This comparative analytical study of 15 months was done at PHRC, SRCCH, NICH and Provincial TB Lab Ojha Institute of chest diseases Karachi. Convenient Sampling Technique was used to recruit the subjects
Inclusion Criteria: Probable cases of pulmonary TB suggested by multiple criteria like history of contact, ATT, radiological findings, immunological reaction, biopsy, sign and symptoms etc) having PPA score 4 or more than 4, age 15 or below 15 years were included in the study. Exclusion Criteria: All those patients who either refused to participate in the study or aged more than 15 years were excluded from the study
Study instrument: Structured Performa was used for data collection including demography, socio-economic status, clinical history, scoring chart and diagnostic tests (annexure-2).
Sputum samples were collected preferably outdoor in open air or in a separate ventilated room.
Patients were suggested to clean mouth with water rinse, Inhale and exhale for 2-3 time, keep both hands on hips, cough forcibly and collect sputum in the mouth, spit the sputum carefully into a wide-mouthed, unbreakable leak proof container and close the lid tightly to avoid spills or spilling outside the container.
Each specimen was labeled with the name of patient and local lab register number that matched with information on request form.
Gastric aspirates were collected by physician according to clinical laboratory standards institute (CLSI), National tuberculosis program (NTP) and WHO protocol. Patient was kept on fasting (NPO) at least for 6 hours .Details of the procedure was explained to the parents/caregiver and consent was obtained. After that, an appropriate sized feeding tube (10-12G) was inserted through one nostril till it reached the stomach. The position of tube was checked by insufflation of air into stomach. The contents of stomach were aspirated completely, kept in sterile container. Usual volume collected was around 10 ml. Samples were transported to laboratory for further processing within 1–2 h of sample collection.
Storage/transport of specimen
All specimens’ were transported as soon as possible and were kept in cool temperature/refrigerator between collection and shipment.
Packaging of specimen for transportation:
The basic packaging system for local surface transport of sputum specimens was considered as follows:
Primary receptacle- the specimen container
Secondary packaging zip lock vinyl bags (plastic bags) compatible to the size of specimen container so the vinyl bag could be sealed to avoid leakage and cross contamination.
Outer packaging- Transport box specimen container packed in vinyl bags were placed in transport box with suitable cushioning material.
Each transport box was placed inside with frozen ice packs for every shipment.
ZN staining for smear microscopy was done according to WHO recommended protocol14.
Xpert MTB/RIF assay: Sample reagent was added in a 2:1 ratio to unprocessed specimen in 15 ml falcon tube and the tube was agitated twice during 15 minute incubation period at room temperature. Then 2 ml of the inactivated material was transferred to the test cartridge by a sterile disposable pipette (provided with kits). Cartridges were loaded into the Gene expert. The interpretation of data from TB/RIF test is software based15. Culture on Lowenstein–Jensen (L.J) media: After decontamination of the sputum samples they were inoculated on LJ media slopes.
MGIT: Added 0.8 mL of the supplements mixed above to each MGIT tube using a sterile pipette. Then, added 0.5 ml specimen to the appropriately labelled MGIT tube. Immediately recapped the MGIT tube tightly and mix well by inversion several times.
MGIT recorded the date the tube was flagged as positive and the number of days and hours taken to reach positivity (TTP = time to positive, also known as TTD = detection) 16.Taking LJ culture as Gold standard, sensitivity, specificity, positive predictive value and negative predictive values of Xpert assay were calculated by following formula.
True positive (TP____________________×100
True positive (TP) + False negative (FN)
True negative (TN) _________________x 100
True negative (TN) + False positive (FP)
Negative predictive value (NPV): True negative x 100
False Negative+ True negative
Positive predictive value (PPV): True positive (TP x 100
False positive+ True positive
Sample size: Sample size was 143 for the proposed study calculated on the basis of previous study; in that study, prevalence of TB among children was estimated to be 10.41% in developing countries including Pakistan2 .Sample size was calculated at 95% confidence interval with 5% precision using EPI info version 6.