could be a valuable tool for diagnosing CNS candidiasis (Frans et al ., 2004). E-Biochemical tests : The supernatant part of centrifuged CSF tested for glucose , protein , lactate , C-Reactive protein , adenosine deaminase (ADA) content , estimation of the level of cytokines , lysozyme tests,total and differential leucocytic count ( Salmaso et al ., 1997). (a)- Glucose content : Simultaneous estimation of blood and CSF glucose levels is the most discriminatory test of the nonspecific CSF parameters to differentiate between bacterial and viral meningitis. CSF contain 2.2-4 mmol glucose/liter .Normal CSF glucose is about 60% of serum glucose value. If CSF glucose is <50% of serum glucose this will raise the possibility of bacterial meningitis. Glucose level is usually reduced in bacterial meningitis but may be normal or slight decreased in viral meningitis (Cinque et al ; 1996) . (b)- Protein content : Protein concentration which is below 0.4 g/L in normal CSF is usually more elevated in bacterial meningitis but may be normal or mild elevated in viral meningitis. In purulent (septic) meningitis , the glucose concentration is reduced and the protein concentration increased but in AM , the glucose concentration is normal and the protein concentration either normal or raised a little ( Cinque et al ; 1996). (c)- CSF lactate : The best test to differentiate bacterial from viral meningitis is the measurement of CSF lactate . Lactate levels are particularly important when CSF Gram staining is negative and there is a predominance of PMNs with low glucose in the CSF. CSF lactate concentrations greater than 3.5 mmol/L are characteristic of acute bacterial meningitis. As the lactate concentration in the CSF is independent of that of serum, there is no necessity to test the serum level (Cunha,2004). (d)- Acute phase reactants : Hansson et al.( 1993) found that determination of concentrations of alpha-¬1-acid glycoprotein (AAG) and C-reactive protein (CRP) in serum and alpha-2-ceruloplasmin (CER) in CSF are useful in differentiation between bacterial and viral meningitis. In children younger than 6 years of age , a discriminatory level of serum CRP of 20 mg/L can be used to distinguish between bacterial and viral meningitis but for older patients, a discriminatory level of 50 mg/L is more appropriate. Determination of AAG, CRP in serum are good markers for treatment efficacy and infectious complications in case of bacterial meningitis (Kwaik et al; 1995 and Paradowski et al; 1995) . (e)-Estimation of cytokines : Interleukin 6 (IL6) in CSF was reported to be as a diagnostic marker in the differential diagnosis of meningitis.It can be measured using monoclonal antibody enzyme immunoassay or Radioimmunoassay(RIA). CSF IL6 concentrations were found to be elevated in pyogenic meningitis in 100% of cases and in >50% of viral and other subarachnoid space infections and rarely in patients without CNS infections. Though, patients affected by pyogenic meningitis show the highest levels of CSF IL6 (Lopez-Cortes et al;1997).

(f)- Lysozyme test :

The principle of this test is to add polymexin M sulfate into
the gel bacterial medium. Rapid differential diagnosis of bacterial and viral meningitis with the use of this test is based on different time of the appearance of the lyses areas in bacterial meningitis. The CSF lysozyme activity is detectable within 15-120 min whereas in viral meningitis it manifests 40-50 min later or does not manifest at all. The results are dependant on the time of the CSF collection as more positive results are obtained when CSF samples are early collected (Babich et al ;1992).