Laboratory diagnosis of meningitis

 

Preface

Meningitis is an inflammatory condition of the meninges (membrane surrounding brain and spinal cord) with an infectious aetiology.

            A typical case of meningitis usually presents with headache, high grade fever, projectile vomiting, nuchal rigidity and positive Kernig's & Brudzinski’s sign. Altered mental status, irritability are also common and early presentations in some cases.

Learning objectives:

1. Different types of meningitis and their etiologies.

2. Laboratory methods for analyses of CSF to evaluate a case of meningitis

            - Cytology

            - Chemical analysis

            - Bacteriological analyses by staining and culture

            - Antigen and antibody detection

3. CSF findings in different types of meningitis

Types of meningitis:         

            Meningitisis categorized into three different types depending upon their aetio-pathogenesis andthe cytology of the Cerebro Spinal Fluid (CSF).

1. Acute pyogenic meningitis:It is an acute inflammation of the meninges caused mainly by bacteria. The CSF will typically have large number of pus cells, predominantly Polymorphs.

2. Tuberculous meningitis: It is caused by Mycobacterium tuberculosis. It is one of the most common causes of meningitis in developing countries. The CSF cytology shows presence of large number of pus cells with predominant lymphocytes.

3. Aseptic meningitis: It is caused by viruses, fungi, parasites &some bacteria. The CSF cytology shows absent or few  pus cells with lymphocytic or mixed cellularity.

 

Causative agents of meningitis

A) Acute pyogenic meningitis

Bacteria causing meningitis vary according to the age groups

1. Newborn (neonatal meningitis):

            Group B beta haemolytic Streptococci (Streptococcus agalactiae): Usually acquired from vagina      while passage of baby through the birth canal.

Escherichia coli, Klebsiellapneumoniae, other coliform bacilli: Usually the source of bacteriais the digestive tract. 

            Pseudomonas aeruginosa, Listeria monocytogenes etc.

2. Children:

            Haemophilusinfluenzae type b (Hib): Common in young children (2 months to 5 years of age)

            Neisseria meningitidis (Meningococcus)

            Streptococcus pneumoniae (Pneumococcus)

3. Adults:

            Neisseria meningitidis (Meningococcus)

            Streptococcus pneumoniae (Pneumococcus)

            Listeria monocytogenes (less common)

4. Hospital acquired or iatrogenic meningitis:

            Various hospital strains i.e. Pseudomonas aeruginosa, Staphylococcus spp. may be introduced during invasive procedures like lumbar puncture, surgery or V-P shunts and may lead to meningitis.

 

B)Tuberculous meningitis: It is generally secondary to tuberculous lesions elsewhere in the body. It is caused by Mycobacterium tuberculosis.

 

C) Aseptic meningitis: It is associated with increased lymphocytes &pleocytosis in CSF with negative bacterial culture.

1. Viruses: Herpes simplex viruses (type I more common), Japanese encephalitis virus, Enteroviruses(i.e. Echo, Coxsackie, Polio) etc.

2. Fungi: Cryptococcus neoformans

3. Parasites:Toxoplasma gondii, Acanthamoeba, Naegleria (may cause pyogenic meningitis)

4. Bacteria:T.pallidum, Leptospira

5.Meningitis may be  one of the presentations of certain non-infectious diseases i.e. cancers, connective tissue disorders, sarcoidosis etc.

 

Laboratory diagnosis

Sample collection:

1.      The main specimen is CSF, which is analyzed to confirm clinical diagnosis, to find the type of meningitis and to evaluate aetiology.

2.      CSF is collected by lumbar puncture with strict aseptic precautions to prevent iatrogenic infection as wel as contamination of the specimen.

3.      Sample should be transported immediately to the laboratory& if possible hand delivered.

4.      If delay in transport of specimen is unavoidable, it should be kept at 37° C  or at room temperature and should never be kept in refrigerator. If CSF is to be processed only for viral studies, it can be refrigerated.

5.      Blood sample may be taken for culture examination. The bacteria causing pyogenic meningitis can usually be isolated from blood culture.

            To increase the isolation rate, nowadays, it is recommended that the CSF and blood should directly be collected separately in the bottles of automated culture system (Bactec or BacTAlert ) at bedside and transported immediately to the laboratory.

Laboratory examination of CSF:

CSF may be processed for,

1. Cytological study to confirm the diagnosis of meningitis and to evaluate its type and aetiology:

Total cell number up to 1-3 / cmm is considered normal in CSF. This will increase in case of meningitis.

a.     Acute pyogenic meningitis: Total cells rise up to 1000-10000/     

cmm; Predominant cells are neutrophils.                          

b.    Tuberculosis meningitis: Total cells rise upto 50-500/cmm;  

Predominant cells are lypmhocytes.

c.     Viral meningitis: Total cells rise upto 10-100/cmm; predominant

cells are lymphocytes.

            2. Biochemical tests

            In case of meningitis, total protein will increase and sugar will diminish than normal level.

            3. Microscopic examination for identification of microorganism

                        CSF is centrifuged and deposits are examined by following methods  

                     to identify the aetiological agent

                        a. Gram stain: to identify pyogenic bacteria

                        b. Z N stain: to detect M.tuberculosis

                        c. Negative stain(India ink or nigrosin): to detect Cryptococcus       

                        neoformans

d.    Wet film: to detect parasites like Naegleriafowlerii,

Acanthamoeba, Toxoplasma gondii

 

            4. Culture examination for isolation of pathogenic organism

            There are three ways to culture the CSF sample

1.    A set of culture plates including blood agar, chocolate agar and mac conkey agar are inoculated directly from sample and incubated at 37° C  in candle jar (or  CO₂incubator).
2.    CSF is centrifuged with precautions to avoid contamination and the deposit is inoculated in a similar set of culture plates.
3.   1 - 2 ml of CSF is inoculated in a liquid medium ( i.e. Glucose broth ) or in automated culture system bottle. This will allow growth of even minute number of bacteria if present. When the growth is indicated or observed in liquid medium, it is sub-cultured in similar set of plates to isolate them.

             The culture plates are observed for growth after 24-48 hours of incubation. The isolated bacteria are processed for their identification and antibiotic sensitivity testing.

 

            5. Detection of antigens or antibodies

             This is generally done from the supernatant of the centrifuged CSF sample.

            Detection of antigen:

a.     Capsular polysaccharide of aetiological agent may be detected by latex agglutination.

The following capsulated organisms may be detected by this method,

                        - N.meningitidis(Meningococci),

                        - Haemophilusinfluenzae,

                        - Streptococcus pneumoniae(pneumococci)

                        - Cryptococcus neoformans

                        b. Agglutination test may be useful to detect the surface antigens of

                        - Group B Streptococci or E.coli which are responsible for meningitis  

                       in neonates.

                       

            Detection of antibody:

Antibodies are demonstrable in the following infections, which can be detected by ELISA or immunofluroscence

                        - Herpes simplex virus

                        - Japanese encephalitis virus

                        - T. pallidum( neurosyphilis )

 

 

Exercise:

An 8 month child presented with fever, neck rigidity and projectile vomiting and suspected for meningitis.

Q.1     What are the microscopic findings in case of different kind of meningitis? Which stains are used to examine the smear prepared from centrifuge of CSF?

Q.2     Which culture media are used for plating?

Q.3     Which are the common agents suspected in this case?

Laboratory diagnosis of bone and joint infections

Laboratory diagnosis of bone and joint infections

Infection of Bone (Osteomyelitis)

The patient might develop osteomyelitis from:

·       Hematogenous spread of an infectious agent

·       Invasion of bone tissue from an adjacent site eg. joint infection, dental infection

·       Breakdown of tissue caused by trauma or surgery

·       Lack of adequate circulation followed by colonization of skin ulceration with microorganisms.

Infection of bone can progress towards chronicity, particularly if blood supply is insufficient in the affected area.

Causative organisms:

a.     Staphylococcus aureus

b.    Salmonella spp.

c.     Haemophilus spp.

d.    Enterobacteriaceae

e.     Pseudomonas spp.

f.      Fusobacterium necrophorum

g.     Yeasts

Note:

-        Parasites or viruses rarely, if ever, cause osteomyelitis.

-        Human bite may lead to infection with Eikenellacorrodens osteomyelitis whereas animal bite may lead to Pasteurellamultocida osteomyelitis.

-        Poor oral hygiene may lead to osteomyelitis of the jaw with Actinomyces spp., Capnocytophaga spp., and other oral flora, particularly anaerobes.

-        Pelvic infection in the female may result in a mixed aerobic and anaerobic osteomyelitis of the pubic bone.

-        Patients with neuropathy in the extremities, notably patients with diabetes, who may have poor circulation, may experience an unrecognised or notable trauma. They develop ulcers on the feet that do not heal, become infected, and may eventually progress to involve underlying bone. These infections are usually polymicrobial, involving anaerobic and aerobic bacteria.

 

Laboratory diagnosis:

-        To identify the etiologic agent of osteomyelitis, a small piece of bone / pus collection / scrapping material may be sent to the microbiology laboratory.

-        Microscopy – Gram stain

-        Culture – Organism can be isolated by culturing in routine aerobic media (i.e. Blood agar & Mac Conkey agar) & anaerobic media (i.e. Robertson’s cokked meat broth)

 

Infection of Joint ( SepticArthritis / synovitis)

·       Arthritis is an inflammation in a joint space. Infectious arthritis may involve any joint in the body.

·       Infection of the joint usually occurs secondary to hematogenous spread of bacteria, or less often fungi, as a direct extension of the infection of the bone.

·       It may also occur after injection of material, especially corticosteroids, into joints or after insertion of prosthetic material (eg total hip replacement).

·       Although infectious arthritis usually occurs at a single site (monoarticular), a pre-existing bacteremia or fungemia may seed more than one joint to establish polyarticular infection, particularly when multiple joints are diseased, such as in rheumatoid arthritis.

·       In bacterial arthritis, the knees and hips are the most commonly affected joints in all age groups.

·       Sterile, self limited arthritis caused by antigen antibody interactions may follow an episode of infection, such as meningococcal meningitis.

·       Occasionally the causative agent might not be detected due to either absence of viable agents at the site or faults in laboratory procedure.

·       Non-specific test results such as increased WBC, decreased glucose or elevated protein may indicate that the infective agent is present.

Causative organisms:

Causes of septic arthritis:

a.     Staphylococcus aureus is the most common etiological agent

b.    Neisseria gonorrhoea (In adults younger than 30 years)

c.     Hemophilus influenzae (In children less than 2 years)

d.    Streptococcus pyogenes

e.     Streptococcus agalactiae

f.      Pneumococci

g.     Viridans streptococci

h.    Bacteroides fragilis

i.       Fusobacterium necrophorum

Causes of chronic monoarticular arthritis:

a.     Mycobacteria

b.    Nocardia asteroides

c.     Fungi

Causes of arthritis in prosthetic joints:

It is most commonly caused by skin flora including the following

a.     Staphylococcus aureus

b.    Staphylococcus epidermidis

c.     Other coagulase negative staphylococci

d.    Corynebacterium spp.

Laboratory diagnosis:

·       Diagnosis of joint infections requires an aspiration of joint fluid for culture and microscopic examination.

·       Inoculating the fluid directly into blood culture bottles may prevent the fluid from clotting.

·       Some of the fluid may be gram stained and inoculated onto blood as well as chocolate and anaerobic media. The use of AFB and fungal media must also be considered. 

 

Extra note:

Bone marrow aspiration or biopsy may be indicated sometimes for detection of organism causing systemic infections

·       Detection of organisms in the bone marrow can be done for diagnosis of some diseases like brucellosis, histoplasmosis, blastomycosis, tuberculosis and leishmaniasis.

·       Many etiological agents associated with disseminated infections in AIDS patients, can be visualised or isolated from bone marrow, eg Cytomegalovirus, Cryptococcus neoformans and Mycobacterium avium complex.

 

Exercise:

Group discussion on the case of Bone / Joint infection

 

 

Laboratory diagnosis of skin and soft tissue infections

 

Skin and soft tissue infections

Learning objectives:

Different types of Skin & soft tissue infections & their aetiological agents

Laboratory methods to identify the aetiology of various SSTI to guide management          

 

Skin and soft tissue infection (SSTI) is a comprehensive term which includes all the kinds of infections which involve skin, muscle and connective tissues. These may be associated with involvement of deeper parts of body like bones and joints.

Various types of SSTI and their aetiology

Type of infection	Description	Aetiological agents
1. Folliculitis	Infection and inflammation of hair follicle	Bacteria: Staphylococcus aureus, Pseudomonas aeruginosa Fungus: Candida albicans, T.rubrum, Malassezia
2. Furuncles (Boil)	Skin infection with painful swelling and collection of pus.	Staphylococcus aureus
3. Carbuncles	Individual boils clustered together leading to large infected mass filled with fluid, pus and dead tissue	Staphylococcus aureus
4. Cellulitis	Diffuse inflammation of connective tissue with involvement of dermal and subcutaneous layers of skin	Staphylococcus aureus & other staphylococci Streptococcus pyogenes
5. Impetigo	Highly infectious bacterial skin infection commonly seen in pre-school children	Streptococcus pyogenes Staphylococcus aureus
6. Erysipelas	Acute infection of skin and superficial lymphatics. Lesions are typically raised and demarcated.	Streptococcus pyogenes
7. Necrotizing infections (Necrotising fascitis)	Also known as flesh eating disease. It is a rapidly progressing infection of skin and subcutaneous tissue which can spread across the fascial plane.	Streptococcus pyogenes, Staphylococcus aureus, Clostridium perfringens ( may lead to gas gangrene). Majority times it is a polymicrobial infection.
8. Surgical site infections	Infection at the site of surgery which occurs within 30 days after operation.	Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, members of enterobacteriaecae family, etc.
9. Pustule / Abscess	Collection of pus due to inflammatory processes	Staphylococcus aureus is most common. M.tuberculosis is associated with cold abscess.
10. Myositis and pyomyositis	Infection of muscle with or without pus collection.	Only myositis in absence of other surrounding site involvement is more commonly associated with autoimmune disorders rather than directly due to infection. Pyomyositis is usually caused by Staphylococcus aureus
11. Infection following animal contacts		B. anthracis (cutaneous anthrax), Bartonella henslae (Cat scratch disease), E.rhusiopathiae (Erysipeloid)
12. Infection after animal bite		Mixed aerobic and anaerobic bacteria, Rabies virus
13. Infection after human bite		Mixed aerobic and anaerobic bacteria
14. Mycetoma	Chronic subcutaneous infection characteristically associated with multiple sinuses which discharge typical grains	Eumycetoma: Caused by fungi (e.g. Madurella mycetomatis)   Actinomycetoma: Caused by bacteria (e.g. Nocardia sp.)
15. Cutaneous ulcers	Loss of epidermal & part of dermal tissues.	B. anthracis, M. marinum,
16. Burn wounds	Generally associated with bacteremia	Pseudomonas aeruginosa, members of Enterobacteriaceae family.
17. Systemic infections with skin manifestations		a) Petechiae in meningococcaemia. b) Cutaneous ulcers & bullae in V. vulnificus bacteremia.
18. Leprosy	Systemic disease primarily involving skin, peripheral nerves and nasal mucosa.	Mycobacterium leprae
19. Macule	Flat, non-palpable discoloration of skin (<5 cm size). If size exceeds 5 cm, is called as patch	Dermatophytes Viral rashes (e.g. enterovirus, measles)
20. Papule	Elevated lesions usually <5 mm in size that can be felt or palpated	·       Virus:  Molluscum contagiosum,  Warts (Human Papilloma virus) ·       Parasite scabies (Sarcoptes scabiei)
21. Plaque	Multiple papules my become confluent to form plaque which are palpable lesions >5 mm
22. Nodule	Firm lesions >5 cm size	·       Bacteria Staphylococcus aureus, Mycobacterium marinum ·       Fungi sporothrix
23. Vesicle	Fluid-filled lesions with a diameter less than 0.5 cm	Herpes simplex virus,  varicella-zoster virus
24. Bulla	Fluid-filled lesions with a diameter more than 0.5 cm	·       Bacteria Clostridium Staphylococcus aureus ·       Virus Herpes simplex virus
25. Scale	Excess dead epidermal layer	Dermatophytes Streptococcus pyogenes

 

Laboratory diagnosis:

Sample collection:

            1.        If there is subcutaneous or deep pus collection and pus is to be collected through intact skin, It should be done with sterile syringes and needle. Prior to collection, the surface area should be cleaned with alcohol. Sample collected in syringe should be immediately transported to laboratory in it.

            2.        If the pus is discharged through the skin, it should be collected with sterile swab. Prior to collection of sample, the surface area should always be cleaned with sterile saline (never use antiseptics). The pus is squeezed and collected with swab.

            3. Skin scrapings or biopsy may be required to collect particularly in dry lesion. In case of leprosy, skin smear with nasal smear is required or biopsy from nodular skin lesion and thickened nerves is recommended in some cases.

 

Laboratory procedures:

 

1. Microscopic examination:

            a. Gram stain: To screen for bacteria

            b. KOH examination: To screen for fungus

            c. Z N stain: For demonstration of M. tuberculosis Atypical mycobacteria. Modified acid fast   stain required for diagnosis of suspected case of leprosy.

 

3. Culture examination:

            According to clinical presentation & type of lesion, the Samples should be cultured in one or more of following culture media to identify the aetiology of lesion

                        a. Simple media: Nutrient agar

                        b. Differential media: Mac-Conkey agar

                        c. Enriched media: Chocolate agar / Blood agar

                        d. Anaerobic media: Robertson's cooked meat broth

                        e. L J medium: For atypical Mycobacteria

                        f. Sabouraud's dextrose agar

 

Exercise

A 5 year child presented with multiple painful swellings over the leg and thigh. Pus is discharging from few lesions.

Q.1     How the samples are collected? (In practical / exam students should identify the materials used for sample collection and transportation)

Q.2     Which culture media are used for isolation of possible aerobic and anaerobic bacterial pathogen (In practical / exam students may be asked to identify these media and bacterial growth)

Q.3     Which antibiotics should be tested for its sensitivity against isolated bacteria? (In practical / exam students may be asked for selection of appropriate antibiotic combination circle from the tray; and materials required for sensitivity testing)