Con el departamento de Moquegua

2.2.1 Environmental sample collection from Study A, TB case and control households and Study B, rural village houses and livestock

Study A: the household dust for the NIH project was household dust only was collected by the NIMR/SUA integrated team.

Study B: Collection of the environmental samples chosen in this study were faeces from livestock including cattle and goat, boma soil, dust from households, water and sediment sample from selected water sources.

For household dust collection, the top 3 cm of dust was removed from food preparation area, washing facilities and livestock housing area in three different domiciles including their first house with food preparation area and washing facilities, second house is a chamber for men as well as third one for women and children

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(Figure 2.5). The six dust samples were collected in each house including three from inside of house and another three from outside, using the sterilised spatula into the plastic sample bags for each household and pooled immediately to make a mixed sample to represent each individual household. The bag was shaken and three biological replicates of 2 g selected in the Eppendorf tubes and stored at 4 ˚C. All samples were labelled and blinded for shipment to the UK.

Figure 2.5 Sampling schematic of boma soil and household dust collection points per household. The picture on the right was satellite image to show the structure of household. (http://enduimet.org/wp-content/uploads/2013/05/Maasai-boma.jpg)

2.2.2 Sample collection from pastoral homesteads of dung and boma soil

For environmental sampling the number of replicate samples required from a given location to ensure a < 1% probability of a false negative will be calculated in line with our previous protocols in the UK (Courtenay, Reilly et al. 2006), calculated by equation 2.1. ΨN

(

)

p

−

=

1

Where ΨN is the probability that an Mb positive site tests negative (i.e. a false

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of samples tested per site. Required values of m from our published studies in the UK were between 6 and 8 to give ΨN < 0.01. We evaluated spatial and temporal factors

to calculate m for our study populations, and conducted a pilot study to determine the value of p by testing 15 replicate samples from 10 spatially independent sites in low and high incidence regions.

The top 1 cm3 _{of dried faeces exposed to the sun was removed prior to sample} collection to reduce the effect of UV irradiation. At each of site depending on number of livestock and size of livestock enclosure, ten faecal samples were collected from cattle faeces and four from goat faeces, approximately 2 g faeces were collected using sterilised spatula into the Eppendorf and stored in cooled boxes with an ice pack at 4 ˚C. The same procedure was applied on the boma soil collection but the nine soil samples were selected in each cattle and goat boma area. The soil samples were collected using the sterilised spatula into the plastic sample bags from each boma area and pooled immediately to make a mixed sample to represent individual homestead. The bag was shaken and three biological replicates of 2 g selected in the Eppendorf tubes and stored at 4 ˚C. These sample were deep frozen using liquid nitrogen then shipped to the UK in a dry shipper under appropriate biohazards labelling UN3373.

The moisture of soil was detected using the Tecpel pH 707 Soil and Moisture Tester (Tecpel, Taipei, Taiwan) for 10 min equilibration after inserting in the soil around 5 cm deep from surface. The range of moisture content in the cattle and goat boma soil in the dry season was compared to the wet season (Figure 4.13)

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2.2.3 Other environmental samples: water and sediment samples

The water sample was collected from the surface of running water facing upstream into the current and > 500 ml volume was collected from each sampling site close to villages and each 250 ml water was filtered using 100 ml sterilised plastic syringe and MicrofilV filtration device (Thermo Fisher Scientific, Leicestershire, UK) with 0.22 µm mixed cellulose esters white gridded filters (Millipore, MA, USA).After collection the filters were removed from the plastic holder using sterile forceps and air-dried. The filters were then rolled and folded and stored in 2 ml Eppendorf in a cool box with ice packs.

Representative mixtures of sediment consisting of sedimentary rock, mud and sand were collected using the sterilised plastic spatula scooped along the bottom of surface river body < 1.5 M deep in the upstream direction. The sediment sample was placed into the 50 ml sterilised plastic universal tube (Scientific Laboratory Supplies Ltd, Nottingham, UK) and homogenised. Excess water was removed from the container prior to storage.

The pH value for the river was tested using New Hydrion® pH indicator strips (Micro Essential Laboratory, New York, USA) (Table 2.3).

All samples were deep frozen using liquid nitrogen then shipped to the UK in a dry shipper under appropriate biohazards labelling UN3373.

2.2.4. 16S rRNA sequencing results from livestock and wildlife animal in Tanzania

The livestock lesion samples from slaughterhouse compared to wild buffalo tissue samples from Ruaha national park were tested using 16S rRNA sequencing at Sokoine

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University of Agriculture in Tanzania (Table 2.4). M. intracellulare was detected in tissue samples of wildlife compare to M. lentiflavum which present in most of the wild buffalo and cattle lesions samples. These two SGM species belongs to opportunistic mycobacteria (Mwikuma, Kwenda et al. 2015) and can be identified in drinking water supplies as a possible source of NTM infection in humans (Falkinham, Norton et al. 2001, Marshall, Carter et al. 2011).

Table 2.4 Comparison of different species identification results from livestock lesion (A) and wild buffalo tissue (W) using 16S rRNA sequencing at Sokoine University of Agriculture in Tanzania.