Central nervous system infections Central nervous system infections.

Central nervous system infections

Anatomy

BackgroundA 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).

Samplesminimum clinical information: age, immune

status, clinical epidemiological contextif 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

photophobiaheadachevomitingstiff neckflexed 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 month

Enterobacteriaceae Pneumococi(E. coli K1 ) Haemophilus influenzae / bStreptococcus agalactiae P. aeruginosaStaphylococcus aureus Listeria monocytogenes

Children Neisseria meningitidis Mycobacterium tuberculosis2)

Pneumococci LeptospiraH. influenzae tip b1)

Adult Pneumococci L. monocytogenes4)

N. meningitidis LeptospiraS. aureus Cryptococcus neoformans4)

Gram negative bacili Candida albicans4)

Non spore forming anaerobs3) Coagulase negative staphylococci 5)

M. tuberculosis2) Viridans streptococci

1. Rare after 5 years; 2. Frequence incresing 3. At patients with otic focus 4. Immunodepressed patients 5. Iatrogen meningitis.

New-born and infant

Enterobacteriaceae (E.Coli, K1, Salmonella, non – typhic serotype) Streptococcus agalactiae Staphylococcus aureus Streptococcus pneumoniae Haemophilus influenzae Pseudomonas aeruginosa Listeria monocytogenes

Neisseria meningitidis Streptococcus pneumoniae Haemophilus influenzae Mycobacterium

tuberculosis Leptospira interogans

Children

AdultStreptococcus 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 pneumoniaeNeisseria meningitidisListeria monocytogenesStreptococcus agalactiaeHaemophilus influenzaeStaphylococcus aureusBacili gram-negativi (Enterobacteriaceae, P. aeruginosa şi alţi BGN nonfermentativi)Anaerobic bacteriaBacillus anthracis

Acute meningitis(corelated with CSF shunt )

Coagulase negative staphylococciStaphylococcus aureusPropionibacterium 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 of lymphocytes)

Nocardia asteroidesBrucella spp.Leptospira interrogansMycobacterium tuberculosisTreponema pallidumBorrelia 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

BacterioscopyAntigenic detection

Neisseria meningitidis: microscopy

Latex agglutination test

Cytological and bacteriological examination

Macroscopic appearanceQualitative and quantitative cytologic

analysis of the CSFBiochemical testing Microscpic examination: Gram stain,

other stainsCulture and identificationAntigensSerologyPCR

Results and interpretationReporting 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

TestulMeningitis type

Bacterial Mycobacterial Viral Fungi

Leucocyte/mm3 200 – 20000 PMNs

25-100PMN

50-1000Ly

100-500Ly

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: 35 mg/dl

Ehtilic alcohol - - - Present

Proteins

Chloride

increased: 100-500 mg/dl

increased: 100-200 mg/dl

decreased:<600 mg/dl

15-100 mg/dl

Increased: ~ 100 mg/dl

Typical CSF findings in various CNS diseases

Case 2T.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: cloudy400 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 MIC Penicillin = 0,03 μg/mLAmpicillin – S Ceftriaxone -SCiprofloxacine – SRifampin – R

Case 3G.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/dLCulture – M. tuberculosis

Antibiogram: HIN - SRifampicin - SPirazinamide - SStreptomicin - R

Case 4D.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 : Culture: Streptococcus pneumoniaeOxacilin – R Eritromicin- S Clindamicin- STetraciclin- RCloramfenicol- SCotrimoxazol- RVancomycin –S

Quantitative antibiogram:CMI penicillin – 1,5mg/L (I) CMI cefotaxim- 0,5 mg/L (S)

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.

ConclusionsRT-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.