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Molecular diagnosis of childhood tuberculosis and infection with Bacilli-Calmette-Guerin in Taiwan

  • Ruwen Jou

      Affiliations

    • Reference Laboratory of Mycobacteriology, Research and Diagnostic Center, Centers for Disease Control, Department of Health, Taipei, Taiwan
    • Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
    • Corresponding Author InformationCorresponding author. Reference Laboratory of Mycobacteriology, Research and Diagnostic Center, Centers for Disease Control, Department of Health, 161 Kun-Yang Street, Nan-Kang, Taipei 115, Taiwan.
    • ,
  • Wei-Lun Huang

      Affiliations

    • Reference Laboratory of Mycobacteriology, Research and Diagnostic Center, Centers for Disease Control, Department of Health, Taipei, Taiwan

Received 8 November 2011; received in revised form 10 November 2011; accepted 10 November 2011. published online 20 January 2012.
Corrected Proof

Article Outline

Molecular techniques along with clinical evaluation have been demonstrated to be effective for differentiating childhood tuberculosis (TB) and for establishing an enhanced survey of adverse reactions of Bacilli Calmette-Guerin vaccination in Taiwan. Future development and evaluation of new diagnostics should be prioritized to help strengthen the management of childhood TB.

Keywords: Bacilli-Calmette-Guerin, BCG infection, pediatrics, tuberculosis, vaccination

 

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Introduction 

Tuberculosis (TB) causes 9 million cases annually, of which at least 15%–20% cases occur in children.1, 2 In 2010, the TB incidence rate was 58 per 1,000,000 populations and the absolute number of new TB cases was 13,237 in Taiwan. Of the 13,237 new cases, 0.6% of cases involved children (0–14 years).3 Clinical presentations of childhood TB are different from adult cases. They tend to have a shorter and more frequent transition from primary infection to obvious disease and a higher proportion of cases develop noncavitary pulmonary TB or extrapulmonary TB. It is assumed that most children respond well to anti-TB treatment and have fewer adverse events.4 Pediatric TB cases do not contribute to immediate transmission, however, so they may contribute to a future epidemic. Detection of childhood TB is based on epidemiologic, clinical, and radiologic findings. Due to difficulties in obtaining microbiologic diagnosis and less infectiousness, children generally receive lower priority in TB control programs. Bacilli-Calmette-Guerin (BCG) immunization is recommended to prevent serious miliary TB and disseminated TB in children.5 In Taiwan, neonatal BCG vaccination has been included in the National Immunization Program since 1965.

Accurate diagnosis of childhood TB was hindered by improper specimen and inadequate diagnostics. In general, only a few children cases are confirmed using standard bacteriologic laboratory tests since children are less likely to expectorate, and the bacterial load is lower in young children. To overcome the obstacle of laboratory diagnosis, the reference laboratory of mycobacteriology at the Taiwan Centers for Disease Control (Taiwan CDC) has set up several streamlined algorithms using various molecular techniques for diagnosing suspected childhood TB cases and for confirming BCG-related adverse effects. Nevertheless, conventional bacteriologic tests include acid-fast-stain microscopy, cultivation, nonmolecular-based identification and drug susceptibility testing (DST) have been suggested. Taiwan CDC implemented policies regarding childhood TB cases without lung involvement in children younger than 5 years of age since 2008, and children older than 15 years of age from 2010 to 2011 are required to submit their specimens for further identification.

Types of specimens for bacteriologic diagnosis include expectorated sputum, induced sputum, nasopharyngeal aspirate, gastric washing, stool, string test, lymph node aspiration, pus, cerebrospinal fluid, bone, and biopsy samples, among others. Various molecular diagnostics including real-time polymerase chain reaction (PCR), multiplex PCR, line-probe assays, genotyping, and gene sequencing have been applied in our routine services. A real-time PCR was designed using an IS6110 probe for Mycobacterium tuberculosis complex detection.6, 7 Indeterminate real-time PCR results are further confirmed by IS6110-nested PCR.8, 9 Multiplex PCR was performed for differentiating M tuberculosis complex, M bovis, and M bovis-BCG, and PCR products are then further analyzed using sequencing.10 Two line-probe assay kits (Hain Lifescience GmbH, Nehren, Germany) are used according to the manufacturer’s suggestions: (1) GenoType MTBC (Nehren, Germany, Hain Lifescience GmbH) for differentiating BCG from other M tuberculosis complex species; (2) GenoType MTBDRplus (Nehren, Germany, Hain Lifescience GmbH) for detecting M tuberculosis complex and its rifampicin and isoniazid resistance.11 Besides, genotyping method, mainly spoligotyping, is adopted for differentiating M bovis, M bovis-BCG, and M tuberculosis complex.12 DNA sequencing of 16S13 or pncA gene14 can be used for mycobacteria identification and M bovis family differentiation, respectively.

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Diagnosis 

Children with symptoms suggestive of pulmonary TB cases and those with no symptoms but with identifiable TB contact and with normal immune status are suggested to have rapid TB diagnosis and/or genotyping for identifying infectious sources. In 2007, we confirmed a mother-infant transmission case according to matched genotyping results of the mother’s M tuberculosis isolate and an intestinal specimen of the 90-day-old infants.15 In 2009, the Taiwan CDC implemented a rapid diagnosis policy on multidrug-resistant TB (MDR-TB) cases using the GenoType MTBDRplus assay directly on clinical sputa of high risk populations, such as retreated cases (relapse, treatment after failure, and default) and contacts of MDR-TB cases. For example, we timely identified a family MDR-TB cluster within 3 working days, and two young girls were able to be promptly cared for in our MDR-TB treatment consortium without waiting for results of lengthy bacterial culture and DST.

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Survey of BCG adverse reactions 

We have established the laboratory diagnosis program for identifying of BCG adverse effects since 2003. During 2005–2007, we received 19 clinical specimens from 19 childhood TB cases and found 15 (78.9%) cases were infected with M bovis-BCG.16 The estimated incidence of BCG osteitis/osteomyelitis was 12.9 per million vaccinations in Taiwan during 2005–2007 and that of world was 1 in 700.17 In 2008, the Taiwan CDC initiated a laboratory-based comprehensive BCG adverse events following an immunization surveillance program to monitor local adverse events and severe complications. During 2008–2009, 53 childhood TB cases without lung involvement were notified, and specimens of 41 (77.4%) cases were submitted for differential diagnosis. Of the 41 cases, 24 (58.5%) were infected M bovis-BCG (Ruwen Jou, 2011). Therefore, contact investigation of the index TB case could be halted if rapid bacteriologic clarification was applied. Nevertheless, the causes and risk factors of adverse events are merited to further identification.

Rapid and differential diagnosis of childhood TB using modern molecular techniques is crucial for prompt treatment and appropriate management. Nevertheless, specimen collection remains a major obstacle in the diagnosis of childhood TB. Improved sputum and multiple anatomic site specimen collection can improve sensitivity and shorten the time of diagnosis.18 Advances in the diagnosis of TB have included molecular methods including in-house PCR, integrated real-time PCR, sequencing, and genotyping. Along with clinical evaluation, we have successfully employed molecular techniques for differentiating childhood TB and for establishing an enhanced survey of adverse reactions of BCG. The development and evaluation of new diagnostics should be prioritized in strengthening laboratory diagnosis of childhood TB.

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References 

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PII: S0929-6646(11)00166-5

doi:10.1016/j.jfma.2011.11.021

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