The GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA) test was performed as per the manufacturer’s instruction. Aliquots of decontaminated samples were taken out of 4 °C storage, together with the sample reagent buffer containing NaOH and isopropanol were mixed at the ratio of 1:3 followed by incubation at room temperature for 15 min. Two milliliters of the sample were then transferred into the GeneXpert MTB/RIF cartridge (Cepheid, Sunnyvale, CA) and loaded into the GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA). The results were generated after 2 hours, reported as both M. tuberculosis negative or positive, and whether those positive were RIF susceptible or resistant.
Genexpert mtb rif
Manufactured by Cepheid
Sourced in United States, Germany
The GeneXpert MTB/RIF is a diagnostic instrument developed by Cepheid. It is designed to detect the presence of Mycobacterium tuberculosis (MTB) and identify resistance to the antibiotic Rifampicin (RIF) in clinical samples. The GeneXpert MTB/RIF utilizes real-time PCR technology to provide automated and integrated sample processing, nucleic acid amplification, and detection of the target sequences.
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50 protocols using genexpert mtb rif
1
Rapid Diagnosis of Tuberculosis and Rifampicin Resistance
2
Mycobacterial Identification and Susceptibility
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A direct smear fluorescence microscopy was performed, after auramine-rhodamine staining of the samples, Lowenstein-Jensen medium and Mycobacteria Growth Indicator Tube (MGIT) (Becton, Dickinson, and Company) were used for cultures, and samples were incubated for 60 or 42 days, respectively. Mycobacterium species were identified using Line Probe Assay (Hain), and antibiotic susceptibility was assessed through MGIT. PCR was performed to detect the Mycobacterium tuberculosis complex genome in GAs. During the study period, several assays for molecular detection were used (Cobas-Roche, Artus-Qiagen, Gene Xpert MTB/RIF and Gene Xpert MTB/RIF Ultra- Cepheid).
3
Screening for Rifampicin Resistance in Refugee Camps
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To detect rifampicin (RIF) resistance, all acid-fast bacilli-positive sputum samples were subjected to additional processing using the GeneXpert MTB/RIF (Cepheid Gene Xpert system), following national guidelines and manufacturer’s instructions.
HIV screening: Informed consent and assent were obtained from all enrolled participants for an HIV screening at the Providing Initiative Counselling and Testing (PICT) clinics located within the refugee camps. The National Tuberculosis and Leprosy Control Programme (NTLCP) guidelines advised refugees with positive sputum smears to begin anti-TB treatment, while patients without a positive sputum smear were given a ten-day course of broad-spectrum antibiotic treatment.
The flowchart of sampling techniques and laboratory methods is summarized inFigure 2 .
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HIV screening: Informed consent and assent were obtained from all enrolled participants for an HIV screening at the Providing Initiative Counselling and Testing (PICT) clinics located within the refugee camps. The National Tuberculosis and Leprosy Control Programme (NTLCP) guidelines advised refugees with positive sputum smears to begin anti-TB treatment, while patients without a positive sputum smear were given a ten-day course of broad-spectrum antibiotic treatment.
The flowchart of sampling techniques and laboratory methods is summarized in
Schematic illustration of the sampling techniques and laboratory methods.
Distribution of study participants and smear positivity in four refugee camps.
4
Multidrug-Resistant Tuberculosis Treatment Protocol
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Subjects will be recruited at the outpatient and inpatient departments of the sentinel hospitals. Patients who meet all the following inclusion criteria will be considered for recruitment: (1) M.tb strain positive diagnosed with culture from sputum or bronchoalveolar lavage fluid as identified using a BACTEC MGIT 960 automated mycobacterial detection system (Becton, Dickinson and Company, Franklin Lakes, NJ, US. BD960). (2) GeneXpert MTB/RIF (Cepheid, Caribbean Drive Sunnyvale, California, United States) positive for sputum or bronchoalveolar lavage fluid sample. (3) M.tb strain are simultaneous resistance to IHN and RIF. (4) Cases fulfilling the diagnostic criteria for Qi-yin deficiency syndrome or Yin deficiency lung heat syndrome. (5) Patients age between 18 and 65 years. (6) No history of allergies to drugs involved in the trial. (7) Patients must agree to the treatment voluntarily and provide signed informed consent.
5
GeneXpert Diagnosis of Rifampicin-Resistant TB
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Adigrat General Hospital TB clinic operates under the national TB- and leprosy-control program of Ethiopia, in which the diagnosis of TB is followed by GeneXpert™ MTB/RIF assay for rifampicin resistance. Samples were processed by GeneXpert™ MTB/ RIF (Cepheid) assay according to the manufacturer’s manual.
6
Diagnosis of Active Tuberculosis
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A total of 102 patients at Dr. Soetomo Hospital, Surabaya, Indonesia between September 2017 and June 2018 with a respiratory problem suspected of TB were included in the study. TB suspects were described as patients who required screening for active TB and had clinical symptoms such as cough, dyspnea, chest pain, abnormal chest X-ray, such as lung auscultation, or cavitary lesions. Those patients underwent a mycobacterium diagnostic test, and 59 patients who were positive for the GeneXpert MTB/RIF (Cepheid, USA) test or/and acid-fast bacilli (AFB) were retrospectively selected as the active TB group.21
A total of 102 serum samples from people without TB symptoms as a HC group were also collected. The required age range for the healthy controls was matched to those of the active TB group. Healthy samples were confirmed not to be infected with TB based chest X-ray examinations. The study was approved by the ethics committee in health research of Dr. Soetomo Hospital with ethical clearance number 0410/KEPK/VII/2018.
A total of 102 serum samples from people without TB symptoms as a HC group were also collected. The required age range for the healthy controls was matched to those of the active TB group. Healthy samples were confirmed not to be infected with TB based chest X-ray examinations. The study was approved by the ethics committee in health research of Dr. Soetomo Hospital with ethical clearance number 0410/KEPK/VII/2018.
7
Sputum Screening for Tuberculosis
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The spot- morning-spot sputum samples collected from presumptive TB patients were examined using either Zihel Neelsen microscopy or light emitting diode (LED) fluorescence microscopy (FM) for acid fast bacilli (AFB) at respective health facilities following the manufacturer’s procedures (Zeiss, Germany). Split sputum samples of all smear positive TB patients were further examined using the Gene Xpert MTB/RIF (Cepheid, USA) following the standard procedure to confirm TB positive study participants.
Study participants were considered TB positive, if their sputum samples are positive for TB by GeneXpert or both GeneXpert and smear microscopy. Study subjects were considered TB negative controls, if household contacts of smear positive TB cases had no symptoms suggestive of TB. TB negative study participants were included in this study as a control to compare the association between TB and vitamin D deficiency.
Study participants were considered TB positive, if their sputum samples are positive for TB by GeneXpert or both GeneXpert and smear microscopy. Study subjects were considered TB negative controls, if household contacts of smear positive TB cases had no symptoms suggestive of TB. TB negative study participants were included in this study as a control to compare the association between TB and vitamin D deficiency.
8
Molecular Identification of Mycobacterium tuberculosis
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Sputum samples were submitted to microscopy (12 ) and solid culture in according to PKO method (initials in tribute to Petroff, Kudoh, and Ogawa) (13 (link)). A smear of culture, stained by Kinyoun method, was carried out to confirm if the culture was composed of acid-fast bacilli. Later, the colony aspect (not pigmented and rough morphology) was evaluated. The DNA extraction from pure culture was made as described elsewhere (14 ). So, the molecular identification of M. tuberculosis complex was performed by PCR, amplification of 123 bp fragment from IS6110 insertion sequence, as described (15 (link)). If, the PCR for IS6110 was negative, the differential diagnosis of mycobacteria was performed as established (12 , 16 (link)). This study also included samples (32/263) with diagnosis confirmed for TB by the molecular test, GeneXpert MTB/RIF [M. tuberculosis/rifampin (RIF) resistance] assay (Cepheid, Sunnyvale, CA, USA).
9
Sputum Testing for Tuberculosis Diagnosis
10
Sputum Testing for Tuberculosis Diagnosis
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All sputum samples were processed in the accredited laboratories of the South African
National Health Laboratory Services (NHLS) where auramine sputum smear and
mycobacteria growth indicator tube (MGIT) liquid TB culture were performed.
Cultures were kept for 42 days before being classified as negative. For quality
control (QC) sterile mock sputa were sent weekly to the laboratory. None of 209
mock sputa sent to the NHLS laboratory for TB culture between August 2011 and
June 2014 were found to be positive for Mtb, demonstrating that cross
contamination within this laboratory was very low. When the study began
recruitment, sputum samples underwent auramine smear and MGIT culture, from 2012
MGIT culture was replaced by the GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA)
nucleic acid amplification test.
National Health Laboratory Services (NHLS) where auramine sputum smear and
mycobacteria growth indicator tube (MGIT) liquid TB culture were performed.
Cultures were kept for 42 days before being classified as negative. For quality
control (QC) sterile mock sputa were sent weekly to the laboratory. None of 209
mock sputa sent to the NHLS laboratory for TB culture between August 2011 and
June 2014 were found to be positive for Mtb, demonstrating that cross
contamination within this laboratory was very low. When the study began
recruitment, sputum samples underwent auramine smear and MGIT culture, from 2012
MGIT culture was replaced by the GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA)
nucleic acid amplification test.
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