Características generales de las Operaciones Formales

All the reagents were commercially purchased and the manufacturers’ standard operating procedure (SOP) was strictly followed.

Ziehl-Neelsen (ZN) sputum smears to detect AFB (as described by Cheesbrough, 2004) Principle of Ziehl-Neelsen

The sputum smears were stained with carbol fuchsin combined with phenol. The stain was bound to the mycolic acid in the mycobacterial cell wall which resists decolorization by 3% v/v acid

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alcohol decolorizing solution and upon counterstaining with malachite green or methylene blue provided a background contrast colour against the red AFB.

Test procedure

Smears were spread evenly on slides covering a diameter of about 20mm using wooden spatula.

The smear was air dried completely and then fixed with 2 drops of 70% v/v methanol. The methanol was left on the smear for 3 minutes. The slide was rapidly passed; smeared uppermost, three times through the flame of a bunsen burner. The smear was allowed to cool before staining.

The smear was covered with carbol-fuchsin stain and the stain was heated until vapor begins to rise. The heated stain was left on the slide for 5 minutes and stain was washed off with clean water. The smear was covered with 3% v/v acid alcohol (decolorizing solution) for 3 minutes and was washed with clean water. The smear was then covered in methylene blue for 60 seconds, and washed with water. The back of the slide was wiped and placed on a draining rack to air dry.

The smear was examined microscopically, using 100 oil immersion objectives.

Result

AFB: Red, straight or slightly curved rods, occurring singly or in a group and background material appeared blue. The numbers of bacteria present was reported per high power field as follows;

more than 10 AFB/FIELD:+++

1-10 AFB/FIELD:++

10-1000 AFB/100 FIELDS:+

1-9 AFB/FIELDS:exact number was reported

3.9.6 GeneXpert methods for detection of mycobacterium tuberculosis and rifampin resistance (GeneXpert MTB/RIF) (As reported by Blakemore et al, 2010).

Principle

GeneXpert MTB/RIF assay is a catridge based nucleic acid amplification (NAA) test which identifies DNA sequences specific for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF) (i.e. mutation of the rpoB gene) in less than 2 hours by polymerase chain

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creaction. The Xpert® MTB/RIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples, isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR (Danica et al., 2010).

Procedure

The sputum samples were batched to a maximum of four per run. The patient(s) details were entered on theXpert MTB/Rif worksheet.

Lid of leak proof sputum collection container were unscrewed. The sputum volume was measured using a graduated plastic disposable pipette.The volume was recorded on the Xpert/Rif worksheet. (Note: the maximum volume on the graduated disposable pipette is 3ml). Carefully the pipette was discarded. The sample reagent was added at 2:1 (v/v) ratio to the sputum sample by using separate plastic disposable pipette. The lid of sputum cup was replaced carefully and leakages on the cup were avoided. The sputum cup was shaken vigorously 10-20 times using back and forth movements in a single shake and incubated for 15 minutes at room temperature. It was also shaken at least once, as described above during incubation. After incubation, the sputum sample should be liquefied with no visible clumps of sputum. Particulate matter may exist that is not part of the sample. Each Xpert MTB/RIF cartridge was labelled with the Laboratory number on the front side bottom of the cartridge as the same given on the sputum cup with marker pen.

Preparing the cartridge

The liquefied sample was drawn into the transfer pipette until the meniscus of pipette was above the minimum mark by using the sterile transfer pipette provided in theXpert/Rif kit. (Sample (s) with insufficient volume(less than 2ml) was not processed further) . Sample was transferred into the open port of the Xpert MTB/RIF cartridge and dispensed slowly to minimize the risk of aerosol formation. The transfer pipette was discarded into bio-hazard waste bin. The cartridge lid was closed by making sure the lid snaps firmly into place.The remaining liquefied sample was kept for up to 12 hours at 2-8°C (for repeat testing). The cartridge was loaded into the GeneXpert Dx instrument. The system was confirmed attached to a working uninterrupted power source, then the computer and GeneXpert Dx instrument were turned on. The Sample ID and Laboratory number were recorded in the gene Xpert window. The Laboratory number must match the

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number on the cartridge and on sputum cup.The test was started within 30 minutes of adding the sample to the cartridge by clicking start test. The instrument module door would open and displayed the blinking green light, and the cartridge was loaded. The door of the module was closed firmly (an audible click sound was heard). The test started and the green light stopped blinking. When the test was finished, the light turned off. Next cartridge was loaded following the steps described above. Result was printed automatically once the run was completed. It took around 1 hour 55 minutes to complete run. The cartridge was removed when the system released the door lock at the end of run. The used cartridge was disposed in the biohazard waste container.

Quality controls

Each Xpert MTB/RIF cartridge includes a Sample processing control (SPC) and Probe Check control (PCC). Print out of the test result indicates the validation of controls.

Interpretation of results

The results were interpreted by the GeneXpert Dx system from measured fluorescent signals and embedded calculation algorithms. Result was displayed in the “View Results” window. Lower Ct values represented a higher starting concentration of DNA template; higher Ct values represented a lower concentration of DNA template.

MTB detected

If MTB target DNA was detected- the MTB result displayed High, Medium, Low or Very Low depending on the Ct value of the MTB target present in the sputum sample. Below table lists the Ct value ranges for the displayed MTB results (Boehme et al.,2011).

MTB result Ct range

High <16

Medium 16-22

Low 22-28

Very Low >28

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Rifampicin resistance result types, when MTB is detected:

Rifampicin resistance DETECTED: a mutation in the rpoB gene has been detected that falls within the valid delta Ct setting.

Rifampicin resistance NOT DETECTED: no mutation in the rpoB has been detected.

Rifampicin resistance INDETERMINATE: the MTB concentration was very low and resistance could not be detected.

MTB not detected

MTB target DNA is not detected. Sample Processing Control (SPC ) meets acceptance criteria.

MTB NOT DETECTED-MTB target DNA is not detected

SPC- Pass; SPC has a Ct value range and endpoint above the endpoint minimum setting.

Probe Check- PASS: all probe check results pass.

RIF not detected

Rifampicin target DNA is not detected, SPC meets acceptance criteria.

RIF NOT DETECTED- Rifampicin target DNA is not detected.

SPC- Pass; SPC has a Ct value range and endpoint above the endpoint minimum setting.

Probe Check- PASS: all probe check results pass.

INVALID

Presence or absence of MTB cannot be determined, repeat test with extra specimen. SPC does not meet acceptance criteria, the sample was not properly processed, or PCR is inhibited.

MTB INVALID- Presence or absence of MTB DNA cannot be determined

SPC-FAIL; MTB target results is negative and the SPC Ct is not within valid range.

Probe Check- PASS; all probe check results pass.