,

CME REVIEW

Microbiological aspects of bacterial lowerrespiratory tract illness in children:atypical pathogens

Joshua Wolf and Andrew J. Daley*

Department of Microbiology and Infectious Diseases, The Royal Children’s Hospital andThe Royal Women’s Hospital, Melbourne, Australia

PAEDIATRIC RESPIRATORY REVIEWS (2007) 8, 212–220

KEYWORDS

pneumonia;

pediatric;

pertussis;

Mycoplasma pneumoniae;

Chlamydophila;

Legionella

Summary ‘Atypical’ lower respiratory tract pathogens often cause a distinct identifi-able syndrome in adults, but in children the clinical presentation of atypical, typical andviral pneumonia is less well differentiated. Specific microbiological investigations areusually required, but an understanding of their strengths and weaknesses is necessary tomake interpretation possible. This review examines clinical presentation, microbiologyand current evidence surrounding diagnostic techniques for Mycoplasma pneumoniaeChlamydophila pneumoniae, Chlamydophila psittaci, Bordetella pertussis and Legionellaspecies. Applying an understanding of the investigations to the diagnosis of pneumoniain children may lead to more appropriate patient management by ensuring that theyclarify rather than further obscure the diagnosis.� 2007 Elsevier Ltd. All rights reserved.

EDUCATIONAL AIMS

� To discuss the characteristics of available tests for atypical lower respiratory tract pathogens as they apply tochildren.

� To provide evidence-based recommendations for the use of investigations for atypical pathogens in individualchildren with pneumonia.

INTRODUCTION

The so-called ‘atypical’ pathogens were named for a distinctpresentation of clinical disease in adults. In adults, thesepathogens are commonly associated with non-respiratorysymptoms and bilateral lung disease compared with theclassical presentation of pneumococcal lobar pneumonia.

* Corresponding author. Department of Microbiology, TheRoyal Children’s Hospital, Flemington Road, Parkville, 3052,Victoria, Australia. Tel.: +61 3 9345 4850; Fax: +61 3 9345 5764.

E-mail address: andrew.daley@rch.org.au (A.J. Daley).

1526-0542/$ – see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.prrv.2007.07.004

This contrasts with the presentation in children which isfrequently indistinguishable from that caused by ‘typical’bacteria and by viruses, as each can cause isolated lobarconsolidation and similar systemic responses. This makesidentification of the causative organism of lower respiratorytract infection (LRTI) in children even more important andemphasizes the need for appropriate microbiological inves-tigations.

The microbiological diagnosis of respiratory infectionwith atypical organisms in children is challenging. First, all ofthe problems applying to typical bacteria remain relevant,

LOWER RESPIRATORY TRACT ILLNESS IN CHILDREN: ATYPICAL PATHOGENS 213

KEY POINTS

� Diagnosis of atypical pathogens is challenging inchildren.

� Serological assays rarely provide diagnostic cer-tainty in acute illness.

� Culture is not generally feasible for atypical patho-gens.

� Nucleic acid amplification tests are likely to be themost useful tests for atypical pathogens.

including the difficulty of obtaining an appropriate sample.Second, routine culture of these organisms is rarely a viableoption. Third, the investigations for each of these pathogensoften have limited sensitivity and specificity.

Despite these challenges, the appropriate use of clinicaland laboratory information can shed light on the micro-biological diagnosis of LRTI in many children. This reviewexamines the current status of diagnostic techniques forMycoplasma pneumoniae, Chlamydophila pneumoniae,Chlamydophila psittaci, Bordatella pertussis and Legionellaspecies.

MYCOPLASMA PNEUMONIAE

M. pneumoniae is the smallest known free-living organism. Itis implicated in a wide spectrum of clinical disease involvingmultiple body systems. The most common presentation iswith respiratory illness. Although other species of myco-plasma are described, the term mycoplasma here refersonly to M. pneumoniae. Although there is no conclusiveevidence that specific antibiotic treatment of mycoplasmarespiratory infection affects the outcome of disease, it iscommon to treat symptomatic infection with macrolide or,in children over 8 years of age, tetracycline antibiotics.

Clinical spectrum of disease

The spectrum of M. pneumoniae disease includes self-limit-ing upper respiratory tract infection (URTI), otitis mediaand LRTI. Mycoplasma URTI is usually indistinguishablefrom common viral infections. Pneumonia probably onlyoccurs in 3–13% of infections.1 In lower respiratory tractdisease the clinical examination may reveal wheeze, crepi-tations or both.2 Chest radiograph in patients with acutemycoplasma pneumonia demonstrates bilateral or unilat-eral opacity, which may be dense or patchy. Contrary to thefindings in adult patients, children often have lobar con-solidation or effusion.3 Empyema is rarely seen and oftenrepresents infection with another organism.

Even in primary respiratory tract disease other systemsare commonly involved; diarrhoea, headache, malaise andskin rash are frequently reported. Although bullous myr-ingitis had been thought to be pathognomonic of M.pneumoniae infection, this is unlikely to be the case.4

Neurological infections such as meningitis and encephalitishave been reported and often occur without respiratorysymptoms. Acute infection may be associated with coldagglutinin disease and haemolysis. Arthritis and arthralgiahave also been described.

Epidemiology

M. pneumoniae infection occurs in all age groups but isespecially common in children. Recent evidence suggeststhat infection often occurs in the preschool age population,although it was previously thought unusual in this group.2

The disease is endemic in most populations with super-imposed epidemics occurring in 3–5 year cycles. Out-breaks, with attack rates of up to 70%, also occur infamilies and close social groups such as military recruits.1

Diagnostic tests

All available diagnostic tests for M. pneumoniae performpoorly when their clinical application is scrutinized. Eachtest has unique limitations which are discussed below. It isimportant to note that studies of diagnostic testing for M.pneumoniae are difficult to assess or compare becausethere is no gold standard; consequently, each study usesdiffering diagnostic criteria for assessment of the sensitivityand specificity of the method. Clinical acumen, in conjunc-tion with judicious use of diagnostic tests, is required toidentify respiratory infection with M. pneumoniae in childrenand to institute appropriate therapy.

Culture

M. pneumoniae has been cultured from throat swabs,nasopharyngeal specimens, sputum and middle ear fluid,and occasionally from other sites where it is presumed tocause disease. However, culture of the organism is difficult,requiring expertise with special media and culture techni-ques and an incubation period of up to 4 weeks.1 Thesensitivity is often <60% even in the most expert labora-tories. The specificity of culture for diagnosis of acuteinfection has been challenged because of the demonstra-tion of prolonged carriage after active infection. Given thepoor sensitivity, difficult technique, inability to providetimely results and possible lack of specificity, routine diag-nostic culture for M. pneumoniae is not recommended.

Nucleic acid amplification testing

Polymerase chain reaction (PCR) testing for M. pneumoniaeis now widely available but suffers from limited sensitivity.For LRTI, sensitivity is maximized by testing sputum sam-ples, but young children are rarely able to produce sputumreliably. Nasal swabs, nasopharyngeal aspirates (NPAs) orthroat swabs are more commonly obtained from children.The sensitivity of PCR on each of these specimens in one

214 J. WOLF, AND A. J. DALEY

study of young adults was 69% for sputum, 50% for NPAand 38% for throat swab.5

Similar to culture, there are concerns that PCR testingmay fail to distinguish between colonization and trueinfection, markedly limiting the diagnostic value of the test.This is especially true given the qualitative nature of mostavailable PCR methods. However, a Chilean study found acolonization rate of only 2% in healthy children, suggestingthat a specificity of 98% is possible.6 In contacts of diag-nosed cases the rate is higher at about 15%.7 Afterresolution of infection, prolonged carriage may also occur.However, taking these data into account, a positive result ina patient with a consistent clinical picture may be useful.

Serology

A number of commercial assays are available for theserological diagnosis of mycoplasma infection. The assayis usually regarded as positive if it detects IgM specific to M.pneumoniae or a significant rise in specific IgG betweenacute and convalescent sera (usually a fourfold rise over 2–4 weeks). The sensitivity and specificity of serological testsvary considerably between children and adults, betweendifferent assays and with time elapsed since the onset ofillness. A number of different techniques to detect antibodyare available, including enzyme immunoassay (EIA), com-plement fixation and particle agglutination tests.1 Eachtechnique suffers from similar benefits and pitfalls and theseare discussed together below.

A significant rise in mycoplasma-specific IgG or IgM overa 2–4 week period seems to have a high specificity andsensitivity if the first sample is collected early in the illness.Although this is useful for epidemiological studies, wherediagnosis can be performed retrospectively, it is not suffi-cient for clinical situations where a timely result is requiredto influence acute management.

Detection of mycoplasma-specific IgM on a single speci-men has often been postulated as an effective test todiagnose acute infection. This test suffers from a numberof limitations. First, the sensitivity is limited and false nega-tives certainly do occur. More importantly, the specificity issimilarly poor and may be as low as 60% in children. Oneinteresting study from Israel showed high rates of IgMpositivity at around 30% in healthy children attending forelective surgery.8 IgG was also raised in a large proportionof these children. Such data suggest that single-specimenmycoplasma serology has a very limited role in the diagnosisof acute mycoplasma infection in children.

Other diagnostic tools

A number of direct antigen detection techniques have beenproposed, but sensitivity is limited by the small number oforganisms relative to the detection limit of the test, andspecificity is limited by cross-reactivity with upper respira-

tory tract commensal organisms.1 These tests cannot berecommended.

Measurement of cold-agglutinins was used historically todiagnose M. pneumoniae infection before more specifictests became available. Cold-agglutinins are present in only�50% of patients with mycoplasma infection and alsofrequently occur in other diseases which may have a similarclinical picture, such as adenovirus or Epstein–Barr virusinfection. The test may be useful if cold-agglutinin asso-ciated disease is suspected, but it performs poorly as adiagnostic test.

Summary

The available diagnostic technology for M. pneumoniaeinfection in children performs relatively poorly. Single-speci-men serology, direct fluorescent antibody (DFA) and PCRare able to provide rapid answers, but these are of limitedpredictive power. Paired serology probably provides accu-rate information well after it is clinically useful. Given the lackof conclusive evidence for specific treatment of the organism,the effectiveness of mycoplasma-active agents against con-ventional organisms and the poor function of these tests,many clinicians choose to make a clinical diagnosis of myco-plasma infection rather than relying on diagnostic testing.

If testing is thought necessary, PCR is suggested as thebest test for M. pneumoniae infection in children. Its abilityto provide rapid diagnostic information and relativelysuperior sensitivity and specificity make it more acceptablethan the other options. Sputum or bronchoalveolar lavage(BAL) would seem to be ideal specimens, but throat swabor NPA are viable alternatives.

CHLAMYDIA AND CHLAMYDOPHILA

Chlamydia and the newly differentiated Chlamydophila areobligate intracellular organisms. Three species are promi-nent human pathogens. Chlamydophila pneumoniae andChlamydophila psittaci most commonly cause respiratorydisease. In the developed world Chlamydia trachomatis ismost commonly a urogenital tract pathogen, which alsocauses neonatal pneumonia and conjunctivitis. In the devel-oping world it is the cause of trachoma.

Immunity after primary infection with Chlamydia speciesis generally poor. This is perhaps best illustrated by C.trachomatis causing ‘ping-pong’ re-infection as sexual part-ners re-expose each other after successful treatment.

CHLAMYDOPHILA PNEUMONIAE

C. pneumoniae is a primary human pathogen without anyknown animal reservoir. It causes LRTI but is also docu-mented as frequently causing mild or asymptomatic infec-tion. It causes invasive disease by entering cells, reproducingwithin cytoplasmic inclusions and being released with orwithout destruction of the host cell. Treatment for diag-

LOWER RESPIRATORY TRACT ILLNESS IN CHILDREN: ATYPICAL PATHOGENS 215

nosed infection is usually with macrolides or tetracyclines(in children over 8 years);9 however, there is no goodevidence that this treatment is superior to b-lactam anti-biotics.

It has been difficult to tease out the role of C. pneumo-niae infection in acute lower respiratory tract disease. Manystudies have identified it by serological or direct methods inthe context of disease, often in conjunction with anotherrespiratory pathogen. Further, it has been identified forprolonged periods after acute infection and very frequentlyin the absence of symptoms.

Studies on diagnosis of C. pneumoniae infection are verydifficult to interpret. There is no gold standard and availabletests have been applied differently with widely disparateresults. For example, studies have commonly demonstratedan absent antibody response in the context of positiveculture or nucleic acid identification and consistent illness.Conversely, studies which have looked for microbiologicalevidence of infection in the context of ‘positive’ serologyhave often failed to find it. Lastly, microbiological techniquesfor directly identifying the organism in clinical specimensremain rudimentary.

Clinical features

Apart from asymptomatic infection, the usual clinical pre-sentation of significant C. pneumoniae infection in children isprobably with mild pneumonia indistinguishable from thatcaused by other organisms. It does not seem to cause‘atypical pneumonia’ as in adults. Severe disease and pleuraleffusion are rare in immunocompetent children. C. pneu-moniae infection may be responsible for up to 20% ofpresentations with acute chest syndrome in children withsickle cell disease.10

Epidemiology

C. pneumoniae infection is probably distributed through allage groups and geographical areas. Because of the difficultyof establishing an effective diagnostic strategy, estimates ofthe frequency of this organism in community-acquiredpneumonia (CAP) vary from 0 to 44%.11 This is an addi-tional challenge to diagnosis as, even if sensitivity andspecificity of the available tests were known, extrapolationto positive and negative predictive value is impossible in theabsence of reliable information about pre-test probability.

C. pneumoniae can spread within families and amongstclose social groups. The role of colonization or prolongedcarriage is unclear. Carriage of C. pneumoniae in asympto-matic adults is probably uncommon. One study of subjec-tively healthy adults in Japan using both PCR and culturefound positive results in only 1.8% of participants.12 Twoexceptions are carriage after acute infection and asympto-matic infection after recent exposure. In the setting of anoutbreak, most cases of acquisition of the organism areasymptomatic.

Diagnosis

Culture

As C. pneumonia requires eukaryotic cells for growth,culture of the organism is a difficult process requiringinoculation into cell culture. It requires specialized equip-ment and expertise. As with any technique which requiresthe isolation of a live fastidious organism, the sensitivity ofculture is limited. Because of all these problems, culture isnot routinely recommended for diagnosis of C. pneumoniae.However, if performed and found to be positive in theappropriate clinical context, culture is strongly suggestive ofinfection as false positive results are rare.12

Nucleic acid amplification testing

PCR is the most promising diagnostic technique for C.pneumoniae. At present, however, a number of issuesrequire attention. Different PCR targets have been devel-oped, each of which is likely to perform differently. Whenthe tests have been run on identical samples they haveoften correlated poorly with each other. Further, none ofthe tests has been validated on varying clinical specimens.11

No PCR test has yet been licensed for this purpose by theUnited States Food and Drug Administration. As a result ofthese issues, there is insufficient evidence to recommendany test for clinical use. It is likely that a commercial PCR forC. pneumoniae will soon become available for routine use.

Serology

The Infectious Diseases Society of America recommendsthe use of microimmunofluorescence (MIF) as an accep-table diagnostic test for C. pneumoniae. The Society recom-mends accepting a fourfold increase in IgG titre or a singlepositive IgM (�1:16) as a positive result. It does not statewhat period should be left between the paired sera.13

These recommendations should be received with caution.Serology is not a straightforward test for the acute diagnosisof C. pneumoniae infections. Single-specimen serology haspoor sensitivity and specificity and even testing of pairedsera fails to diagnose a large number of infections.

The sensitivity of serology is very poor, especially inchildren. The majority (�70%) of children with goodevidence of C. pneumoniae infection have negative serologyboth at the time of infection and for months afterwards.Even if seroconversion does occur after primary infection,IgM does not appear until 2–3 weeks after onset, and IgGmay not rise for 6–8 weeks, both of which are too late to beof diagnostic value.11 Specificity is similarly limited as highlevels of C. pneumoniae-specific IgM have been documen-ted in subjectively healthy adults without any other evi-dence of infection. The use of a single IgG level �1:512 hasbeen postulated as diagnostic of acute infection, but this isnot recommended because of false positive results in anumber of studies.

216 J. WOLF, AND A. J. DALEY

Summary

There is no perfect diagnostic test available for acute diag-nosis of C. pneumoniae infections. The most routinely avail-able test, serology, has a number of pitfalls which limit itssensitivity and specificity. PCR and culture are likely to be themost specific tests but culture is insensitive and difficult andPCR has problems with validation. We await the develop-ment of validated and effective PCR tests which are the mostlikely candidates for a useful diagnostic system for this disease.

CHLAMYDOPHILA PSITTACI

C. psittaci is the cause of psittacosis in humans. The naturalreservoir for C. psittaci is birds, in which it can be sympto-matic or asymptomatic. The bacterium is likely spread bythe respiratory route. Humans are generally infected bydirect contact with birds or their excreta or indirect con-tact, such as aerosolization of dead birds with a law-nmower.14 Person-to-person transmission is rare but canprobably occur as psittacosis has been documented inhealthcare workers exposed to patients with the disease.15

In adults, psittacosis presents in protean ways. It canpresent with non-specific symptoms suggestive of viralinfection, as a typhoidal illness or as atypical pneumonia.In children the common presentation is with headache,cough and fever. Chest radiograph usually demonstratespulmonary infiltrates and may also show pleural effusion.Liver function test abnormalities are common.

The diagnosis of psittacosis is usually based on a con-sistent clinical presentation with known bird exposure.Testing is generally with serology demonstrating a fourfoldrise in titre or, less convincingly, a single high titre of C.psittaci-specific IgG. The sensitivity and specificity of thesetests remains somewhat unclear. DFA, EIA and PCR testingof sputum have been described as effective in some caseseries but have yet to undergo full evaluation. Culture isdifficult and is not routinely available or recommended.

LEGIONELLA

Legionella species are a cause of both pulmonary andextrapulmonary infection. The most common manifesta-tion is acute pneumonia, which may be either community-or hospital-acquired. The genus and the pulmonary infec-tion, Legionnaires’ disease, were named after an outbreakthat involved delegates at an American Legion conventionin Philadelphia in 1976.16 Infection with Legionella speciesmay produce significant morbidity and mortality if notrecognized and treated promptly.

Legionella are small Gram negative obligate anaerobicbacilli. They are widespread in the environment. They growoptimally between 20 and 42 8C and have a propensity togrow in water, including cooling towers and plumbed warmwater systems. They can replicate and survive within free-living amoeba and remain in a dormant state within biofilms,

which allows them to survive in unfavourable environmen-tal conditions.

Epidemiology

Over 49 species of Legionella are described with manysubgroups and subspecies. Of the 20 species that causeinfection in humans, L. pneumophila is the most common,responsible for 50–90% of infections. Infection occursfollowing inhalation of aerosols or micro-aspiration ofcontaminated water. Person-to-person transmission doesnot occur. Community outbreaks have been associatedwith many sources of contaminated water from air-con-ditioning cooling towers and fountains to showerheads andrespiratory therapy devices.

Most infections occur in the elderly or susceptible adultswith risk factors such as chronic pulmonary or cardiac con-ditions, smokingand immunesuppression. Legionellosis is lesscommon in children. Some have suggested that Legionellaspecies are responsible for up to 5% of CAP in immuno-competent children,17 but the very small number of definitereported cases suggests that it is much rarer than this.18 Mostcases have been described in neonates and children withacquired (chemotherapy, antirejection medications, chronicpulmonary disease) or congenital (chronic granulomatousdisease, severe combined immunodeficiency) immunodefi-ciency. Neonatal legionellosis can present either as pneumo-nia or non-specific sepsis and is usually seen in the setting ofprematurity or congenital cardiac disease.19

Nosocomial Legionnaires’ disease has been described inseveral paediatric institutions with approximately 80% ofaffected children being immunocompromised in somefashion.20 Environmental surveillance is indicated in hospi-tals performing organ transplantation and managing immu-nocompromised patients if warm water systems or air-conditioning cooling towers are in use. Nosocomial Legion-naires’ disease is largely preventable and various controlmethods are available to disinfect warm water systems,including superheating, chlorination, ozone, metal ioniza-tion and ultraviolet light. Other simple approaches toreducing exposure include drawing baths before the childenters the room, avoiding showers for high-risk patientsand using sterile water to clean respiratory equipment.

Clinical presentation

The clinical presentation ranges from a non-specific con-solidating pneumonia to a non-pneumonic illness calledPontiac fever (named after a cluster of cases described inPontiac, Michigan) to a range of extrapulmonary manifesta-tions (including sinusitis, pyelonephritis and endocarditis)and even subclinical infection. Pneumonia manifests as anacute febrile illness with a non-productive cough. Theconstellation of symptoms may help to differentiate Legion-naires’ disease from more common bacterial infectionssuch as pneumococcal pneumonia, but the clinical pre-

LOWER RESPIRATORY TRACT ILLNESS IN CHILDREN: ATYPICAL PATHOGENS 217

sentation is frequently non-specific. Myalgia, abdominalpain, diarrhoea, headache and confusion, anorexia andchest pain may be present. The fever is not associatedwith significant tachycardia and radiographic changes mayappear more severe than the respiratory symptoms wouldsuggest. Laboratory testing may demonstrate hyponatrae-mia, hypophosphataemia and abnormal liver function tests(principally raised aminotransferase levels).

Diagnosis

Chest radiographs often show unilateral changes, whichmay extend to bilateral involvement, with or withoutpleural effusion, as the infection progresses. There maybe patchy alveolar or interstitial infiltrates or nodularlesions, which may cavitate, particularly in neonates orimmunocompromised patients.21

The laboratory diagnosis of legionellosis relies on theisolation of the bacterium in culture, direct detection inclinical specimens or the measurement of serologicalresponses to infection. Legionella is best isolated on buf-fered charcoal yeast extract (BCYE) agar on which growthcan be expected within 2–5 days, but cultures should bekept for up to 14 days.

Lipopolysaccharide antigen from L. pneumophila ser-ogroup 1 is detectable in urine within a few days of onsetof the illness. Several commercial assays are available andsensitivity is around 80%, although this varies depending onthe severity of the illness.22 Specificity of the Legionella urinaryantigen test approaches 100%.Antigenuria persists for weeksto months and cannot be used to determine the response totherapy. Direct identification of Legionella in respiratoryspecimens using direct immunofluorescence is rapid buthas a sensitivity of only 60%. Nucleic acid amplification tests(NAAT) have the highest sensitivity and specificity.

Serological diagnosis may be useful for epidemiologicalstudies and is often retrospective. A fourfold rise in titre isdiagnostic but seroconversion can take several weeks andcross-reaction with other organisms such as C. psittaci andCoxiella burnetii has been described.23

Although the diagnostic gold standard remains culture,urinary antigen detection or molecular methods and ser-ology are the most practical tools for routine diagnosis.

PERTUSSIS

Pertussis (whooping cough) is caused by the Gram negativepleomorphic bacterium Bordetella pertussis. Occasionally,similar disease can be caused by B. parapertussis. Althoughpertussis is part of the routine vaccine schedule in mostdeveloped countries, the organism remains a significantcause of disease.

Clinical features

The clinical spectrum of pertussis is broad. In preschool andschool aged children it most often causes a coryzal pro-

drome (the ‘catarrhal phase’) followed by a coughing illness(the ‘paroxysmal phase’). The cough is often associatedwith a high pitched inspiratory ‘whoop’ or with post-tussivevomiting. After this, there is a term of ongoing less severecough (the ‘convalescent phase’) which lasts several weeks.Adults and adolescents tend to present without a ‘whoop’or post-tussive vomiting, and may have no catarrhal phase,but simply have a persistent cough lasting for many weeks.Complications of the disease are uncommon in olderchildren and adults, but infants have much higher rates.24

The clinical presentation of pertussis in infants may benon-specific and the range of presentations includes acuteor chronic cough as well as life-threatening apnoea as thefirst indicators of infection. Infants bear the greatest burdenof pertussis complications. In the USA, between 1992 and1994, infants under 6 months old with pertussis demon-strated: hospitalization in 70%, pneumonia in 15%, seizuresin 2% and death in 0.6%. In neonates, the death rate is evenhigher at 1.3%.25

Epidemiology

Pertussis most commonly occurs in adults or adolescents,unimmunized children and infants too young to havecompleted a full course of pertussis vaccine. Infants pre-senting with pertussis have acquired the disease from acoughing sibling or parent in at least 80% of cases.26 InAustralia in 2006, 10 975 cases of pertussis, including 1163children, were reported, although this is very likely asignificant underestimate of the burden of disease. Pertussisvaccine is effective at preventing disease in childhood, butthree doses are required to achieve high efficacy andprotection does not last well into adolescence.27 Herdimmunity or ring immunity is required to protect infants asthey are not protected by vaccine.

Diagnosis

The diagnosis of pertussis infection is based on appropriatediagnostic testing in a patient with a consistent illness. Themost appropriate test will depend on available resources,the age of the patient and the timing of testing with respectto the onset of the illness. Commencement of antibiotictherapy may be appropriate prior to results of diagnostictesting being available. Pertussis may be suggested by asignificant lymphocytosis, but this is non-specific, especiallyin children. Specific diagnostic tests can be divided into twogroups: direct and indirect. Direct testing includes NAAT,culture and DFA. This group of tests relies on the presenceof the bacterium and is therefore relatively insensitive late inthe illness or after antibiotic treatment. Indirect testinginvolves serological tests looking for the immune responseto infection. Serology is often insensitive early in the illnessand becomes much more sensitive over ensuing weeks. It isnot affected by antibiotic administration. In general, serol-ogy is useful in adolescents and adults who tend to present

218 J. WOLF, AND A. J. DALEY

late in the illness, whilst direct techniques are useful inyounger children who may present earlier.

Nucleic acid amplification testing

PCR-based testing of nasopharyngeal secretions, obtainedby NPA or nasopharyngeal swab is now considered themost sensitive and specific test for pertussis infection,especially early in the illness.27 There are a number ofdescribed PCR targets, and each different test will demon-strate different characteristics.

The sensitivity of pertussis PCR is difficult to determine,but cases in which B. pertussis culture is positive and PCR isnegative are rare using current technology. Furthermore,the test picks up significantly more positive results thanculture, so calculating specificity is similarly challenging.28

The reported sensitivity for clinical use of B. pertussisPCR ranges from 73 to 100%, which is much higher thanculture. It is clear that the sensitivity of both culture andPCR falls with time and antibiotic administration, but theexact nature of the fall off is not well described. PCRremains positive significantly longer in pertussis diseasethan culture and is less affected by antibiotic administration.In one study, PCR remained positive 4 days into antibiotictherapy in 89% of cases and 7 days in 56%, but culture waspositive at those times in only 56% and 0%, respectively.

The specificity of pertussis PCR using current metho-dology approaches 100%. Occasionally, false positiveresults may occur because of unvalidated tests, laboratorycontamination or because of cross-reactivity with B. hol-mesii or B. bronchoseptica which can be nasopharyngealcommensals and uncommonly cause disease.29 Most PCRtargets will miss B. parapertussis as a cause of a pertussis-likeillness.

Culture

Pertussis culture should be performed on specific media toencourage the growth of this fastidious organism anddiscourage the growth of commensal organisms. The idealspecimen is nasopharyngeal secretions obtained by aspirateor swab. Sensitivity of culture varies greatly with time sinceonset of illness, method of specimen collection and trans-port and culture technique.

Culture is a relatively poor test in comparison to PCR;the time taken to obtain a result may be many days andsensitivity is often lower than 50%. Late in the illnesssensitivity is very low, falling to approximately 0% at 3weeks in most studies.29 Specificity is significantly higher,and unless contamination occurs, is regarded as 100%. Theprinciple benefit of culture is the epidemiological dataavailable from antibiotic resistance testing and typing ofthe organism which is unavailable from other diagnostictechniques. Therefore, from a public health perspective,culture should be performed on all specimens, but it rarelyadds value from a clinical viewpoint.

Immunofluorescence

DFA testing involves placing a fluorescent labelled mono-clonal antibody specific to pertussis antigen directly onto aclinical specimen, washing off the unbound immunoglobulin,and then identifying any antibody which remains. The sensi-tivity of this test is limited (�40% in one large study)30

meaning that it will miss many cases of pertussis if used as theonly test. Specificity may be up to 99.6%, but in a populationwith low rates of pertussis this will likely translate to relativelyfrequent false positive results. The major benefit of this test isthe rapidity with which results are available.

Serology

The diagnosis of pertussis by serological techniques generallyrequires the testing of acute and convalescent sera or testingof serum weeks into the illness. Serology is, therefore, rarelyuseful in the acute setting. These tests are useful for epide-miological purposes and may be helpful to confirm thediagnosis in adolescents and adults who tend to presentafter a prolonged coughing illness. Different antigens areutilized with pertussis toxin (PT) being the most specific for B.pertussis, but cross-reaction with other Bordatella speciesoftenoccurs. Titres are generally elevated in the secondweekof the illness and a single high IgG to PT may be suggestive ofrecent infection in a patient with a compatible clinical illness.

Summary

PCR is the most sensitive and specific test for pertussisinfection early in the disease; however, the sensitivity fallsrapidly over time. Serological testing is the most sensitivetest later in the illness and is the most appropriate test whena patient presents with a prolonged cough. Culture is stillrecommended for public health purposes but cannot beregarded as a useful method for diagnosis.

CONCLUSION

In children, atypical pathogens often cause LRTI indistin-guishable from that caused by other organisms. This makesspecific microbiological investigation very important in thisgroup. A very large range of novel investigations is available,and the clinician requires an understanding of the relativestrengths and weaknesses of these tests for the identifica-tion of each pathogen when investigating LRTI. This reviewprovides recommendations to help navigate the quagmireof available tests and to apply them in a way which ensuresthey clarify rather than further obscure the diagnosis.

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Educational questions

Answer true or false to the following questions:

1. With regard to the diagnosis of Mycoplasma pneu-moniae infection:

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nosis. Lancet Infect Dis 2006; 6: 529–535.

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review. J Perinatol 1998; 18: 287–290.

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in a children’s hospital. Pediatr Infect Dis J 2000; 19: 228–234.

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cavitating pulmonary nodules in a 7-month-old infant. AJR Am J

Roentgenol 1993; 161: 367–368.

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Orenes MC, Segovia M. Sensitivity of urinary antigen test in relation to

clinical severity in a large outbreak of Legionella pneumonia in Spain.

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Finegold SM. Immunologic diagnosis of Legionnaires’ disease: cross-

reactions with anaerobic and microaerophilic organisms and infections

caused by them. J Infect Dis 1980; 141: 652–655.

24. Halperin SA, Wang EE, Law B et al. Epidemiological features of

pertussis in hospitalized patients in Canada, 1991–1997: report of

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Dis 1999; 28: 1238–1243.

25. Cherry JD, Heininger U. Pertussis and other Bordetella infections. In:

Feigin RD, Cherry JD, eds: Textbook of Pediatric Infectious Diseases.

Philadelphia: Saunders, 1998; pp. 1423–1440.

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French hospitals in 1993 and 1994: thirty years after a routine use of

vaccination. Pediatr Infect Dis J 1998; 17: 412–418.

27. Munoz FM. Pertussis in infants, children, and adolescents: diagnosis,

treatment, and prevention. Semin Pediatr Infect Dis 2006; 17: 14–19.

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30. Hallander HO. Microbiological and serological diagnosis of pertussis.

Clin Infect Dis 1999; 28(Suppl 2): S99–S106.

a. M. pneumoniae usually causes lower respiratorytract infection in children.

b. M. pneumoniae PCR is rarely positive in asympto-matic children in the community.

c. M. pneumoniae PCR is frequently positive inasymptomatic close contacts of patients withMycoplasma disease and for some weeks aftersymptomatic infection.

d. M. pneumoniae serology is positive in >20%of children without evidence of Mycoplasma dis-ease.

e. M. pneumoniae culture can be easily and rapidlyperformed in most microbiology laboratories.

2. With regard to the diagnosis of Chlamydophila infec-tion:a. Microimmunofluorescence is a sensitive and spe-

cific test for C. pneumoniae infection in children.b. There is no reliable sensitive and specific test

currently available for the diagnosis of C. pneumo-niae infection.

220 J. WOLF, AND A. J. DALEY

c. There is no adequate randomized controlled trialevidence that specific treatment of C. pneumoniaeinfection causes improved outcome.

d. Chlamydophila psittaci is most frequently spreadfrom person to person.

e. C. psittaci infection can be acquired from aeroso-lization of infected dead birds with a lawnmower.

3. With regard to the diagnosis of Legionella infection:a. Legionella frequently causes infection in children.

b. Although it is more common in immunocompro-mised hosts, most Legionella infections occur inimmunocompetent hosts.

c. Legionella urinary antigen is a very specific test forinfection with this organism.

d. Legionella longbeachae is the most common infect-ing organism.

e. Legionella is best isolated on buffered charcoalyeast extract agar.