Study area
Kebbi State is located in the North-Western geopolitical zone of Nigeria. It is mostly populated by Hausas and Fulanis. The main occupations of the peoples of the area are farming, fishing and trading It is bordered by Niger Republic, Zamfara State and Sokoto State. It is mostly populated by Hausas and Fulanis. According to the National Population Commission, population figures stand at 3,256,541 persons spread over an area of 36,800 square kilometer of land (NPC, 2006).
Study population
The study was a descriptive cross sectional study conducted in Kebbi State, Nigeria. The research was conducted in some designated health zones in the Kebbi state which serve as reference laboratories for various local governments within the State, namely: Birnin-Kebbi, Kamba, Argungu, Zuru and Yauri. Study population includes all patients that have been suspected of having TB who visited the designated health zones, serving as reference laboratories.
Eligibility criteria
Inclusion criteria
All patient’s samples that are positive for tuberculosis and showed rifampicin resistant within Kebbi State designated health centers were included for this research
Exclusion criteria
Patient’s that are negative for tuberculosis were excluded for this research.
Sample collection and processing
Sputum samples were collected in a wide-mouth, dry, clean, leak-proof container (CDC, 2016). Trained National Tuberculosis and Leprosy Control Programme (NTBLCP) staff assisted in the collection of the sputum samples used during the research. The sputum sample was collected as an early morning on the spot specimen.
It was subjected to Gene Xpert assay to check for presence of Mycobacterium tuberculosis and rifampicin resistance. A positive result indicating resistance was recorded and then a molecular assay to assess mutation of the katG and inhA gene were performed.
DNA Extraction: Using a sterile graduated pipette 0.5 ml (500 µl) of the decontaminated sputum sample was transferred into microcentrifuge tube. This was done for all the samples, after which the tubes were closed and centrifuged for 15 minutes at 10000xg. The supernatant was discarded and 100µl lysis buffer was added and re-suspended by vortexing gently for 30 sec. The tubes were arranged in a floater inside the BSC II and incubated for 5minutes in a water bath at 950C. Then, 100µl neutralisation buffer was added and vortexed for 30 seconds and the tubes were centrifuged at maximum speed (10,000xg). The heavier debris formed the pellet and the lighter DNA (free from impurities) was suspended in the supernatant which was transferred into clean labeled micro-centrifuge tubes for further use.
PCR amplification of the extracted DNA: The master mix preparation was done according to manufacturer’s specification (HainLifescienceGmbH, 2015) and World Health Organization (WHO) (WHO, 2008). The master mix was made up of 10µl of the AM-A and 35µl of AM-B Reagent which was placed in a PCR tube labeled with sample number and mixed very well. This was prepared inside dead air box in a clean DNA free room. Then 5µl of each sample (containing the extracted DNA from above) was added to the corresponding tube containing the master mix and then mixed gently by pipetting up and down a few times. The PCR tubes were then placed in a 30 cycle (10 + 20) thermal cycler program for amplification. After amplification the DNA contained in the amplicons were denatured in the TwinCubator® which was pre-warmed to 45 oC and 20µl of denaturation solution (NaOH) was added to each labeled well of the TwinCubator® tray followed by the addition of 20µl of the amplicons respectively. The mixture was mixed gently by pipetting up and down five times and then incubated at room temperature for 5mins.
Hybridization and Detection: Hybridization and detection procedures were carried out according to according manufacturer’s specification (HainLifescienceGmbH, 2015) and WHO (WHO, 2008). After denaturation of the amplicons, 1ml of the pre-warmed hybridization buffer (HYB) was carefully added to the wells using a pipette and thoroughly mixed. The tray was placed on the TwinCubator® and labeled strips were added to each well ensuring that the strips were completely covered by the liquid and incubated at 45oC for 20mins. After incubation, the HYB buffer was aspirated completely from each well and 1ml of the pre-warmed red stringent wash buffer (STR) was then dispensed into the tray. After 10 minutes’ incubation at 45 oC in the TwinCubator®, STR buffer was aspirated and was washed off with 1 ml of Rinse solution (RIN) for 1 minute. Then 1ml of the Conjugate solution was dispensed into each well and incubated for 20 minute on the TwinCubator®. The strips were washed twice with 1 ml of Rinse solution (RIN) for 1 minute in the TwinCubator®. Then sterile distilled water was added and a 1-minute wash performed on the TwinCubator® to wash off the RIN solution after which the distilled water was completely decanted. One (1) ml of the Substrate solution was then dispensed into each well and incubated for 10 minutes on the TwinCubator® after which the Substrate solution was aspirated and the strips washed twice with sterile distilled water. A pair of clean tweezers was used to remove the strips from the TwinCubator® tray and placed onto absorbent paper. The developed strips were partially dried and transferred to the GenoType® MTBDRplus score sheet for interpretation (HainLifescienceGmbH, 2015).
Statistical Analysis
Results obtained from line probe assay were analyzed using Microsoft Excel 2016 and Statistical package for social sciences (SPSS).