Tuesday, August 16, 2011

Laboratory Diagnosis of Meningitis: Microscopic examination


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.

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. Table (2) Characters of CSF in normal and pathological alterations:- Cunha ,(2004) & Puccioni-Sohler et al.,(2002).

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 (S√łgaard 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).

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