Chapter 3 Overview of study cohort and methods
3.6 Study measures
Clinical and laboratory investigations were completed in a rigorous standard manner using SOPs. All investigations and measures were undertaken by trained research personnel.
Table 3.1. Schedule of investigations in all children enrolled
Enrolment Month
1 Month 2
History (including evaluation and
quantification of TB exposure) x x x
Clinical examination, including
anthropometry x x x
Tuberculin Skin Test (Mantoux) x
CXR (antero-posterior and lateral) x x
HIV test x
Respiratory specimens (smear, Xpert and
culture*) x x x
Stool x
Evaluation of TB treatment adherence x x
Urine (biorepository) x x x
Serum§ (biorepository) x x
* 2-4 specimens of at least 2 different types collected over 2 days at enrolment in all children; at follow-up, 2 specimens of 2 types were collected only in TB cases (clinically diagnosed or confirmed); §Collection of blood at the 2-month visit was only completed for TB cases;
CXR: chest X-ray.
Visit windows: The baseline visit was defined as the date of starting
antituberculosis treatment in TB cases, and the date of the enrolment visit for children not diagnosed with TB. For TB cases, a visit up to 14 days before TB treatment initiation was considered to be the baseline visit, in cases where the decision to start antituberculosis treatment may have been delayed. The month 1 visit was allowed a window 15 to 42 days after the baseline visit. The month 2 visit was allowed a window of 43 to 120 days after the baseline visit. Different methods were used to trace participants for follow-up visits (See Appendix B: SOP 9).
Signs and symptoms were systematically recorded. Details on the duration
and severity of symptoms, TB exposure history, HIV exposure and disease were collected. M.tb exposure was ascertained from caregivers using a standard data
instrument to determine the extent of contact between the child and the TB index case in the child’s household, or a non-household member in close contact with the child, treated for active TB within the preceding 12 months 74. The sputum status
(smear or culture positive), drug susceptibility pattern and the type of disease (pulmonary vs. extrapulmonary) of any adult TB source case was recorded if known (See Appendix D: SDOC25).
Symptoms were classified as follows:
a) Well-defined symptoms of PTB in children:
− Prolonged unremitting cough: daily cough reported for ≥14 days. − Prolonged fever: fever ≥7 days, not responsive to antibiotic treatment − Prolonged poor feeding in infants or poor appetite in older children for
≥14 days
− Unexplained lethargy/ lack of playfulness: child unusually tired/sleepy, not engaging in usual daily activities and play
− Failure to thrive: clear deviation from the child’s previous growth trajectory (evident in the child’s growth card) or static growth or weight loss in the preceding 3 months; alternatively, WFAZ ≤-2 in children with no previous weight measurements
b) Additional symptoms (not included as the “well-defined” symptoms in the literature and NIH case definitions), but reported on in this analysis:
− Prolonged wheeze: wheeze reported for ≥14 days. The presence/absence of wheeze was only documented in 352 children (the case report form did not include wheeze as a symptom initially)
Anthropometry was assessed at each study visit (See Appendix B: SOP 11).
Measurements included weight, recumbent length (infants) or height and mid-upper arm circumference using calibrated equipment. UK Z-score charts were used as reference, as they include WFAZ for all ages 176.
HIV infection and measures of immune function.
The provincial policy for early neonatal testing of HIV-exposed infants: From 2012 to 2015, the 2010 National ART Guidelines were followed 177, which
recommended all HIV-exposed infants to have an HIV DNA PCR test at 6 weeks of age, or earlier in case of severe illness or symptoms suggestive of HIV infection. A confirmatory viral load (VL) test was completed on all HIV PCR-positive infants, with fast-tracking to start ART if HIV infection was confirmed. All breastfed infants testing HIV-negative at 6 weeks received an age-appropriate HIV test (see HIV-testing below) 6 weeks or more after cessation of breastfeeding or if clinically indicated during breastfeeding. In 2015, guidelines were updated 178 to recommend HIV PCR testing at
birth for all HIV-exposed newborns, with a second confirmatory PCR for infants who tested positive. VL testing as a confirmatory test was discontinued due to the successful implementation of the B+ Prevention of Mother-to-Child Transmission (PMTCT) program, which resulted in undetectable VL even in infected infants due to maternal and neonatal ART. An HIV PCR test was recommended again at 18 weeks for all HIV-negative infants receiving postnatal nevirapine to 12 weeks of age.
HIV testing for children enrolled in the study: In children not documented to be HIV-infected, an HIV DNA PCR test (Roche Cobas AmpliPrep/Cobas TaqMan) was completed at the virology laboratory, National Health Laboratory System (NHLS), TBH, in children <18 months of age, and an HIV ELISA antibody test (Roche HIV Combi PT Elecsys Cobas e100) for children ≥18 months. If phlebotomy was insufficient, a rapid HIV test was performed, followed by a confirmatory test if positive. HIV medical records were abstracted in children with known HIV-positive status. In HIV-positive children with no CD4 count or VL, both assays were completed. WHO clinical and immunological staging was determined.
HIV status was defined as follows:
− HIV- uninfected: a child not exposed to HIV perinatally and testing negative on a confirmatory test (see HIV testing above).
− HIV-exposed uninfected: a child exposed to HIV perinatally and testing negative on a confirmatory test
− HIV-infected: a child confirmed to be HIV-infected on a confirmatory test. Not all children had confirmed perinatal HIV exposure, mostly due to absent records in older children and maternal demise.
HIV-infected children who were not receiving ART at the time of enrolment were referred to the Infectious Disease service at TBH or KBH (depending on the recruiting hospital) for expedited ART initiation and HIV care.
HIV treatment. During the course of the study, ART guidelines for children
changed. Between 2012 and 2015, the 2010 South African National ART guidelines were followed 177. These recommended ART to all HIV-infected children <1 year of
age (not restricted by clinical stage or CD4 count); children 1-5 years with WHO clinical stage 3 or 4, or CD4 count ≤ 25% or ≤750cells/µl; and children> 5 years with WHO clinical stage 3 or 4 or CD4 count ≤350 cells/µl. Recommended first-line ART regimen for children <3 years of age or <10kg body weight was abacavir, lamivudine and lopinavir/ ritonavir; and abacavir, lamivudine and efavirenz for older children weighing >10 kg. Second-line treatment for efavirenz-based regimens was zidovudine, didanosine and lopinavir/ ritonavir; while referral to a specialist centre was recommended in case of failed lopinavir/based regimen. In 2015 these guidelines were updated 178 to expand eligibility to ART for all children <5 years of
age regardless of clinical stage or HIV count, and to children 5-15 years of age with WHO clinical stage 3 or 4 or CD4 <500 cells/µl. The first-line ART regimen was changed for children >3 years of age previously exposed to perinatal nevirapine, where lopinavir/ ritonavir replaced efavirenz as the third drug. The second-line regimen after 2015 was zidovudine, lamivudine and lopinavir/ ritonavir for failed efavirenz or nevirapine-based regimen, while a regimen based on genotypic resistance-testing was recommended for failed lopinavir/ritonavir-based regimen. Both guidelines recommended ritonavir boosting of lopinavir/ritonavir-based regimens if a rifampicin-based antituberculosis treatment was co-administered.
TST: (Mantoux, 2 Tuberculin Units of PPD RT-23, Statens Serum Institute,
Copenhagen). TST was injected intradermally in the volar aspect of the left forearm and read 48-72 hours after placement. A TST reaction was considered positive if the wheal measured ≥10mm if HIV-negative and BCG-vaccinated, ≥5mm if HIV-positive or not BCG vaccinated. Evidence of BCG vaccination was determined by written
record in the immunization card or evidence of BCG scar in the right deltoid area (see Appendix B: SOP 7).
Close TB exposure: Exposure to any identified adult TB source case in the
preceding 12 months, where exposure was either within the household; or involved the child’s primary caregiver; or occurred for >4 hours per day during the period of exposure.
Chest radiographs
Antero-posterior and lateral films were completed at enrolment and at the month 2 visit, unless clinically indicated before the 2 month follow-up. Films (digital or hard copy) were independently interpreted by two of three paediatric TB experts, blinded to all patient clinical and laboratory data and to the other readers’ reports, using a standard form for recording and reporting 91,179 (See Appendix B: SOP 8).
Radiographs were considered ‘unreadable’ if one reader classified the quality as technically unacceptable.
a) Radiological diagnostic certainty of TB and observed disease spectrum: If the quality of the film was acceptable, each reader documented any key radiological features and classified the film according to radiological diagnostic certainty of PTB disease. The following features were classified as being ‘typical of TB’: presence of soft tissue shadowing suggestive of perihilar or paratracheal lymphadenopathy, airway compression or deviation, expansile pneumonia, a Ghon focus, pleural effusion, miliary infiltrates and cavities (excluding bronchiectasis). The following features, reported without any of the ‘typical’ features described above, were classified as being ‘not typical of TB’: alveolar consolidation/collapse (lobar,
segmental or bronchopneumonic), interstitial infiltrates (including perihilar infiltrates), generalised hyperinflation and bronchiectasis.
After independent dual reading of the CXRs, consensus criteria were applied to classify each film as ‘normal’, ‘abnormal, typical of TB’ or ‘abnormal, not typical of TB’. Where agreement on diagnostic certainty was not reached, a third reader reviewed the film; consensus between 2 of the 3 readers was used to reach a final classification. For reporting patterns of disease, only patterns for which there was complete agreement were reported.
b) Radiological disease severity:
Each CXR was subsequently assessed for radiological severity of disease, using a pragmatic modification of a published classification 99. Radiographs were classified
as reflecting severe disease if there was perfect consensus between 2/2 or 2/3 readers of 1) complicated PTB disease, 2) extensive parenchymal involvement or 3) evidence of dissemination (miliary pattern).
For CXRs typical of TB, severity was defined as either extensive, complicated or disseminated disease:
− Complicated TB was defined as airway compression, expansile pneumonia or cavitating disease (including adult-type PTB disease, cavitating Ghon focus or TB bronchopneumonia).
− Extensive disease was defined as bilateral parenchymal involvement (alveolar consolidation), or involvement of >2 zones of the lung or a total lung surface area considered greater than the surface area of the right upper lobe.
For CXRs not typical of TB (TB cases or symptomatic controls – reviewers were blinded to TB disease status), the extent of parenchymal disease (therefore excluding interstitial patterns and hyperinflation) was documented as it was done for “typical TB” (see above) and complicated disease was defined as the presence of non-TB cavities (e.g. classified as being due to bronchiectasis).
Additional imaging and other investigations: results, if routinely
completed as part of clinical care (e.g. computed tomography of the chest or brain, abdominal ultrasound and bronchoscopy), were systematically recorded and were incorporated into the final classification to describe the full spectrum of disease. The spectrum of TB disease was recorded based on collated imaging, laboratory and clinical data.
Respiratory specimen collection (See Appendix B: SOP 1-3). The study had
distinct phases during which different diagnostic strategies were evaluated (See Table 3.2). At a minimum, the specimen collection schedule required one specimen each of GA (in children <5 years of age unable to expectorate spontaneously) or ESP (older children) and IS (with or without nasopharyngeal suctioning depending on the child’s age), collected daily for two consecutive days.
NPAs were introduced in August 2013 (from participant number 101 onwards), in order to evaluate the diagnostic utility of this potentially less invasive specimen type.
If any of the respiratory specimens required by the study had already been collected by the hospital personnel, these were not collected again by the research team, and their bacteriological results were documented. Some children also had
FNAB of peripheral lymph nodes and BAL, collected by the hospital personnel if clinically indicated.
SOPs were followed for all study measures. Specifically, minimum volume requirements for respiratory specimens were prescribed: 5mL for GA, 2mL for ESP, and 1mL for IS and NPA. If greater volumes could be collected, this was encouraged. Specimens were not discarded if the minimum volume could not be achieved.
Table 3.2. Schedule of collection of respiratory specimens Enrolment Day Day
1 2
M1 M2
Younger children (< 5 years) unable to expectorate sputum
GA for Smear, Xpert, MGIT x x x
IS for Smear, Xpert, MGIT x x x
NPA for Smear, Xpert, MGIT (for study patients 101 to 540) x x x Respiratory specimens pooled for Smear, Xpert, MGIT (for study
participants 234 to 621) 1. GA
2. NPA
3. Sputum induction then suction
x
Older children able to expectorate sputum
Expectorated Sputum early morning after long fast (4hr min) for
Smear, Xpert, MGIT x x
Expectorated Sputum spot (2h fast) for Smear, Xpert, MGIT x x x Induced Sputum (IS) for Smear, Xpert, MGIT x x x Respiratory specimens pooled for Smear, Xpert, MGIT
1. Expectorated Sputum early morning 2. Expectorated Sputum spot
3. Sputum induction then expectorated sputum (no suction used)
x
M1: month 1; M2: month 2; GA: gastric aspirate; IS: induced sputum; NPA: nasopharyngeal aspirate; MGIT: Mycobacteria Growth Indicator Tube liquid culture
Stool collection. Stool was collected within 7 days of enrolment. Caregivers
were given verbal and written instructions on how to collect stool (Appendix B: SOP 4).
Specimen handling and transport: After collection, GA specimens were
titrated to neutral pH at the time of collection by the study nurse, using 4% sodium bicarbonate solution and gradated pH strips 180. All respiratory specimens were kept
refrigerated and transported to the laboratory in a cool box within 4 hours of collection. Stool specimens collected at home from children discharged from hospital and stools that could not be processed immediately were stored at 2-8°C for maximum 72 hours before processing.
Laboratory processing of respiratory specimens: Respiratory specimens
were processed at the NHLS Microbiology Laboratory at TBH according to national standard laboratory procedures (See Appendix B: SOP 13). For digestion/ decontamination, NALC-NaOH was used (final NaOH concentration=1.25%), before concentrated fluorescent Auramine-O smear microscopy 128, Xpert and liquid MGIT
(Becton Dickinson, Sparks, MD, USA) culture 181. The SOP for processing pooled
respiratory specimens for the sub-study described in Chapter 9 is detailed in the relevant paper and is provided in Appendix B (SOP 14). Smears were graded according to the WHO/ International Union Against Tuberculosis and Lung Disease classification. Cultures were incubated for up to 42 days. If no growth was observed, cultures were declared negative. For positive cultures, the TTP in days was noted and Ziehl–Neelsen (ZN) stain was performed on the culture. If ZN positive, mycobacterial identification and drug susceptibility for isoniazid and rifampicin were completed using MTBDRPlus LPA (Hain LifeScience, Nehren, Germany). The cycle threshold (Ct) values for Xpert-positive specimens were recorded, as well as the semi-quantitative results reported by the laboratory. Rifampicin-resistant strains detected by Xpert or
LPA were transported to the NHLS in Green Point, Cape Town, for phenotypic DST for ofloxacin and amikacin using the agar proportion method. If growth of bacteria/fungi was observed on blood agar plates and/or non-acid-fast bacteria were seen on the ZN smear, the MGIT culture was considered contaminated. Contaminated cultures from respiratory specimens were re-decontaminated and re-incubated once only. Invalid or error Xpert results were repeated if sufficient concentrated specimen was available for second testing.
Laboratory processing of stool specimens: Stool specimens were processed
by the study laboratory technician. Different stool processing methods were used in different sub-studies and are detailed in chapters 6, 7 and 8, and in Appendix B in SOPs 4, 15 and 16.
Microbiological confirmation status, for the overall reporting of the cohort:
− Smear positive: a child with at least one respiratory specimen with a positive smear result (including scanty positive). Smear-positivity was not considered a criterion for microbiologically confirmed TB in the absence of a positive Xpert or culture/LPA result.
− Xpert-positive: a child with at least one respiratory specimen with a positive Xpert result.
− Culture-positive: a child with at least one respiratory specimen with a positive culture result, confirmed to be M.tb on LPA.
− Microbiologically negative: a child with all microbiology test results negative (non-positive). Non-positive includes indeterminate/error/invalid results and contaminated cultures.
− Overall microbiologically confirmed TB: M.tb confirmed by Xpert or culture/LPA, from any number of respiratory and non-respiratory specimens. Smear positivity in the absence of Xpert or culture confirmation was not regarded as microbiologically confirmed TB.
Reporting of microbiology results: microbiology results are defined and
reported in detail in the individual diagnostic sub-studies which form part of this dissertation (Chapters 6-9), including all non-valid results (error/invalid Xpert; contaminated cultures; indeterminate DST results). For the description of the clinical cohort (Chapter 4) and the overall microbiology results (Chapter 5), non-valid results were considered non-positive (i.e. analysed as negative) and are not reported separately. The DST results reported in Chapters 4 and 5 are those which were considered as final by the NHLS after investigation and resolution of any potential discrepancies between tests (e.g. between Xpert and LPA rifampicin resistance results), and were used to inform treatment decisions.
Antituberculosis treatment decision: The decision to treat for TB was made
by the attending clinicians, based on clinical/epidemiological assessment and the results of all laboratory investigations and was not based on research case definitions. All results from study samples, including stool, were available to clinicians. Children were typically prescribed treatment at the time of TB diagnosis in hospital for the duration of their hospital stay. Children diagnosed with DS-TB were discharged to the
local clinic to receive ongoing medication according to South African National TB Programme (SA NTP) guidelines 182. Referral to care at the local clinic was reinforced
through a local initiative linking children diagnosed in hospital to the appropriate primary care clinic 183. Children with neuro-tuberculosis and miliary TB were
typically managed at Brooklyn Hospital for Chest Diseases (BCH), a medium- and long-term TB care facility for patients with complicated forms of TB. Some children with stage 1 TB meningitis were managed as outpatients through a dedicated service established for such patients at TBH in close collaboration with local community TB clinics 184.
Antituberculosis treatment regimens
DS-TB: A child receiving only drugs used for DSTB. Excludes children who received a combination of DSTB and DRTB regimens, because the diagnosis changed, and children who received therapy for DSTB and additional preventive therapy for DRTB disease. Routine first-line antituberculosis drugs at standard WHO- recommended doses included rifampicin (15 mg/kg/day), isoniazid (10-15 mg/kg/day) and pyrazinamide (35 mg/kg/day), with ethambutol (20 mg/kg/day) added in cases of severe or HIV-associated TB, or with ethionamide (15-20 mg/kg/day) added in cases of neuro-tuberculosis or miliary TB. The local TB clinic was responsible for administering antituberculosis treatment.
DR-TB: A child receiving any drugs used to treat DR-TB (any form, including isoniazid mono-resistance, rifampicin resistance or with any additional resistance), for any duration of time, was classified as having received a DR-TB regimen. Children diagnosed with DR-TB were typically admitted to BCH, at least initially, to receive