How can your microbiologist help you manage ...

SYMPOSIUM: IMMUNITY AND INFECTION

How can your microbiologist help you manage paediatric infection?

Interpreting preliminary microbiology results Paediatric trainees are often phoned with preliminary results from the microbiology laboratory describing a Gram stain result. Table 1 is a reference guide for the common organisms that cause paediatric infections, divided into Gram-positive and Gramnegative organisms and those that are identified by alternative methods.

Common paediatric microbiology consultations

Ciara O’Connor

The eye Conjunctivitis is an inflammation of the conjunctiva of either one or both eyes and is frequently described as ‘red or sticky eyes’ by parents or carers. Occasionally, conjunctivitis may occur in association with infection of the cornea (keratoconjunctivitis) or eyelid (blepharoconjunctivitis). The most common causes of bacterial conjunctivitis are Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae, while the most common causes of viral conjunctivitis are adenoviruses, herpes simplex virus (HSV)-1/-2 and varicella zoster virus (VZV). Neisseria gonorrhoea, Haemophilus parainfluenzae and Group B Streptococcus (GBS) can cause infection in the neonatal period. When conjunctivitis is suspected, eye swabs should be sent for culture and/or viral PCR analysis. Orbital cellulitis is an infection of the orbital tissue that can occur secondary to trauma, surgery or following the spread of infection from the paranasal sinuses. The most common causes of orbital cellulitis are S. aureus, anaerobes and various streptococci. Eye swabs are of limited value in the investigation of orbital cellulitis. Intra-operative aspirates from infected tissues should be sent to the laboratory. Table 2 shows the spectrum of cover of commonly prescribed topical ophthalmic antimicrobials.

Richard J Drew

Abstract The presentation of a child to hospital with an acute illness is distressing for the child and his/her parents or carers. The clinical aim of the admitting paediatric team is to identify the cause of the illness, to treat it effectively and to discharge the child home quickly and safely. Multi-disciplinary care between the paediatric and the clinical microbiology team, who oversee and support the laboratory work of skilled scientists, is essential to manage paediatric infection. In this review, we will focus on current diagnostic methods for common paediatric microbiology consultations, with a focus on newer molecular technology to reduce laboratory turnaround time, and discuss the emergence of multi-drug resistant organisms that are impacting on antimicrobial prescribing practices. We will also highlight useful infection prevention and control advice that will be beneﬁcial to the on-call paediatrician.

Keywords clinical microbiology; consultations; laboratory; molecular diagnostics; paediatrics

Introduction The brain and spine Encephalitis is an inflammatory process in the brain accompanied by cerebral dysfunction manifesting as an altered level of consciousness. Seizures are common. Encephalitis is predominantly caused by viruses including VZV, Ebstein Barr virus (EBV), cytomegalovirus (CMV), HSV-1/-2 and enteroviruses. Meningitis is defined as inflammation of the meninges. From neonates to babies up to 2 months of age, GBS, Escherichia coli, Listeria monocytogenes, N. meningitidis are commonly isolated. In older children viral meningitis is more common than bacterial meningitis. Other bacterial causes of meningitis include S. pneumoniae, N. meningitidis and H. influenzae type b (Hib), in unvaccinated children. In taking the child’s history it essential to discuss potential risk factors for meningitis including the previous diagnosis of a cerebral tumour, the presence of cerebrospinal fluid (CSF) shunts or cochlear implants, whether the child has a meningomyelocoele or other spinal congenital malformations, preceding infections of contiguous sites such as the orbit, paranasal sinuses, middle ear cavity or recent trauma such as a basilar skull fracture. Chronic meningitis is defined by the continued signs and symptoms of meningitis for greater than four weeks with abnormal CSF findings, most commonly caused by Mycobacterium tuberculosis. Rare non-infectious causes of meningitis include sarcoid meningitis, post intravenous immunoglobulin (IVIG) administration or treatment with co-trimoxazole or non-steroidal anti-inflammatories (NSAIDs).

In approaching any child with a potentially infectious illness it is worth considering the following: (1) what is the likely source of infection, what are the usual organisms that either colonise that site or can readily gain access to the site to cause infection, what are the optimal specimens to send and can molecular technology such polymerase chain reaction (PCR) be utilised, (2) does the child have any past microbiology results and/or is the child colonised with a multi-drug resistant organism (MDRO) that may limit prescribing practices, (3) is the child potentially harbouring a transmissible organism and if so what infection prevention and control precautions do I need to implement pending confirmation, (4) is prophylaxis needed for parents/carers or staff who have come into contact with this child and (5) is a vaccination or vaccination booster required.

Ciara O’Connor B. Ed MB MRCPI MRCPUK MD FRCPath is a Clinical Microbiology Specialist Registrar in the Department of Clinical Microbiology, Temple Street Children’s University Hospital, Dublin 1, Ireland. Richard J Drew MB FRCPI FRCPath DipHIC MD PGDip PID (Oxon) is a Consultant Microbiologist in the Department of Clinical Microbiology, Temple Street Children's University Hospital, Dublin 1 and in the Department of Microbiology, Royal College of Surgeons in Ireland.

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The diagnosis of meningitis is established by the examination of CSF. It is essential to write on the request form if a CSF shunt (ventriculo-atrial or ventriculo-peritoneal) and/or extra ventricular drain (EVD) are in-situ. CSF should be collected into three or more containers numbered consecutively. No more than 2 h should elapse between CSF collection and laboratory microscopy and culture as cells can disintegrate rapidly. Never place a CSF sample in any hospital refrigerator until microscopy and culture have been performed. Laboratory examination of CSF includes a complete cell count, differential leucocyte count, examination of a Gram stained smear and culture. Normal CSF values

are detailed in Table 3. In-house testing of CSF using multiplex PCR panels, with the capability to identify bacteria, viruses and fungi simultaneously, such as the FilmArrayÒ Meningitis/Encephalitis (Biomerieux, France), are increasingly utilised. Blood cultures, pharyngeal swabs and stool specimens should also be sent when meningitis and/or encephalitis are suspected. The ear Otitis externa is defined as infection of the external auditory canal. Acute localised otitis externa is usually caused by S. aureus. Acute diffuse otitis externa, also known as “swimmer’s

Classification of common organisms causing paediatric diseases Organisms detected via Gram stain Typical shape of the organism when Gram stained and viewed under the microscope Coccus (sphere shaped)

Bacillus (rod shaped)

Cocco-bacillus (variable appearance)

Vibrio (curved rod/comma-shaped)

Nocardia spp. Bacillus spp. Clostridium spp. Listeria spp. Corynebacteria spp. Gram-negative Kingella spp. Pseudomonas spp. Haemophilus influenzae Vibrio spp. Neisseria spp. Bordatella spp. Moraxella spp. Legionella spp. Stenotrophomonas spp. Pasteurella spp. Capnocytophagus spp. Bacteroides spp. Fusobacterium spp. Acinetobacter spp. Shigella spp. Campylobacter spp. Helicobacter pylori Salmonella spp. Citrobacter spp. Enterobacter spp. Klebsiella spp. Escherichia coli Organisms not detected using Gram stain method. If suspect infection due to microorganisms listed below contact the clinical microbiology team to discuss local methods of diagnostic testing performed. Samples may be referred to a reference laboratory. Spirochaetes Treponema spp. Leptospira spp. Borrelia spp. Non-culturable as no cell wall Mycoplasma spp. Obligate intracellular pathogens Rickettsia spp. Chlamydia spp. Coxiella spp. Special staining process performed Mycobacteria spp. to detect presence of acid fast bacilli (AFB) Gram-positive

Staphylococcus spp. Streptococcus spp.a Enterococcus spp. Micrococcus spp.

a

Streptococcus spp. can be classified by their appearance on blood agar after overnight incubation. A greenish appearance on the blood agar plate signifies the presence of a-haemolytic Streptococcus spp., usually S. pneumoniae or a member of the viridans group, which encapsulates a broad range of Streptococci including S. oralis, S. sanguis, S. mutans, S. mitis, which are commensals of the oral cavity and upper respiratory tract. A clear zone of complete haemolysis on the blood agar plate around visible colonies after an overnight incubation demonstrates the presence of b-haemolytic Streptococcus spp., which includes Groups A, B, C, G Streptococci.

Table 1


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(inflammation of the lung parenchyma), bronchiolitis (infection of the small airways), lung abscess (where lung parenchyma is replaced by pus filled cavities) and empyema (where pus occupies the pleural space). When documenting in the child’s medical notes, the term ‘LRTI’ should be avoided and the anatomical site of the suspected infection clearly specified. The aetiology of pneumonia and its management varies as to whether it had been acquired in the community or nosocomial (present greater than 48 h post-hospitalisation). S. pneumoniae accounts for 60% of all cases of community acquired pneumonia (CAP). In-house multiplex PCR assays, such as the FilmArrayÒ Respiratory Panel (Biomerieux, France), for the detection of a wide range of bacteria and viruses using nasopharyngeal aspirates or swabs are now widely used. The sensitivity of expectorated sputum culture is poor for many pathogens due to contamination with commensal oropharyngeal flora and saliva. Early-morning sputum samples are preferred as they contain pooled overnight secretions. Bronchoalveolar lavage (BAL) samples have greater sensitivity and specificity compared to expectorated sputum. Pertussis, also referred to as “whooping cough” is a high infectious respiratory disease caused by the Gram-negative organism Bordatella pertussis. Laboratory confirmation of clinically suspected pertussis is made via culture or more commonly now DNA detection on molecular assays using nasopharyngeal aspirates or nasopharyngeal swabs/pernasal swabs. PCR is increasingly utilised for the diagnosis of pertussis as it has better sensitivity compared with culture-based methods. As with all PCR methods, culture is still necessary in order to perform susceptibility testing so it is beneficial to send samples for culture and PCR testing simultaneously.

Topical antimicrobials Antimicrobial name

Topical antiviral Aciclovir

Organisms covered (in the absence of resistance mechanisms)

Herpes simplex virus Varicella zoster virus

Topical antibacterials Most Gram-positive organisms Chloramphenicola Most Gram-negative organisms but no cover for Pseudomonas spp. Fusidic acid Staphylococcus aureus Also has streptococcal activity a

Most frequently prescribed topical antimicrobial for superficial eye infections as the majority are caused by Staphylococcus aureus.

Table 2

ear”, is associated with anaerobes, S. aureus and Pseudomonas aeruginosa. Chronic otitis externa can caused by fungi such Candida or Aspergillus species. Malignant otitis externa is an invasive form of necrotising otitis externa almost always caused by P. aeruginosa. Otitis media is defined by the co-existence of fluid in the middle ear and signs and symptoms of acute illness. It occurs when oropharyngeal flora ascend the eustachian tube. Typical causative organisms include S. pneumoniae, H. influenzae and Moraxella catarrhalis. Superficial external ear swabs are not helpful in the investigation of otitis media unless there is perforation of the ear drum as ordinary resident or commensal flora will be cultured. Deeper swabs of pus or exudate from the affected ear are the most useful laboratory specimens. If a fungal infection is suspected, scrapings of material from the ear canal are needed for laboratory culture.

The gastrointestinal tract Peritonitis is inflammation of the peritoneum, which is the serous membrane lining the abdominal cavity and the abdominal viscera. Primary bacterial peritonitis, without evidence of intra-abdominal organ peforation, is rare accounting for less than 1% if all cases of bacterial peritonitis. It can be seen occasionally in children with nephrotic syndrome. Secondary bacterial peritonitis arises following gastrointestinal leakage within the peritoneal cavity following perforation of viscera or abdominal trauma. In paedatrics, secondary bacterial peritonitis commonly complicates the perforation of an acute gangrenous appendix. Samples of pus collected into a sterile containers are preferred to swabs of pus.

The throat Pharyngitis or inflammation of the pharynx is usually caused by viral pathogens. Typical bacterial pathogens include Group A streptococci (GAS), also known as S. pyogenes, various Corynebacterium spp. and occasionally Fusobacterium necrophorum. Epiglottitis is an inflammation of the epiglottis characteristically associated with stridor. Throat swabs should never be taken if there is any concern for epiglottitis as doing so may precipitate an acute airway obstruction. Blood cultures should be sent. Where there is strong suspicion for GAS infection, an anti-streptolysin O titre (ASOT) should be checked by drawing a blood sample. Tonsillitis is frequently viral in aetiology. Peritonsillar abscess or quinsy is a rare bacterial complication of tonsillitis where a unilateral collection of pus develops between the capsule of the palatine tonsil and the superior constrictor muscle. It is usually associated with infection by S. anginosus group, GAS and F. necrophorum and F. nucleatum. Intra-operative pus should be sent for culture in the laboratory. Laryngitis or inflammation of the larynx (voice box) typically presents with hoarseness and is generally viral in aetiology. When taking throat swabs, always avoid the tongue and uvula and aim to swab the tonsillar area and/or posterior pharynx.

The urinary tract There is a wide spectrum of urinary tract infections (UTIs) but the two most common presentations in paediatrics are cystitis (bladder infection) and acute or chronic pyelonephritis (infection of the kidney and renal pelvis). Confirmation of a UTI in a child is dependent on the quality of the specimen. Mid-stream urine (MSU) samples and clean-catch urine specimens are preferred. Bag urine and pad urine sampling is discouraged as these are easily contaminated with the child’s own faecal flora. The probability of a UTI is increased by the isolation of the same organism from two specimens. Generally a pure growth of between 107 - 108 cfu/L (104 - 105 cfu/mL) is indicative of a UTI. E. coli is the most common organism associated with UTI accounting for nearly 70% of all isolates cultured. It is worth remembering that the National Institute for Health and Care Excellence (NICE) guidelines on the management of UTIs in

The lower respiratory tract The term lower respiratory tract infection (LRTI) encompasses a wide spectrum of clinical presentations including pneumonia


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Normal CSF values. Adapted from Public Health England, UK Standards for Microbiological Investigations: Investigation of Cerebrospinal Fluid (issue date 31.05.2017). Always check with local laboratory with regard to cut-off values for CSF analysis. Always rule out any contraindications to performing an acute lumbar puncture before proceeding and if in doubt seek senior advice Opening pressure

Normal opening pressure is 10e20 mmHg. When measuring opening pressure the child must be laid on his/her side and should not be placed sitting upright. Bacterial meningitis Elevated Viral meningitis Usually normal Fungal meningitis Variable Tuberculous meningitis Variable

Erythrocytes Leucocytesb,c

No red blood cells should be present in normal CSFa Neonates Infants (less 28 days of life) (1 to 12 months old)

Protein

d

Glucose

Children (older than 1 year)

0e30 cells x 106/L

0e15 cells x 106/L

0e5 cells x 106/L

Neonates (less 28 days of life)

Infants (29-56 days of life)

Children (2 months to 18 years old)

0.65e1.5 g/L

0.5e0.9 g/L

0.05e-0.35 g/L

Neonates (less 28 days of life) 1.94e5.55 mmol/L

Infants (29 to 58 days of life) 1.55e5.55 mmol/L

Infants (2-12 months old) 1.94e5.0 mmol/L

Children (older than 1 year) 2.22e4.44 mmol/L

a

The presence of red blood cells (RBCs) in the CSF can result from an intracerebral or subarachnoid haemorrhage (SAH) or from a “traumatic” lumbar puncture (LP), in which peripheral blood contaminates the CSF. A traumatic tap occurs in approximately 20% of LPs performed. Peripheral blood in the CSF after a “traumatic tap” will result in an artificial increase in white blood cells (WBCs) by one WBC for every 500 to 1,000 RBCs in the CSF. This correction factor is accurate as long as the peripheral WBC count is not extremely high or low. Examine the RBC count from sequential LP samples labelled 1, 2 and 3. Uniform bloodstaining of all samples suggests previous haemorrhage into the subarachnoid space. Reducing red blood cell counts in sequential CSF samples, with the lowest count in sample 3, suggests bleeding induced by the LP procedure; a “traumatic tap”. Remember normal CSF is crystal clear. Xanthochromia is a more reliable predictor of haemorrhage and is present in greater than 90% of patients within 12 h of SAH onset. It is a yellow, orange, or pink discoloration of the CSF, caused by the lysis of RBCs resulting in haemoglobin breakdown to oxyhaemoglobin, methaemoglobin, and bilirubin. Discoloration begins after RBCs have been in spinal fluid for about 2 h and remains for 2e4 weeks. b A leucocyte or WBC: RBC ratio of 1.500 to 1:1000 is not indicative of infection. CSF obtained more than 12 h after a CT or MRI brain confirmed intracranial haemorrhage may show raised WCC counts of up to 500 x 106/L as a consequence of the normal physiological inflammatory response. c The CSF polymorph:lymphocyte ratio is an unreliable with regard to whether meningitis is bacterial, viral or mycobacterial in origin. This is particularly important in interpreting neonatal CSF results or when total leucocyte counts are less than 1000 x 106/L. In viral meningitis there is a typically a lymphocytic predominance in the CSF, but in the early course of the disease both neutrophils and lymphocytes can be found. Tuberculous meningitis can be associated with a neutrophil rather than a lymphocytic infiltrate early in infection. It is important to remember that neutropenic patients may not produce any polymorph or neutrophil response in the CSF and lumbar puncture results should be correlated with the clinical situation. d CSF glucose is normally two thirds of the serum glucose, measured during the preceding 2e4 h before an LP is performed; can be higher in neonates. This ratio decreases with increasing serum glucose levels. Bacterial meningitis can cause lowered CSF glucose levels. Glucose levels are usually normal in viral infections. Normal glucose levels however do not rule out infection, because up to 50% of patients who have bacterial meningitis will have normal CSF glucose levels. A range of noninfectious inflammatory conditions, SAH, and hypoglycemia also cause hypoglycorrhachia (low glucose level in CSF).

Table 3

infants, children and young people (under 16 years), published in 2013 and updated 2017, classifies all non-E. coli UTIs as ‘atypical infections’ and additional investigations are warranted. Microscopy for the presence of red cells, white cells, casts, cellular components and bacteria is performed in the laboratory. Chromogenic agar plates, which allow the rapid identification of bacteria present after overnight incubation, based on a defined colour scheme of colonial growth, are increasing in popularity and help to guide empiric treatment pending final culture results.

disorder such as psoriasis, eczema or recurrent furuncles/carbuncles, whether there was preceding trauma (indoor or outdoor), burn, bite (human, animal, insect), recent swimming pool/freshwater/saltwater exposure, recent foreign travel, recent surgical procedure/wounds and parental/carer occupation as healthcare worker exposure can increase the risk of colonisation with methicillin-resistant S. aureus (MRSA). The majority of skin/soft tissue infections are due to S. aureus and the betahaemolytic streptococci groups A, C, G and B.

The skin and soft tissues The key to a rapid diagnosis of skin and soft tissue infection is a thorough history. When the skin is broken and infection ensues, it is important to know if there is a history of underlying skin

The bones and joints Osteomyelitis is infection resulting in inflammation of bone. Common causative organisms include S. aureus, coagulase negative staphylococci (CoNS) and Enterococcus species. Osteomyelitis


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Commonly prescribed beta-lactam antimicrobials and their potential spectrum of cover (not all susceptible organisms are listed). Always discuss with your local laboratory regarding resistance patterns, review past microbiology results if available and follow empiric antimicrobial guidelines in your hospital. Susceptibility of an organism to an antimicrobial does not infer that it is the best treatment option; always consider the anatomical site of infection Antimicrobial(s)

Penicillin-based Benzylpenicillin antimicrobialse (penicillin G),

Route

IVa

Clinical use Gram-positive cover

Gram-negative cover

Streptococcus pneumoniae Beta-haemolytic streptococci (A,B,C,G)

Allergic reactions in