Pediatric Pulmonology, Supplement 26:240–242 (2004)

Tuberculosis: Role of Etiologic Diagnosisand Tuberculin Skin Test

Luı́sa Pereira, MD

Tuberculosis is an infectious disease caused by Myco-bacterium tuberculosis. As any other infectious disease itshould be diagnosed accurately, and for this purpose thegold standard is agent identification, generally on cultures.Unhappily this is not always easy for several reasons andwe tend to rely on presumptive diagnosis which can lead towrong diagnosis. On the top of this, when identification ofthe responsible agent is possible, it takes a long time onclassical methods (around 6 weeks on culture).(1,2,3) Inclinical practice it is not suitable to wait for these resultsfor starting treatment, if indicated.

When treating children this problem is even morepronounced, the isolation of the organism being evenmore difficult. In typical childhood tuberculosis tuberclebacilli in endobronchial secretions are relatively sparse,and children, lacking the tussive force of adults areunable to produce adequate sputum samples for bacter-iological exams. (1,4) The necessity of agent identifica-tion, always present to avoid misdiagnosis, become evengreater during the last years, with the increasing presenceof resistant strains. Resistance can obviously be studiedonly if we are able to grow the bacteria involved. On theother hand, in all patients but even more in children, wemust start treatment the sooner as possible, this means onan early phase when isolation and identification of micro-organisms is impossible. At this point we have to rely onthe tuberculin skin test evidence of tuberculous infection.The tuberculin skin test is an evidence of infection, whichcan be a precious help on dealing with tuberculousinfection if well made and interpreted.

ETIOLOGIC DIAGNOSIS - MYCOBACTERIUMTUBERCULOSIS IDENTIFICATION

The first step for organism identification, specimencollection, is not the easiest one in paediatric patients.When considering pulmonary tuberculosis, the most com-mon form of disease, whereas sputum is an easy obtain-able and useful specimen for processing in adults, this isnot the case in children. Children have a relatively lowbacillary load in bronchial secretions, rarely producesputum and lack the tussive force necessary to provide anadequate specimen. (1,4)

Aspiration of early-morning gastric fluid with anasogastric tube, first thing in the morning before thechild has eaten or even awakened enough to start

swallowing saliva and stimulate gastric motility, providesa good specimen of pulmonary secretions. That way wecan pick secretions which have been swallowed duringthe night, after being carried by the respiratory cilia to thelarge airways. Given these requirements, collection isbest achieved in hospital. Gastric washing obtained mustbe processed in 15 minutes or frozen. Collection of speci-mens for 3 consecutive mornings is recommended toimprove sensitivity. (1,4,5,6)

Bronchoalveolar lavage is another option for obtainingspecimens, and if a bronchoscopy is to be performed forclinical reasons, lavage should be performed in order toobtain samples for staining and culture. It has similarsensitivity to gastric aspiration (1), or in some studieseven lower. (4) Cerebrospinal fluid, pleural fluid, lymphnode aspirate (obtained by fine needle aspirate or exci-sional biopsy) and urine are other specimen to be studiedaccording to clinic. (1,4,5)

After collection of the specimen, staining (generally byZiehl-Neelson procedure) followed by microscopicobservation is performed. If the organism is present insufficient number, what is the exception in children, thisis the most rapid procedure for the detection of my-cobacteria (at least 5� 103 to 105 organisms/ml arerequired for detection). (1,3,4)

Identification by staining does not provide informationon identification or viability of the organism - all speciesof mycobacteria are acid-fast. Fluorescent stainingmethod offers the advantage of screening a larger numberof slides in less time. (1,2,4)

A culture positive for M.tuberculosis from any site isdiagnostic of tuberculosis and is easier to obtain; forcultures only 10 to 100 bacilli are needed for a positiveresult. (1,2, 3, 4) Children with primary disease have lownumbers of micro-organisms. Around 95% will havenegative smears and about 60% will have negativecultures even with active disease. Infants with pulmonarydisease have around 70% positive cultures, a value

From the Respiratory Unit, Pediatric Department, Hospital de Santa Maria,

Odivelas, Portugal

Address correspondance and reprint requests to Dr. Luisa Pereira, From the

Respiratory Unit, Pediatric Department, Hospital de Santa Maria, Rua

Ricardo Reis, 1-38B, 2675-239Odivelas, Portugal,E-mail: luisa.pereira@hsm.min-saude.pt

� 2004 Wiley-Liss, Inc.DOI 10.1002/ppul.70117

significantly higher than the 40% found in older children.(1) Adolescents may have reactivation tuberculosis,which is clinically like adult disease, and may be ableto provide adequate sputum specimens for culture. (1,4)In the United States, between 1985 and 1988, 90% oftuberculosis cases in adults were confirmed by culture,whereas only 28% of children had positive cultures. (1)

Traditional mycobacterial culture media include egg-based media and agar based media. The most commonlyused is Lowenstein-Jensen media, an egg based media. Itshould be incubated for 4 to 8 weeks for results, and anadditional 4 to 8 weeks may be necessary for sensitivityplates to grow. (1,2,3)

Newlaboratorymethodshave improvedandhastened thediagnosis of tuberculosis. Radiometric or colorimetricsystems like BACTEC allow for rapid grow within 1 to3 weeks; antibiotic susceptibility testing can be performedfrom this system. For this purpose specimens must beculturedinbrothmedia(liquidMidlebrook7H12containing14C-labeled palmitic acid) for the radiometric detection ofgrowth. Traditional solidmedia should be set up also so thatcolony morphology can be examined. (1,2,3)

Once organisms are detected on culture, DNA probescan be used to hasten diagnosis. The technique cannot beused directly on clinical specimens. The most widelyused methods are nucleic acid hybridization and highperformance liquid chromatography. The procedure isquick, and when in combination with BACTEC culturesmay shorten diagnosis up to 15 to 20 days. (1,2,3)

Another good help for prompt diagnosis comes fromnucleic acid amplification techniques, such as the PCR.Those techniques rapidly replicate specific target sequen-ces of DNA or RNA, which can then be identified byprobe. This can be done directly on clinical specimensand results are generally available within 1 to 2 days.Very few organisms are need for identification (researchlaboratories have reported positive results with only10 bacilli), but some organisms must be present. Studiesin children report the sensitivity of PCR to be about 40%when directly on clinical specimens, and 60% when PCRand culture are combined. (1,2,3)

HISTOLOGY

Histologic and cytolgic studies can also be a valuableaid to diagnosis, specially in tuberculous lymphadenitis,where fine needle aspiration both for pathology andcultures permits cytologic diagnosis in a few hours, andin pleural, pericardial or peritoneal tuberculosis wherepathology is the main form of diagnosis. (4)

TUBERCULIN SKIN TEST

In paediatric patients we have, quite often, to rely ontuberculin skin test results for starting treatment, whilewaiting for cultures. It is also the major method of

diagnosing tuberculous infection besides being a goodhelp when evaluating tuberculous disease on children.Ninety percent of healthy individuals infected withMycobacterium tuberculosis will develop delayed typehypersensitivity within 3 weeks to 3 months. (1) TheMantoux skin test is the standard method for tuberculinskin testing. Tuberculin is composed primarily of tuber-culoprotein obtained from cultures of the tuberclebacillus. An individual infected with M. tuberculosis,will develop sensitivity to these antigens and then, whenthe material is injected intracutaneously, a classic delayedhypersensitivity reaction occurs.(1,7)In order to obtain reliable results, when employing and

interpreting this very useful test, it is necessary to payattention to 2 orders of things: 1- the correct technique forperforming and reading the test, so that the result ob-tained (a value in millimetres) is the right one. 2 - acorrect interpretation of the value found, considering thevaccinal status, eventual underlying diseases and severityof the disease.The test is performed by injecting 0.1 ml of PPD

tuberculin (purified protein derivative) - 5 TU (tuberculinunit) into the skin of the forearm, transition from the volarto the dorsal area. The injection must be intracutaneous,and done with the needle bevel upward. A single-doseplastic syringe is used with a 26 gauge needle. Properdosage is important, weaker doses can produce smallerreactions. If there is any problem while performing thetest, i.e. the child moves and the technique is incorrect, thetest should be repeated at once in the opposite forearm.The test is read in 72 hours by an experienced health careworker. The amount of transverse induration, not erythemais measured by the ballpoint pen technique, in which linesare drawn on the skin toward the site and stopped atthe point of resistance on either side of the site, then thedistance is measured between the ends of the lines. For thispurpose it is better to use a plastic transparent ruler.Results should be recorded in millimetres.(1,5,7)After obtaining the result of the Mantoux test, this one

must be interpreted: - values of 5 or more millimetresinduration are considered significant if the patient hadn’thad BCG vaccine. On vaccinated individuals, values of10 mm or more are considered. Any degree of indurationmay be significant in immunocompromised patients. Thecomplete evaluation and decision to treat should be basedalso on clinical features and contact history; some authorsconsider that values higher than 15 mm should always beconsidered of significance. (1,5,7,8,9,10,11)False-negative reactions may occur because of inaccu-

rate administration or interpretation, recent tuberculosisinfection, anergy, recent viral infection or live-virusvaccination, immunosupressive therapeutics includingcorticosteroids, or severe forms of disease.(1,5,7)When considering the diagnosis of tuberculosis on a

child, we must choose the diagnostic methods that are

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more likely to be useful on that particular case, we mustcollect and process all the specimen available and exhau-stively try to obtain an etiologic confirmation of thediagnosis. That will also allow us to study drug resis-tance. Any way, in my personal experience, we willhardly arrive to 50% micro-organism isolation in child-hood tuberculosis.

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