Download LP 3 Engl VI_24.10.2014

Central nervous system
infections
Anatomy
Background

A CSF microbiological exam is done for:
◦ Meningitis and ventriculitis
◦ Encephalitis and meningoencephalitis
◦ Intracranial suppuration (abscess, empyema, septic
thrombophlebitis)

These infections appear:
◦ in nosocomial / community settings
◦ often life-threatening

The clinician and microbiologyst must work
very closely together in this serious
situation, so that the investigations can be
focused or refocused according to the
clinical epidemiological data.
The objectives of the microbiological
examination

to orientate very quickly the patient’s therapy and
management

to confirm the bacterial, fungal or viral origin by
isolating the microorganism or detecting the
components (Ag, DNA, RNA) or antibodies produced

to establish the antibiotic sensitivity profile of the
involved strain

to notify the cases to the health authorities to initiate
management of index case contacts (e.g., Neisseria
meningitidis).
Samples
minimum clinical information: age, immune
status, clinical epidemiological context
 if there are no contraindications (intracranial
hypertension, severe coagulation disorder,
local infection at the puncture site):
 lumbar puncture – CSF;
 blood cultures;
 skin biopsy – purpura;
 ventricular shunts – proximal drain tap;
 leptomeningeal and brain biopsy – CT
guided puncture aspiration (brain abscess).

CNS infectious – medical emergencies

Purulent meningitis – produced by bacteria and sometimes by
protozoal.

Meningitis with clear CSF and acute evolution are frequently
made by viruses (aseptic meningitis), leptospira. When it is a
chronic evolution, can be a TB etiology, syphilis or fungi. Problems
of difficult diagnostic are the “headless” meningitis, through
incomplete antimicrobial treatment, which can evolve with clear
CSF.

Encephalitis – brain inflammations, are viral more frequent; can
appear through infectious – allergic mechanism.

Poliomyelitis are inflammations of the nervous tissue, which
evolve with selective destruction of motors neurons; more
frequently they are viral.

Supurations (cerebral, sub-dural and epidural abscesses).
Cytological and biochemical changes are unspecific.
Neurologic signs





photophobia
headache
vomiting
stiff neck
flexed legs
Normally CSF is clear, sterile
liquid
 ≤ 3 lymphocyte / mm3
 50 – 70 mg glucose / dl
 15 – 40 mg proteins / dl;
 680 – 730 mg chloride / dl.
Infectious of the central nervous system
can be produced through:

Blood, during of some bactaeremia (especially when there
is more than 104 CFU / ml blood), or viraemia, with different
pathways of entrance.

Naso pharynx, through olfactory lymphatic way
(meningococci, pneumococci, Haemophilus influenzae).

Adjacency, from infectious focus (e.g., otomastoyditis,
sinuzytis).

From exterior:
◦ extern auditory , skin, nasopharynx,
◦ after cranial fracture (traumatic meningitis with the most
varied etiologic spectrum),
◦ lumbar puncture or surgical intervention on neuraxis (iatrogen
meningitis, eventually with hospital bacteria).
In CSF we are looking for:

Infectious microorganisms: bacteria, fungi, protozoal, viruses.

Antigens of infectious microorganism.

Antibodies appearance.

Consequence of the microbial metabolism: decreased value of
glucose in CSF, ethylic alcohol.

Modification which reflect inflammation: increased value of
proteins, increased number of leukocytes.

Modification of the electrolytes in the systemic circulation
(increase of the proteins, sodium chloride).
Lumbar puncture –
strictly aseptic technique
Assay sampling and transport





Specialist: infectious disease, neurologist,
neurosurgeon
Lumbar / ventricle puncture
Aseptic conditions
5-10 ml
◦ minimum 1 mL for pyogenic bacteria
◦ minim 5 mL for M. tuberculosis (centrifugal
action)
3 centrifuge tubes with screwed lid;
◦ biochemistry
◦ microbiological
◦ cytological examinations.

Immediate transport (< 15’), without delay at the
laboratory, without refrigeration
5 – 10 ml of CSF: L4 – L5
Sterile tube
Bacterial and fungi meningitis
New-born and children till 2 Enterobacteriaceae
Pneumococi
month
(E. coli K1 )
Haemophilus influenzae /
b
Streptococcus agalactiae
P. aeruginosa
Staphylococcus aureus
Listeria
monocytogenes
Children
Adult
1.
2.
3.
4.
5.
Rare after 5 years;
Frequence incresing
At patients with otic focus
Immunodepressed patients
Iatrogen meningitis.
Neisseria meningitidis
tuberculosis2)
Pneumococci
H. influenzae tip b1)
Mycobacterium
Leptospira
Pneumococci
L.
4)
monocytogenes
N. meningitidis
Leptospira
S. aureus
Cryptococcus
neoformans4)
Gram negative bacili
Candida
4)
albicans
Non spore forming anaerobs3)
Coagulase
negative staphylococci 5)
M. tuberculosis2)
Viridans
streptococci
New-born and infant
Enterobacteriaceae (E.Coli, K1, Salmonella, non – typhic serotype)
Streptococcus agalactiae
Staphylococcus aureus
Streptococcus pneumoniae
Haemophilus influenzae
Pseudomonas aeruginosa
Listeria monocytogenes
Children
 Neisseria meningitidis
Streptococcus pneumoniae
Haemophilus influenzae
Mycobacterium tuberculosis
Leptospira interogans
Adult
Streptococcus pneumoniae
Neisseria meningitidis
Staphylococcus aureus
Bacili gram negativi
Anaerobi nesporulati
Listeria monocytogenes
Mycobacterium tuberculosis
Leptospira interogans

Viral meningitis
◦ enterovirus(Coxsackie A7, A9, B2-5, ECHO, rare
poliovirus or enterovirus 71)
◦ mumps virus
◦ varicela-zoster virus
◦ influenza virus
◦ paramyxovirus
◦ adenovirus
◦ cytomegalovirus
◦ herpes-simplex virus
◦ arbovirus

Meningitis made by protozoa:
◦ Naegleria fowleri
◦ Acantamoeba-Hartmanella
The etiology of meningitis:
Acute meningitis
(increased value of PMN) Signs and symptoms of less
than 24 hours duration
Streptococcus pneumoniae
Neisseria meningitidis
Listeria monocytogenes
Streptococcus agalactiae
Haemophilus influenzae
Staphylococcus aureus
Bacili gram-negativi (Enterobacteriaceae, P.
aeruginosa şi alţi BGN nonfermentativi)
Anaerobic bacteria
Bacillus anthracis
Acute meningitis
(corelated with CSF shunt )
Coagulase negative staphylococci
Staphylococcus aureus
Propionibacterium spp.
Gram negative bacili (e.g., E.coli, Klebsiella
spp.)
Non-fermentative gram negative bacili (e.g.,
P. aeruginosa, Acinetobacter spp.)
Chronic meningitis
(increased
value
lymphocytes)
Nocardia asteroides
of Brucella spp.
Leptospira interrogans
Mycobacterium tuberculosis
Treponema pallidum
Borrelia burgdoferi
Ventriculo-peritoneal shunt:
surgical creation of a communication between a cerebral ventricle and the
peritoneum by means of a plastic tube to permit drainage of cerebrospinal fluid for
relief of hydrocephalus
Quality criteria

Sampling before anti biotherapy, or, if it is not possible, before
the next drug administration. Specification of the antibiotic
used.

Avoidance of sampling through catheter in meningitis correlated
with shunt.

Never add the buffer in the CSF.

Sufficient volume.

Tubes without fissure, with screwed lid.

Skin decontamination with iodine.

At once sending.

Transporting in isotherm conditions, at 37°C.
Rapid exams
Bacterioscopy
 Antigenic detection

Neisseria meningitidis: microscopy
Latex agglutination test
Cytological and bacteriological
examination
Macroscopic appearance
 Qualitative and quantitative cytologic analysis
of the CSF
 Biochemical testing
 Microscpic examination: Gram stain, other
stains
 Culture and identification
 Antigens
 Serology
 PCR

Results and interpretation
Reporting the results: by telephone, immediately;
written report;
 Interpretation:

◦ A normal CSF does not contains any living
microorganisms;
◦ Any positive microbiology result must be considered as
a marker of infection.
◦ Case of aseptic meningitis:
 enteroviruses
 bacterial meningitis masked by preliminary antimicrobial
treatment
 meningitis with fragile microorganism, difficult to detect on
culture
 true aseptic meningitis;
 inflammatory diseases after drug treatment.
Declaration to the national health
agency (ECMID Manual 2014)

The medical microbiologist should declare
the cases as soon as possible to the
Regional Health Agency according with the
recommendations in force in each country.

Microrganisms:
◦ meningococci, pneumococci: to keep the
epidemiology of bacterial meningitis and
antibiotic resistance up to date;
◦ EV 71, rabies.
Case 1
A 3 year-old girl was brought to the emergency room by her parents because of fever
and loss of appetite for the past 24 hours and difficulty in arousing her for the past 2
hours. The developmental history had been normal since birth. Her childhood
immunization were current.
 Clinical features:
◦ Temperature: 39.5° C, pulse 130/min, respiration 24/min, bloodpresure 110/60 mmHg.

Physical examination: somnolent child, her neck was passively flexed, her legs also flexed
(positive Brudzinski sign, suggesting irritation of the meninges).
 Laboratory findings:
◦ CSF – lumbar puncture: cloudy;
◦ Gram staining: many polymorphonuclear (PMN) cells with cell-associated
(intracellular) gram negative diplococci suggestive for Neisseria meningitidis.
◦ 5000 PMNs / μL;
◦ CSF proteins = 100 mg/dL
◦ Glucose = 15 mg/dL (hypoglycorrhachia)
◦ Culture of blood and CSF: serogrup B / N. meningitidis.

 Treatment: intravenous cefotaxime for 14 days. Further neurologyc examination and
hearing tests were planed for the future.
Waterhouse Friedrichsen Syndrome
Typical CSF findings in various CNS diseases
Testul
Meningitis type
Bacterial
Mycobacterial
Viral
Fungi
Leucocyte/mm3
200 – 20000 25-100
PMNs
PMN
50-1000
Ly
100-500
Ly
Glucose
decreased:
5-20 mg/dl
decreased:
20-40 mg/dl
Normall:
65-70 mg/dl3)
decreased:
20-40 mg/dl
Lactic acid
Crescut: > 35
mg/dl
> 35 mg/dl
Normal:
mg/dl
Ehtilic alcohol
Proteins
Chloride
-
increased:
100-500
mg/dl
-
increased:
100-200 mg/dl
decreased:<60
0 mg/dl
35
15-100 mg/dl
Present
Increased:
~ 100 mg/dl
Case 2
T.O, 19 years old, soldier, after one week after enrolling, present fever, headache.
Clinical examination: fever 39° C, skin rush, neck stiffness
Laboratory finding: 15.000 / mmc, 85% PMN
PLT – 100.000 / mmc
CSF:
cloudy
400 elements / mmc, 80% PMNs,
glucose 30 mg / dL,
proteins 110mg / dL
Bacterioscopy – negative
CSF cultivation and bloodcultures : – meningococci (after 48 hours)
Difuzimetric antibiogram:
Penicillin– S
Ampicillin – S
Ceftriaxone -S
Ciprofloxacine – S
Rifampin – R
MIC Penicillin = 0,03 μg/mL
Case 3
G.M. 32 years old, woman, in convalescence after chickenpox ,
present headache, fever for one week; lose the appetite; in the next
week present eructation. The patients became indifferent; in 4 days
arrive to the hospital.
Clinical examination: paralisy of III, VI nerves
slow expression
verbal aggresiveness
Laboratory findings: Rx – superior lobar pneumonia
CT- acute hydrocephalus
CSF: clear
350 elements / mmc 87% lymphocyte
protein 168 mg/dL
glucose 20 mg/dL
chloride 550 mg/dL
Culture – M. tuberculosis
Antibiogram:
HIN - S
Rifampicin - S
Pirazinamide - S
Streptomicin - R
Case 4
D.P., 3 years old, present fever, headache, neck stiffness, stream eructation, is received
into hospital with suspicion of meningitis.
CSF:
turbid
780 elements/mmc, 75% PMN
Protein 150 mg/dL
Glucose 25 mg/d
Bacterioscopy: gram positive diplococci, flame shape, capsule.
Antibiogram on primary culture :
Oxacilin – R
Eritromicin- S
Clindamicin- S
Tetraciclin- R
Cloramfenicol- S
Cotrimoxazol- R
Vancomycin –S
Quantitative antibiogram:
CMI penicillin – 1,5mg/L (I)
CMI cefotaxim- 0,5 mg/L (S)
Culture: Streptococcus pneumoniae
Case 5:
Diagnosis of meningococcal infection by qPCR:
detection and quantification of
DNA / Neisseria meningitidis

Case of a 22 months old child, male, from a family of average
condition from rural area, transferred from The “St Marie“
Universitary Clinical Children's Hospital, where he was
hospitalized for 24 hours with a diagnosis of "febrile
syndrome, acute nasopharyngitis, dispeptic syndrome".

The onset of the disease was sudden, 12 hours prior to his
admission to Children’s Hospital with fever (39.9°C), chills,
coldness, phenomena which occurred on a background of
vomitting, diarrhea and watery rhinorrhea (which evolved
about 14 days and were treated symptomatically in
ambulatory).
Petechial rash
In pediatrics there was rapid worsening of the general
condition and the petechiaes, rash, exanthema
appearing on the chest, with rapid generalization
throughout the body, having shock and meningeal
contracture syndrome.
 Biohumoral test revealed 6280 leukocytes /mmc with
PMN 72.4%, the decreasing number of thrombocytes
(from 257000/mmc to 83000/mmc), increased TQ and
PTTK clotting time, decreased prothrombin activity and
the lumbar puncture reveals 8 elements / mmc.
 Ceftriaxone was administered (1 g), and then the
patient was referred to the Clinic of Infectious Diseases.

Blood culture, direct bacteriological
culture and latex agglutination (LA) of
cerebrospinal fluid (CSF) were negative.
 A sample of CSF and one of blood
collected on admission were sent to the
Virology Laboratory of the Microbiology
Discipline for RT-PCR analysis

Nano Drop Pearl (a) and Stratagene
MX3005P Thermocycler (b)

Real Time PCR Diagnosis: DNA / Neisseria
meningitidis was purified using the kit
PrimerDesign PrecisionTM Gram Negative Bacterial
DNA extraction.

Detection of DNA / N. meningitidis through the
method qualitative end point PCR.

Quantitative PCR method, which uses the
standard curve. The PrimerDesign™ genesig kit
for N. meningitidis use the TaqMan principle.
a) Amplification plots for positive control, CSF and blood
sample – end point PCR
b) Initial template quantity of DNA/meningococci in CSF
and blood sample – absolute quantification
Under the treatment with Ceftriaxone, 1.5g/day,
10 days, the pathogenetic treatment for shock
and cerebral edema, with symptomatic and
hygienic-dietary regime, the evolution was
favourable, with remission of fever in the 3rd day
of hospitalization;
 the patient became hemodynamically stable
after two days from admission and the
meningeal contracture syndrome disappeared
after the 7th day.

Conclusions

RT-PCR is more sensitive than conventional PCR.

RT-PCR tests allow rapid and accurate quantification of
bacterial load (made of viable and nonviable bacteria) .

The case shown above proves the fact that the RT-PCR
technique is a rapid and sensitive method for the
diagnosis of cases of meningococcal infection with
classical negative diagnosis following the administration
of antibiotics before admission to hospital.

The final diagnosis, based on the previous
clinical data correlated with the laboratory data
(RT-PCR of blood and CSF positive for Neisseria
meningitidis) was that of „Meningococcemia
(acute form) with meningitis”.

The patient was discharged, with improved
status after 10 days of treatment, with the
persistence of a single petechia (faded) on the
trunk.