Wednesday, April 18, 2012

(I) Septic meningitis Septic meningitis is an inflammation of the meninges caused by bacterial pathogens . Acute meningitis is usually bacterial infection caused by one of several organisms. For development of septic (bacterial) meningitis , the invading organism must gain access to the subarachnoid space (Ashwal ; 1995 ). (A) Pathophysiology : After initial colonization in the upper respiratory tract the invading organism can gain access to the subarachnoid space usually via haematogenous spread and less frequently through an injury such as skull fracture which can cause meningeal seeding via direct bacterial inoculation during trauma (Johansson & Bergentoft ,2005). Bacterial meningitis in the newborn is transmitted either vertically from colonized pathogens in the maternal intestinal or genital tract or horizontally from nursery personnel or other contacts with the patient (Kelley ; 1996).Once bacteria reached to the CSF the relative lack of antibody, complement and white blood cells (WBCs) allow the bacterial infection to flourish (Waler & Rathore; 1995). The types of bacteria that cause septic meningitis vary according to the age of patients. In premature babies and newborn up to three months, the commonest bacteria are group B streptococcus especially in the first week of life–and bacteria that normally inhabit the digestive tract such as Escherichia coli. Listeria monocytogenes may affect the newborn and occurs in epidemics ( Sáez-Llorens & McCracken , 2003). Older children are more commonly affected by Neisseria meningitidis , Streptococcus pneumonia and Haemophilus influenza type B (Ginsberg ,2004). In adults, N. meningitidis and S. pneumonia together cause 80% of all cases of meningitis with increased risk of L. monocytogenes in those over 50 years. Staphylococci , Pseudomonas and other Gram-negative bacilli are likely to cause meningitis especially in patients with trauma, neurosurgical interferences and with impaired immune system (van de Beek et al ., 2006 & Tunkel et al ., 2004 ). ( B) Symptoms and signs : The history in children with bacterial meningitis varies with age. The younger the child, the less likely to exhibit the classical symptoms which include fever, headache and meningeal signs. Babies under 3 months may have very non specific symptoms including hyper or hypothermia, changes in sleeping or eating habits, irritability or lethargy, vomiting, high pitched cry or seizures (Nigrovic et al ., 2007). According to Kelley;(1996) , signs of meningeal irritation are diagnostically helpful when present, they include: * Nuchal rigidity or discomfort on neck flexion. * Kernig's sign which is characterized by passive knee extension in supine patient elicits neck pain. * Brudziniski's sign which is characterized by passive neck , single or both hip flexion . * Meningismus and bulging fontanels may be seen. Other manifestations are systemic findings such as extracranial infection as sinusitis, otitis media, urinary tract infection , arthritis , non-branching pitechae, cutaneous hemorrhage and endotoxic shock especially with Neisseria meningitidis (Thomas et al., 2002) . (C) Risk factors : Risk factors include young age, rapid onset of illness, low peripheral WBCs count and high CSF protein (Pfister , 2005 ). (D) Prognosis : Prognosis depend on the virulence of pathogen , age and severity of acute illness (Puopolo et al ., 2005). In general, mortality rates vary with age and pathogen with the highest being for S. pneumonia. Bacterial meningitis also causes long-term sequelae and results in significant morbidity beyond the neonatal period. Mortality rates are highest during the first year of life, decreasing in mid life and increasing again in elderly persons ( Ray et al ., 2007). (E) complications : *Seizures: Persistent , focal or appear late in the course of disease are more likely to be associated with neurological sequelaes (Chavez-Bueno & McCracken, 2005). *Other complications: include subdural effusions and brain abscesses. Subdural effusions are generally asymptomatic and resolve without neurological sequelae (Prasad , Karlupia , 2007 & Nelson , McCracken , 2005).CNS sequelaes include nerve deafness, cortical blindness, hemiparesis, quadriparesis, muscular hypertonia, ataxia, complex seizure disorders, mental motor retardation, learning disabilities, obstructive hydrocephalus, and cerebral atrophy (Nigrovic et al ., 2007). (II) Aspetic meningitis: According to Lee & Davies ,( 2007) AM is an inflammation of the meninges caused mainly by nonbacterial organisms or other disease processes. AM denotes a clinical syndrome with a predominance of lymphocytes in the CSF in absence of bacterial agents in CSF. Also , AM is an acute infection of the central nervous system that occurs most frequently in infants and young children (Berlin, et al.,1993). It could be either viral, fungal , tuberculous meningitis or other causes ( Rorabough et al ;1993). (A) Pathophysiology: Organisms colonize and penetrate the nasopharyngeal or oropharyngeal mucosa , survive and multiply in the blood stream and they invade host immunological mechanisms and spread through the blood-brain barrier. Infection cannot occur until colonization of the host has taken place (usually in the upper respiratory tract).The mechanisms by which circulating viruses penetrate the blood-brain barrier and seed the CSF to cause meningitis are unclear ( CDC ; 2005). In tuberculous meningitis infection begins in the lung and may spread to the meninges by a variety of routes.Blood-borne spread occurs and 25% of patients with miliary TB have TB meningitis by crossing the blood-brain barrier.A proportionof patients may get TB meningitis from rupture of a cortical focus in the brain (Rich focus) and little proportions get it from rupture of a bony focus in the spine. It is rare and unusual for TB of the spine to cause TB meningitis , but isolated cases have been described(Jain et al .,2006). (B) Symptoms and signs: Clinical presentations and courses of AM are markedly variable. Severity of presentations are correlated with prognosis. They usually start by general viral prodroma for several days which is presented by fever, headache, nausea, vomiting , lethargy, myalgia, dysuria and pyuria (Kung., 2007). Specific prodroma in cases of Varicella zoster virus (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Measles and Mumps viruses have been reported such as rash, lymphadenopathy, hepatosplenomegaly and parotid enlargement.Common presentation of viral meningitis and encephalitis is encephalopathy with diffuse neurological symptoms and signs including behavioral and personality changes and/or altered mental status, decreased consciousness, generalized seizures, acute confusional or amnesia states but meningismus and headache are less common. Other nonspecific symptoms may include arthralgia, myalgia, sore throat, weakness and lethargy (Moran et al; 2003). Focal signs such as cranial nerve defects, hemiparesis, focal seizures and autonomic dysfunction are encountered. Other signs of viral meningitis include ataxia , dysphagia and hydrophobia have been reported especially in rabies (Lee et al.2006) . The clinical symptoms of AM are characterized by fever (>38◦C), malaise, vomiting and in some cases petechial rashes. Signs of meningeal irritation include neck stiffness,Kernig’s sign and Brudzinski’s sign .These signs are poorly noted in adults . The nonspecific nature of the symptoms and clinical signs mean that we must confirm the diagnosis (Thomas, 2002) . A variety of clinical manifestations which are associated with enteroviral infections , include respiratory illness, acute hemorrhagic conjunctivitis, myocarditis, neonatal sepsis-like disease, encephalitis and acute flaccid paralysis (Jen-Ren et al;2002) . The clinical features of tuberculous meningitis are non-specific , therefore the discrimination of cases of tuberculous meningitis from other causes of meningitis by clinical features alone is often impossible (Thwaites & Tran, 2005). (C) Epidemiology : The epidemiology of AM and other CNS infections are not fixed and may vary according to location and season (Tyler ,2004). Viral meningitis occurs worldwide as epidemics and as sporadic cases. Cases of enteroviral meningitis are observed increased in the late summer (Tyler ,2004). The incidence of tuberculous meningitis(TBM) is related to the prevalence of TB in the community, and it is still the most common type of chronic CNS infection in developing countries. TB is the seventh leading cause of death and disability worldwide ( Dinnes et al ., 2007). (D) Etiology of aseptic meningitis : Many etiological agents are responsible for AM. Viruses are the most frequent causes. 1-Viruses: a-Enterovirus :Enteroviruses(EVs)belong to the family of Picornaviridae. EVs are small, non enveloped, single-stranded RNA viruses that have been classified into 68 serotypes (Stanway et al.,2005) . EVs include polioviruses and non polioviruses which are including echovirus and coxsackievirus(Andreoletti et al. 1998).All the EVs groups are the common etiological agents of AM (Leite & Barbosa , 2005). Enteroviral meningitis is commonly diagnosed in children less than 5 years of age. It is a febrile illness with anorexia and general malaise or may present as a rather abrupt onset of fever, nausea and headache. It could be followed by meningeal signs with stiffness of the neck or back and muscle weakness may occur which is clinically similar to mild poliomyelitis (Yerly et al; 1996). b-Echovirus : Echovirus is RNA virus that belongs to the genus enterovirus(non polio type) of the picornaviridae family. Echovirus is found in the gastrointestinal tract and hence it is being part of the enterovirus genus (Hauri et al ., 2005). Echovirus is highly infectious and its primary target is children. The echovirus is among the leading causes of acute febrile illness in infants and young children ( Chen et al ., 2005). Echovirus causes a nonspecific exanthemas, herpangina which is a fever and skin rashes that may be maculopapular, morbilliform, macular, petechial or papulopustular in nature ( Pichichero et al ., 1998). Severe forms of disease may be complicated by meningitis especially in infants younger than 3 months and encephalitis, neonatal sepsis and myocarditis especially in patients with altered immunity (Paananen et al ., 2003). c-Herpesvirus : Human herpes viruses include herpes simplex virus (HSV) type I (HSVl ) and type 2 (HSV2),VZV,CMV, Human herpesvirus 6 (HHV6) and EBV(Cardone et al., 2007). HSV AM is most commonly associated with primary genital infection with HSV type 2. Acute AM has also been associated with VZV in patients with or without typical skin lesions. Cases of recurrent Mollaret's meningitis have been associated with HSV type 1, HSV type 2 and EBV( Subramanian & Geraghty, 2007). The structure of herpes virus is spherical in shape. Its lipid envelope encloses the nucleocapsid that arranged in a icosahedral form. Its genome is a single, linear, double-stranded molecule. The capsid is surrounded by a number of loosely associated proteins known collectively as the tegument. Many of these proteins play critical roles in initiating the process of virus reproduction in the infected cell. The tegument is in turn covered by a lipid envelope studded with glycoproteins that are displayed on the exterior of the virion (Mettenleiter et al.,2006). The majority of patients with HSV meningitis present with subacute neurological symptoms developing over 1-7 days and the classic symptoms include: headache, neck stiffness, fever, chills, photophobia with other common meningeal symptoms such as vomiting, seizures and altered consciousness. These manifestations are not evident in infants and elderly ( Fatahzadeh &Schwartz,2007) . Figure 1 :Structure of herpesvirs (Roizman et al ., 2006) d-Cytomegalovirus : Cytomegalovirus (CMV) is a viral genus of the herpesviruses group in humans and it is commonly known as human cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) (Adler , 2005). All herpesviruses share a characteristic ability to remain latent within the body over long periods and its structure is similar to HSV structure (Bennekov et al ., 2004). HCMV infection may be a life threatening condition especially in immunocompromised patients. Active infection in healthy children and adults can cause prolonged high fever, chills, severe tiredness, a generally ill feeling, headache and spleenomegaly.Most infected newborns have no symptoms at birth but in some cases symptoms include poor weight gain, swollen glands, rash, liver, lung and blood involvement (Bottieau et al .,2006). Patients with impaired immune systems are more prone to serious, potentially life-threatening illnesses, with fever, pneumonia, CNS complications as meningitis , encephalitis ,liver involvement and anaemia. Illnesses can last for weeks or months and can be fatal. In persons with human immunodeficiency virus (HIV) infection , CMV can infect the retina of the eye (CMV retinitis) and cause blindness (Griffiths & Walter , 2005). e-Epstein-Barr virus : EBV or human herpesvirus 4 infects more than 95% of the world's population. The most common manifestation of primary infection with this virus is acute infectious mononucleosis(glandular fever) which is a self-limited clinical syndrome and frequently affects adolescents and young adults (Nicholas & Joseph ,2008). A mature infectious viral particle consists of nucleoid , capsid and an envelope. The nucleoid contains linear double-stranded viral DNA. It is surrounded by icosahedral capsid which is constructed of capsomers which are tubular protein subunits.The envelope derived either from the outer membrane or the nuclear membrane of the host cell encloses the capsid and nucleoid ( the nucleocapsid) (Gasser et al., 2007). Classical symptoms of acute infectious mononucleosis include sore throat, fever, headache and myalgia with generalized lymphadenopathy and splenomegaly , mononucleosis with relative and absolute lymphocytosis. Infection with EBV in younger children is usually asymptomatic or mild. However, EBV is also a human tumor virus which is the first virus associated with human malignancy as nasopharyngeal carcinoma and Burkitt lymphoma (Chaganti et al., 2008). Nonfatal complications are encountered as various forms of CNS and hematological affection.CNS affections are in the form of meningitis, encephalitis, hemiplegia and transverse myelitis. Hematological affection as EBV can cause autoimmune hemolytic anemia and various cytopenias ( Lockey et al., 2008). f-Varicella zoster virus : Varicella zoster virus (VZV) or human herpesvirus 3 is a member of eight herpes viruses known to infect humans. It commonly causes chicken-pox in children and both shingles and postherpetic neuralgia in adults (Steiner et al., 2007). VZV is closely related to the herpes simplex viruses (HSV) sharing much genome homology (CDC,1996). Chicken-pox (varicella) may rarely causes complications as meningitis ,encephalitis or pneumonia. VZV remains dormant in the nervous system of the infected person (virus latency) in the trigeminal and dorsal root ganglia. In about 10-20% of cases, VZV reactivates later in life producing a disease known as herpes zoster or shingles. Serious complications of shingles include postherpetic neuralgia, zoster multiplex, myelitis, AM , herpes ophthalmicus (Steiner et al., 2007). g-Measles virus :Measles is one of the typical viral diseases of childhood. However, unlike other common viral diseases , measles often leads to severe complications that may be fatal ( Helfand et al. ,2008). Measles virus is a member of the family of Paramyxoviruses which is enveloped,single strand RNA(ssRNA)with helical symmetry.The envelop has only one glycoprotein type which is haemaglutinin (HA-antigen) (Leonard et al., 2008). Figure 2:Structure of measles virus (MicrobiologyBytes , 2008). The classical symptoms of measles include a four day fever up to 40°C ,cough, coryza (runny nose) and conjunctivitis (red eyes). Pathognomonic koplik's spots are seen inside the mouth.The characteristic rashs are classically described as a generalized,maculopapular and erythematous that begin several days after the fever starts. They begin on the head before spreading to cover most of the body and often cause itching. The rashs change colour from red to dark brown before disappearing (Perry et al., 2004). Complications with measles are relatively common ranging from relatively mild to sever include diarrhea , pneumonia , encephalitis , meningitis, corneal ulceration leading to corneal scarring (Torjesen ,2008). h-Mumps virus : Mumps or epidemic parotids is a viral disease of the human species. It is a paramyxovirus with the same genetic structure as Measles virus (Hviid et al ., 2008). It manifested by painful swelling of the salivary glands (classically the parotid gland) . Painful testicular swelling and rash may also occur. The symptoms are generally not severe in children. In teenage males and adults, complications such as infertility or subfertility are more common due to orchitis (testicular inflammation).Central complications include AM which is commonly occurring asymptomatically with inflammatory cells in CSF 50%–60% of patients.Symptomatic meningitis (headache, stiff neck) occurs in up to 15% of patients and resolves without sequelae in 3–10 days (Kanra et al ., 2004). The disease is generally self-limited and the symptoms can be controlled by painkillers druges (Preveden et al ., 1996). i-Rubella Virus : Rubella is commonly known as German measles because the disease was first described by German physicians in the mid-eighteenth century. This disease is often mild and attacks often pass unnoticed. The disease can last one to five days. Children recover more quickly than adults. Infection of the mother by Rubella virus during pregnancy is serious especially in the first 20 weeks of pregnancy as the child may be born with congenital rubella syndrome (CRS) (Richardson et al ., 2001). Rubella virus is the only member of the Rubivirus genus of the Togavirus family. Unlike most Togaviruses it is not arthropod-borne but is acquired via the respiratory route. It is an enveloped (toga=cloak), non-segmented, positive sense, RNA virus (Dayan et al., 2006 & Stegmann, Carey , 2002). After an incubation period of 14-21 days, the primary symptom is the appearance of a rash (exanthema) on the face which spreads to the trunk and limbs and usually fades after three days and the rash disappears after a few days with no staining or peeling of the skin. Other symptoms include low grade fever, post cervical lymphadenopathy, joint pain, headache and conjunctivitis. ( Atreya et al., 2004). Rubella can cause congenital rubella syndrome in the newly born. The syndrome (CRS) follows intrauterine infection by the virus and comprises cardiac, cerebral, ophthalmic and auditory defects. It may also cause prematurity, low birth weight , neonatal thrombocytopenia, anaemia and hepatitis (De Santis et al ., 2006 ). In most cases there is neural involvement , irritability, motor tone problems, mental retardation,meningitis, encephalitis , abnormal posture and neurosensory hearing loss ( Weisinger & Pesudovs ,2002). j- Lymphocytic Choriomeningitis virus : Lymphocytic choriomeningitis or lymphocytic meningoencephalitis (LCM) is a rodent-borne viral infectious disease that may cause AM , encephalitis or meningoencephalitis. The causative agent is the lymphocytic choriomeningitis virus (LCMV) which is a member of the family Arenaviridae which is ssRNA , enveloped virus (Barton & Mets , 2001). In humans LCMV infection usually causes mild illness such as fever, fatigue, loss of appetite, muscle aches, headache, nausea and vomiting. A small number of individuals may become quite ill and develop meningitis or encephalitis .Other individuals may not have any symptoms (Barton & Mets , 2001). Figure 3 : Diagram and electron micrograph of LCMV. International Committee on Taxonomy of Viruses database Management( ICTVdB) , 2006.

Tuesday, April 17, 2012


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Monday, April 16, 2012

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).

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).