Methods
Study Population and Design
From 2006 to 2008 patients newly diagnosed with active pulmonary TB were consecutively enrolled in this prospective cohort study in the framework of two large randomized, double-blind, controlled nutrition intervention studies, with all patients diagnosed and treated for TB according to international guidelines. All participants were randomized to either an energy-protein study or a multi-micronutrient study comparing the effect of high-doses against low-doses. To be eligible for the energy-protein study, TB patients had to be diagnosed as sputum positive pulmonary TB (PTB+) with HIV co-infection. All other enrolled TB patients were eligible for the multi-micronutrient study.
The study was conducted in an urban setting in Mwanza City, northern Tanzania. Tanzania ranks among the world's 22 high TB-burden countries, with a low prevalence of multi-drug resistant TB, and half of the TB patients co-infected with HIV. The estimated national diabetes prevalence in 2011 in Tanzania was 2.3%. All participants underwent baseline examination of anthropometry, diabetes, and HIV, and venous blood samples were drawn for additional laboratory analyses. To be included in the study testing for HIV was mandatory. Persons below fifteen years of age, pregnant or lactating women, terminally ill, and non-residents of Mwanza City were all excluded.
Measurements
Sputum results were done as part of the routine diagnostic procedure using "spot-morning-spot" samples (method described in ) using Ziehl-Nielsen staining technique in combination with culture of Mycobacterium tuberculosis on Lowenstein Jensen solid media. All participants had verified pulmonary TB (PTB); in this study a positive culture test result was defined as PTB + with the diagnosis relying primarily on culture status; initial microscopy results were only used if the culture result was missing. In case of a negative culture result, the diagnosis was defined as sputum negative pulmonary TB (PTB-), in which case the TB diagnosis was based on clinical suspicion, history of disease, lack of clinical improvement after treatment with a broad antibiotic spectrum as well as a positive x-ray result as suggested by WHO.
Weight (Seca, Hamburg, Germany) and height were measured with the participant barefoot and with minimal clothing (nearest 0.1 kg and 0.1 cm), from which body mass index (BMI) was calculated as weight/height (kg/m). Waist circumference was measured between the lower costae and the iliac crest. The midpoint between the acromion process of the shoulder and olecranon process of the ulna bone was determined and marked on the left arm, on which mark the triceps skinfold thickness (TST) (Harpenden caliper, Baty International, West Sussex, UK) was measured (with arm hanging loosely). Mid-upper arm circumference (MUAC) was measured on the same arm and same mark using a standard tape, but with the arm flexed in a 90 angle. Measuring TST and MUAC allowed for estimation of arm fat area and arm muscle area (methods for calculation described in ). Finally, grip strength (0.1 kg) was assessed using a digital hand dynamometer (Takei Scientific Instruments, Niigata City, Japan). All anthropometric measurements were performed in duplicate.
Fasting blood glucose (FBG) was determined on capillary whole blood using point-of-care diagnostic instruments (HemoCue Glucose System, Ängelholm, Sweden). The test was performed between 8.00–10.00 AM after an overnight fasting period (> 8 hours), and only water was allowed prior to the test. As the FBG in the TB participants might be affected by the infection (non-diabetes stress hyperglycaemia), the range of the FBG for offering a standard two hour (2 h) oral glucose tolerance test (OGTT) was expanded from the commonly used 5.6–6.0 mmol/L; those with a FBG between 5.1–11.0 mmol/L completed the 2 h OGTT (intake of 75 g of anhydrous glucose dissolved in water), whereas those with FBG < 5.1 or >11.0 mmol/L did not. Final diabetes diagnosis was based on either a FBG > 6.0 mmol/L or a 2 h blood glucose >11.0 mmol/L. Since the diagnosis of diabetes was for epidemiological purposes only, we did not repeat the test in those with values suggestive of diabetes. Participants diagnosed with diabetes prior to their TB diagnosis were only classified as such, if the diabetes diagnosis was reproduced within the present study. The diabetes testing was performed as soon as possible after initiation of TB treatment to eliminate the role of adverse drug effects.
Venous blood was drawn in EDTA tubes at local health facilities and transported to the research laboratory, whereupon serum was collected and kept at −80 °C until analysed. HIV diagnosis was based on two rapid tests, Determine HIV 1/2 (Inverness Medical Innovations Inc., Delaware, USA) and Capillus HIV-1/HIV-2 (Trinity Biotech Plc., Wicklow, Ireland). If the HIV test results were discordant, ELISA was used. Cluster of differentiation 4 (CD4) counts were determined by flow cytometry after CD4 immuno-flourochrome staining of the leucocytes (Partec FACS, Partec GmbH., Germany), and haemoglobin levels (g/dL) and white blood cell (10/L) counts, including differentials, were carried out at the research laboratory at the National Institute for Medical Research in Mwanza. Serum concentrations (g/L) of the acute phase reactant alpha-1-acid glycoprotein were determined at the Department of Clinical Biochemistry, Aalborg University Hospital, Denmark.
Information using standardized questionnaires on demographic information, smoking habits, and alcohol intake was collected. Smokers were grouped as either previous or current smokers, and alcohol intake was classified as either no intake or any intake.
Follow-up Visits at Two and Five Months
All anthropometric measurements, grip strength and the biological measurements (haemoglobin, white blood cells, CD4) were repeated at the two and five months visit. The diabetes and HIV testing were not repeated, thus the baseline diagnosis was used throughout the study period.
Statistical Analysis
Data were double entered, and all statistical analyses were performed using Stata 12.0 (StataCorp LP, College Station, USA). The distribution of continuous variables was assessed for normality. The t-test was used to test for differences in means and the Χ-test was used to test for differences in proportions across diabetes status. A linear mixed-effects model was used to assess the changes across diabetes status for anthropometry, grip strength and biological measurements at baseline as well as after two and five months of TB treatment (repeated measurements). The mixed-effects models were adjusted for baseline age, sex, HIV status, alpha-1 glycoprotein, smoking habits, alcohol intake, and nutritional intervention.
Ethical Considerations
Ethical permission was obtained from the Medical Research Coordinating committee of the National Institute for Medical Research (NIMR) in Tanzania, and consultative approval was given by The Danish National Committee on Biomedical Research Ethics. Written and oral information was presented to all eligible participants by the health staff before written informed consent was obtained. Written consent was obtained from parents/legal guardians of any participants under 18 years of age. Counselling prior to HIV-testing was compulsory, and post-test counselling was offered to all who tested HIV-positive. Participants with diagnosed HIV and/or diabetes were referred for follow-up at the respective clinics for care and management.