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Serum Based Antigen Test Detects Childhood Tuberculosis

By LabMedica International staff writers
Posted on 01 Jun 2021
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Image: Computer-generated Mycobacterium tuberculosis bacteria, Ziehl-Neelsen stain. The reagents used are carbol fuchsin, acid alcohol, and methylene blue counterstain Acid-fast bacilli stain red and the background is blue (Photo courtesy of  microbiologyinpictures)
Image: Computer-generated Mycobacterium tuberculosis bacteria, Ziehl-Neelsen stain. The reagents used are carbol fuchsin, acid alcohol, and methylene blue counterstain Acid-fast bacilli stain red and the background is blue (Photo courtesy of microbiologyinpictures)
Approximately one million children develop tuberculosis (TB) and 205,000 die of TB-related causes each year. Eighty percent of these deaths occur in children < 5 years old, with the majority (96%) of deaths occurring among children who did not receive treatment, where missed diagnoses are likely responsible for undertreatment.

Children with TB, particularly infants, frequently have paucibacillary TB, exhibit non-specific symptoms, and are likely to rapidly progress to disseminated or extrapulmonary TB in the absence of appropriate treatment. This clinical presentation, combined with difficulty obtaining respiratory samples, makes it challenging to diagnose pediatric TB and monitor treatment responses using standard sputum-based methods.

A multidisciplinary team of medical scientists led by Tulane University School of Medicine (New Orleans, LA, USA) used a small blood sample that can be easily obtained from children of any age to detect a specific protein (CFP-10) that the bacteria secrete to maintain the infection that develops into TB. Since this protein is present at very low levels in the blood, the assay uses an antibody specific for this protein to enrich it from other proteins in blood and a mass spectrometer to detect it with high sensitivity and accuracy.

The team used this test to screen stored blood samples collected from 284 HIV-infected and 235 children without the virus who participated in a large clinical trial conducted from 2004 to 2008. Serum CFP-10pep was analyzed using a nanoparticle-based immunoenrichment assay read by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) (Bruker Microflex LRF, Bremen, Germany) that detects Mtb-specific CFP-10pep from trypsin-digested serum or EDTA plasma samples.

The group found their test identified children diagnosed with TB by the current gold-standard TB tests with 100% accuracy. The assay also detected 83.7% of TB cases that were missed by these tests, but that were later diagnosed by a standard checklist employing an array of other information collected by each child's physician (unconfirmed TB cases). The test also detected CFP-10 in 77% of the blood samples that were collected 24 weeks before children were diagnosed with TB by other methods, indicating its strong potential for early TB diagnosis. The biomarker from some positive cases can be detected as early as 60 weeks before their TB diseases were confirmed.

Tony Y. Hu, PhD, a Professor of Biochemistry and Molecular Biology and senior author of the study, said, “This is a breakthrough for infants with tuberculosis because we don't have this kind of screening technology to catch early infections among those youngest groups who are most likely to be undiagnosed. I hope this method can be pushed forward quickly to reach these children as early as possible.”

The authors concluded that their results suggest that serum CFP-10pep signal could improve TB diagnosis in children, as it exceeds the WHO-specified sensitivity requirements for new non-sputum diagnostics, and exhibits similar performance for all TB manifestations, including culture-negative TB, HIV/TB co-infection, and extrapulmonary TB, which are normally challenging to diagnose. The study was published on May 18, 2021 in the journal BMC Medicine.

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Tulane University School of Medicine

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