Design and population
This analysis is part of a prospective cohort study conducted between July 2012 and June 2014 at the district level in an HIV-prevalent setting that aimed to evaluate an algorithm including the Xpert MTB/RIF assay to diagnose pulmonary TB (PTB) [23]. The study included adults (≥15 years) with signs and symptoms suggestive of PTB (i.e. presumptive PTB), able to produce sputum, who attended the outpatient TB clinic of Homa Bay County Hospital and were living in the area (maximum 20 km from the hospital). Patients were included consecutively after obtaining written informed consent. Patients who had been treated with fluoroquinolones or anti-tuberculosis drugs in the month prior to the consultation, or who were relocating out of Homa Bay in the near-term following the consultation, were excluded.
Site
The study was conducted at the TB clinic of Homa Bay County Hospital, which is the reference health facility for a county of about 800,000 people. Kenya is a high TB burden country with an incidence of 246/100,000 in 2014; Nyanza Province, where Homa Bay is located, is the area with the highest case load reported in the country [24]. In 2013, overall HIV prevalence among people aged 15–49 years was estimated at 6.0% in the country and 25.7% in Homa Bay [25]. TB and HIV care was offered to the patients free of charge through the support of Ministry of Health and Médecins Sans Frontières.
Procedures
On the first day, patients were screened and those fulfilling the inclusion criteria and having no exclusion criteria were included in the study. Patients included in the study were assessed clinically and requested to produce two sputum samples (one on spot and one early morning on the following day). All samples were processed on the same day of collection or the day after. The spot sample was processed for smear microscopy, Xpert MTB/RIF assay and Mycobacterium tuberculosis complex (MTB) culture. The early morning sample was processed for microscopy and MTB culture. Microscopy and Xpert results were received on the same day or the day after sample collection. Patients with positive microscopy or Xpert were started on treatment. Those with negative results had a chest X-ray performed at the hospital. The study clinical officer on site read and interpreted the films and made a TB treatment decision based on the clinical exam and the chest X-ray. Patients not started on TB treatment were given a broad spectrum antibiotic targeting community-acquired pneumonia and were reassessed clinically after 5 days [26]. During the re-assessment consultation, patients with partial or no clinical response had additional smear microscopy and Xpert assessment on sputum. At this point, the study clinical officer made a TB treatment decision based on the clinical exam and the additional Xpert result. All patients, whether on TB treatment or not, were requested to return at the TB clinic for clinical assessment by the clinical officer 2 and at 6 months after the first consultation. For empirically treated patients, TB treatment was not stopped after receiving a negative culture result.
Patients missing appointments and patients with positive MTB culture result who had not been started on TB treatment were traced by phone and in-person, and asked to return to the clinic. Outcome (alive/dead) at 6 months was assessed through tracing for patients who missed their 6-month consultation appointment. HIV testing with pre- and post-test counselling was proposed to all patients. Patients diagnosed with HIV infection were offered HIV care including clinical follow-up, CD4 monitoring, ART initiation, counselling and other laboratory investigations as necessary.
Following the usual procedures for TB diagnosis in Homa Bay District Hospital, the chest X-rays were interpreted by a clinical officer and not by a medical officer. However, the X-ray films were sent in batches to a radiologist for quality control purposes. The radiologist did not have information about the patient’s symptoms. A tick sheet including pictograms was used for reporting X-ray results by the treating clinical officer and the radiologist.
Sputum samples to be tested with Xpert and culture were centrifuged at 3000 rpm for 15 ± 20 min and decontaminated using 2% NALC-NaOH method. At least 0.5 ml of the sediment was resuspended in a conical tube by adding 1.5 ml of Xpert MTB/RIF sample reagent. The suspension was incubated for 15 min at room temperature before being added to a cartridge and processed. The test was repeated up to two times using a new cartridge in case of an invalid, error or no result. Samples collected for culture were processed on the same day of collection or kept in a fridge until the next day. Culture was performed using 2 methods: Thin Layer Agar (TLA) and Lowenstein-Jensen (LJ). TLA method consists of plates of 7H11 agar-based medium read by conventional microscope [27]. Sputum specimens were decontaminated as described above the re-suspended sediment was inoculated in one TLA plate and in one LJ slant. TLA was incubated at 37 °C in a 5% CO2 incubator and LJ at 37 °C in a standard incubator. Para-nitrobenzoic acid (PNB) was included in the TLA plates for simultaneous non-tuberculous mycobacteria (NTM) detection while the antigen test MPT64 was used for the identification of MTB growth in LJ. Final culture results were reported by the laboratory as positive if any of the 4 culture results was positive, negative if at least 2 results were negative and as contaminated if at least 3 results were contaminated. Negative culture patients included those positive for NTM. Inconclusive culture results comprised contaminated and missing results.
Data collection, sample size and statistical analyses
Patient’s data was collected using hard-copy case report forms specifically designed for the study. The study collected the following information: demographics, HIV status, CD4 count for HIV infected patients, symptoms and findings of the physical exam, antibiotic prescription (if any), microscopy results, Xpert MTB/RIF results, radiological findings, MTB culture results, decision to initiate TB treatment and date, clinical situation and outcome at 6 months, date of death if deceased, and tracing outcome (if tracing done). Data were double entered at the study site using Epi-Data 3.0 software (The EpiData Association, Odense Denmark), with verification of data entry by cross checking of the 2 databases and correction of discrepancies with the case report form.
There was no sample size calculation for this analysis. All patients enrolled in the main diagnostic cohort study were included in this secondary analysis [23].
Patient characteristics were summarized using frequencies and percentages for categorical variables, and median and interquartile ranges (IQRs) for continuous variables. Mortality in the 6 months after the first consultation was the primary endpoint and TB treatment decision the main variable of interest. Patients were divided in 3 groups according to TB treatment decision: “Empirical TB treatment”, “TB treatment with confirmed TB”, “No TB treatment”.
“Empirical TB treatment” was defined as patients who received TB treatment and had no bacteriological confirmation (negative Xpert or culture result). “TB treatment with confirmed TB” was defined as patients who received TB treatment and had bacteriologically confirmed TB (positive Xpert or culture result) [28]. “No TB treatment” was defined as patients who did not receive TB treatment (none of the patients in this category had a positive Xpert or culture result).
Patients were considered severely ill when presenting at least one of the following signs: temperature higher than 39 °C, respiratory rate higher than 30 respirations/minute, heart rate higher than 120 beats minute or unable to walk without help.
Mortality in the 6 months after the first TB consultation was explored using Kaplan-Meier estimates and incidence rates stratified by HIV status and TB treatment decision (categorized as: No TB treatment, empirical treatment, treatment with confirmed TB). Comparisons of Kaplan-Meier mortality curves were done using log-rank test. Univariate and multivariate Cox proportional hazard model was performed separately for HIV-positive and HIV-negative patients to assess the association between mortality and TB treatment decision. Covariates for adjustment included: gender, age (per 1 year increase), clinical severity (severely ill or not), Body Mass Index (< 17 vs ≥17) and TB treatment history. In addition, CD4 (< 200 vs ≥200) and ART initiation (ART treatment started before and ART treatment started during the study vs not started) were included in the model for HIV-positive patients. The result of the chest X-ray interpretation by the clinical officer was included in the univariate analyses only as it could be associated with a decision of empirical treatment. Missing data were not imputed. Proportional hazards (PH) assumption was checked by testing the Schoenfeld residuals. Final multivariate models were fitted using a backward stepwise approach. Statistical significance was assessed with the likelihood ratio test at the 5% level. Data were analysed in Stata® 15.0 software (College Station, Texas, USA).
Ethical considerations
The study protocol was approved by the KEMRI/Scientific and Ethics Review Committee in Kenya and the Comité de Protection des Personnes (CPP), Saint Germain en Laye, France.