Xpert MTB/RIF Testing of Stool Samples for the Diagnosis of Pulmonary Tuberculosis in Children
Mark P. Nicol
2
3
Karalene Spiers
1
2
3
Lesley Workman
0
2
4
Washiefa Isaacs
0
2
4
Jacinta Munro
0
2
4
Faye Black
0
2
4
Widaad Zemanay
2
3
Heather J. Zar
0
2
4
0
Department of Paediatrics and Child Health, University of Cape Town
1
Division of Infection and Immunity, University College London
,
United Kingdom
2
Study Design, Setting,
and Population This was a prospective study in which samples were obtained from an ongoing cohort based at a primary care clinic (Nolung- ile Clinic
, Khayelitsha,
South Africa
) and a tertiary pediatric hospital (Red Cross Children's Hospital
, Cape Town,
South Africa
). Children (age <15 years) presenting with suspected PTB were enrolled consecutively (from 11 July 2011 to 26 March 2012). Criteria for enrollment were a cough lasting longer than 2 weeks and at least 1 of the following: (1) house- hold tuberculosis contact in the prior 3 months, (2) weight loss or failure to gain weight in the previous 3 months, (3) a positive tuberculin skin test,
or
(4) a chest radiograph suggestive of PTB. Children were evaluated at 3 months to assess recovery or response to treatment. We included all children from whom both stool and sputum samples had been collected and where >1.5 g of stool was available (only 0.15 g was used for testing). Children were excluded if they had received treatment for tu- berculosis lasting longer than 72 hours, they did not live in Cape Town, they were unable to attend follow-up visits, informed consent was not given,
or an IS sample was not obtained. Written informed consent was obtained from a parent or legal guardian. The Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town
, approved the study
3
Division of Medical Microbiology and Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, and National Health Laboratory Service of South Africa
,
South Africa
4
Red Cross War Memorial Children's Hospital
, Cape Town,
South Africa
In a pilot accuracy study, stool Xpert testing from 115 children with suspected pulmonary tuberculosis (PTB) detected 8/17 (47%) culture-confirmed tuberculosis cases, including 4/5 (80%) human immunodeficiency virus (HIV)-infected and 4/12 (33%) HIV-uninfected children. Sputum Xpert detected 11/17 (65%) cases. Stool holds promise for PTB diagnosis in HIV-infected children.
Procedures
Routine history and physical examination were performed at
enrollment. All children received baseline chest radiography
and human immunodeficiency virus (HIV) testing (HIV rapid
test followed by confirmatory polymerase chain reaction for
children age <18 months). Patient stool (a single convenience
specimen) and IS (2 specimens) were collected at baseline as
previously described [6]. IS specimens were processed within 2
hours. Stool specimens were stored at 80C within 2 hours;
Xpert testing was performed within 6 months of storage.
Classification of tuberculosis status was as follows: definite
tuberculosis, children culture-positive for M. tuberculosis; not
tuberculosis, children culture-negative for M. tuberculosis who
were not started on tuberculosis treatment and clinically
improved at the 3 month follow-up visit; and possible
tuberculosis, all other children.
Sputum Processing
Samples were decontaminated with N-acetyl-L-cysteine and
1% sodium hydroxide (final concentration) and then
concentrated by centrifugation. Pellet was resuspended in 1.5 mL
phosphate buffered saline (PBS). BACTEC MGIT (Becton
Dickinson) culture was performed using a 0.5-mL aliquot of
the resuspended sample and Xpert using 0.7 mL of the
resuspended sample, to which 1.4 mL of Xpert reagent was added
and processed per the manufacturers instructions.
Stool Processing
Next, 0.15 g of thawed stool (confirmed by weighing) was
retrieved using pediatric FLOQSwabs (Copan Italia, Brescia, Italy).
Swabs were then placed in 2.4 mL PBS and vortexed briefly
before being removed. The sample was left undisturbed for 20
minutes at room temperature to allow large particles to settle
before 2 aliquots of 1-mL supernatant were removed. One aliquot
was tested immediately with Xpert and the other was stored at
4C for later duplicate testing (within 1 week). Prior to Xpert
testing, the sample was centrifuged at 3200 g for 15 minutes. The
supernatant was discarded and pellet was resuspended in 1 mL
PBS. Xpert testing was then performed per the manufacturers
instructions using a 2:1 ratio of Xpert reagent to sample.
Statistical Analysis
The reference standard was a positive liquid culture from at
least 1 IS sample. Statistical analysis was performed with Stata
version 11.0 (StataCorp, College Station, Texas). Diagnostic test
characteristics were determined with 95% confidence intervals
(CIs). A 2-sample test of proportion was used to compare the
sensitivity of Xpert in HIV-infected and -uninfected children.
Testing was performed on 115 children, median age 31 months
(interquartile range 1957 months) of whom 17 (14.8%) were
HIV infected a (...truncated)