Latent tuberculosis in children and youth with type 1 diabetes ... - BMC Infectious Diseases

Study design and population

The study employed a cross-sectional study design to establish the presence of latent-TB in type 1 DM(T1DM) participants. Study participants were recruited from the 5 pediatric and youth diabetes clinics located in Dar es salaam. In each study site, all children and youth with T1DM were included in our study. Recruitment of participants started from Jan 2021 – December 2021. The population included all children and youth aged 1–25 years inclusive attending pediatric diabetes clinics (These clinics include children and youth up to 26 years of age) located in Dar es Salaam, children whose parents gave consent, children aged 12-<18 years, who gave assent on top of parental consent and youth who gave consent. We excluded children with co-morbidity like sickle cell disease, children under one year (who may not be type 1 diabetes) and those above 25 years.

Study setting

The study was conducted in five health facilities with specific pediatric diabetes and youth clinic located in Dar es Salaam. These sites were Mwananyamala in Kinondoni Municipal, Temeke in Temeke Municipal, Vijibweni in Kigamboni Municipal, and Muhimbili in Ilala municipal and one private clinic at Hindu mandal Hospital. Dar es Salaam is the largest city in Tanzania located in the Eastern zone of the country with an estimated population of 5 million inhabitants according to the 2022 census. Dar es Salaam contributes most patients of DM and TB in the entire country. It has a high prevalence of DM in the general population of 9% [26], and had 15% of new Tuberculosis cases diagnosed in 2021 [27].

Data collection

A structured questionnaire was used to collect the required study information, including socio-demographic factors of children and youth, date of birth, number of admissions, amount and frequency of insulin taken, any other medication (e.g., Ant tuberculous), and complications encountered. Physical examination was done including anthropometric measurements, pubertal assessment and eye check using fundopictures. Assessment for puberty was done using tanner staging then grouped into pre-pubertal, pubertal and post pubertal [28].

The first morning urine of the day was collected in an empty clean bottle. Using a multistrip URIT 14 g (Accurex diagnostic, India) determination of presence of proteins, Microalbuminuria, ketones, and sugar in urine was done. Then Albumin creatinine ratio was calculated. A patient was regarded as abnormal if reading was ≥ 30mcg/gm creatinine.

Anthropometric

One well-trained research assistant performed all anthropometric measurements including weight, height and pubertal assessment. Weight and height machine (Tanita BC-553 digital 150 kg scale and a portable stadiometer- Seca 214). The weighing scale was firmly put on a flat floor, the child/youth removed his/her shoes and heavy clothes like sweaters, remaining with one light cloth. The child/youth stood at the center of the scale with both feet. Weight was then read from the weighing scale and rounded to the nearest 100 g. The child moved from the weighing scale to the height scale (a portable stadiometer- Seca 214). In addition to footwear removal to measure height, any hair ornaments (e.g., bands, hats, turbans) that could interfere with the measurement were removed. The head of the stadiometer was then raised to allow room for participant to stand underneath. The research assistant checked if the child/youth is standing at the center of the base of the plate, heels together, arms to the sides and legs straight, shoulder relaxed and head positioned in the Frankfort horizontal plane, the patient was instructed to keep their eyes focused straight on a point ahead and to stand as upright as they can, then the head piece was lowered to the highest place of the head with enough pressure to compress the hair. The participant was then told to step off the stadiometer when reading the measurement. The height was recorded to the nearest 0.1 cm. BMI was then calculated using the Quetelet equation (weight in kilograms divided by the square of height in meters).

Blood pressure

The child /youth was seated on a chair with his/her arm rested on the table. Then the screw was turned to remove the air out of the cuff, which depending on the size of the child was placed on the upper arm above the elbow. The "on" button switched on and the reading appeared automatically on the screen of the machine (Dinamap).

Eye examination

A nurse trained to do eye examination in patients with diabetes took the fundal pictures which were analyzed by an ophthalmologist. At the start of the examination, the child was examined for visual acuity using Snellen's chart and standing at 6 m. This was followed by dilating the eyes of the children using one drop of tropicamide eye drop in each eye and left for 10 to 30 min for the pupil to dilate, after which the fundus pictures were taken by a nurse trained in fundo picture techniques, using a fundus Camera (Topcorn TRC 50 EX, Japan). The ophthalmologists read the fundus pictures for accuracy and quality control.

Screening for tuberculosis

Then children and youth were screened for symptoms of Tuberculosis as per NTLP guideline, if they had TB symptoms, samples for gene- X- pert was taken. All those who did not have symptoms underwent QuantiFERON testing (QuantiFERON TB Gold plus Interferon Gamma Release Assay (IGRA), a whole blood assay developed to detect the IFN-γ produced in vivo by sensitized T cells after in vitro stimulation with mycobacterial antigen and is not affected by environmental mycobacteria or BCG vaccine [29] (Fig. 1). These children and youth were also screened for retinopathy and nephropathy.

Laboratory investigations

We collected blood samples from the anterior cubital fossa (about 4.5 mls) following standard procedures. We immediately used one drop of blood on the glucostick for determination of random/fasting blood glucose, using a glucometer (Gluconavii, home health, (UK) LTD Unit A, Greatham Road, industrial estate Greatham Road, Bushey, Hertfordshire WD23 2NZ, United Kingdom), another drop was put on Hemocue® HbA1c 501, (full automated point of care machine (Radiometer group, Angelholm-Sweden) for HbA1c determination, after the drop of blood was put on an HbA1C machine, with a wait of 6 min the results displayed on the screen of the machine and was recorded. The remaining 4 mls were put in the QuantiFERON gold bottles, one ml for each bottle (Negative control (Grey), TB1(green), TB2 Yellow and Mitogen (Purple). These samples were taken to the Hindu-mandal hospital on the same day of collection at room temperature in Dar es salaam 25-28^oC, and incubated at 37^oC for 24 h. After the 24-hour incubation at the laboratory, the samples were centrifuged to get plasma. Elisa test was performed, absorbance value obtained Using a QuantiFERON analysis software, the results were analyzed. All the samples were taken and analyzed at the Hindu Mandal hospital laboratory except for point of care blood glucose and HbA1c. All the 42 participants with latent tuberculosis were linked to the tuberculosis services in respective hospitals. Quality of care check and standardization of the tests, few samples were taken in both the Shree Hindu Mandal Hospital (The study main laboratory) sample and Muhimbili National Hospital (The main referral hospital in the country) except for the QuantiFERON test which were taken to the main government chemists Laboratory.

Testing for microalbuminuria and urine creatinine

About 2 mls of the 1st morning spot urine collected in an empty sterilized bottle was used. Using a multistics for microalbuminuria, the strips were URIT 14 g (Accurex diagnostic, India) were dipped into the urine container and weighted for 60 s. Then the color of the uristrips was compared with the color scale on bottle, The strips measured the presence of protein, microalbumin, creatinine, ketone and sugar. Albumin Creatinine ratio was then calculated from the two readings. The minimum sensitivity of the test strip was 30mcg/dl of Albumin in urine.

Data management and statistical analysis

A structured questionnaire was prepared to ascertain the participants' demographic and clinical information. Data was analyzed using SPSS version 25 (IBM SPSS, Armonk, NY, USA). Mean with standard deviation (SD) or Median with 25th and 75th interquartile range (IQR) were used to summarize parametric and non-parametric continuous variables (e.g. Age, weight, height, HbA1c levels). Comparison of categorical variables such as frequencies and proportion of children and youth screened and diagnosed with or without latent TB were performed using the Pearson chi-square or Fisher's exact test. A multivariate logistic regression analysis was used to calculate the odd ratio (OD) with 95% confidence interval (CI) for the risk factors that were associated with latent TB and DM complications such as retinopathy and nephropathy. A p-value of < 0.05 was considered statistically significant.

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