Agreement Between Direct Smear Microscopy and GeneXpert MTBIRIFI in Diagnosis of Pulmonary Tuberculosis Among Patients Attending Consultary TB Clinic, Kirkuk

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Bushra Mohammad Mahmoud (1)
Suheila Shamse Elden Tahir (2)
Mohammed Abdul-Aziz Kadir (3)


(1) Bushra Mohammad Mahmoud, MS.c, Microbiology,
Consultary Clinic for Chest and Respiratory Disease, Kirkuk, Iraq.
(2) Suheila Shamse Elden Tahir, MS.c , Family & Community Medicine,
Consultary Clinic for Chest and Respiratory Disease, Kirkuk, Iraq
(3) Mohammed Abdul-Aziz Kadir, Ph.D., College of Medicine,
Kirkuk University, Kirkuk, Iraq.


Correspondence:
Bushra Mohammad Mahmoud, MS.c, Microbiology,
Consultary Clinic for Chest and Respiratory Disease, Kirkuk
Iraq
Email: gkirkuk@gmail.com

Tuberculosis is one of the most deadly diseases worldwide; about 2 billion people in the world carry latent TB; more than nine million will become ill and about three million will die (1).

Poor health system, limited laboratory capacity for case detection, treatment barriers and complications make TB a major challenge for public health programs (2).

Detection of Mycobacterium species is mainly detected by Ziehl-Neelson stain which employs acid-fast staining technique. First the sample is inversed in hot carbolfuchsin that stains red, then the sample is stained with acid-alcohol and counter stained with methylene blue. On examination, the bacteria appear bright red against a blue background (3).

The Xpert MTB/RIF assay is a fully automated molecular diagnostic test for TB disease, developed in partnership with Cepheid Inc, the foundation for innovative new diagnostics (FIND); the university of medicine and dentistry of New Jersey (UMDNJ) and the national institutes of health (NIH). It can detect rifampin resistance from sputum specimens in less than 2 hours and minimizes staff manipulation and biosafety risk (4).

The study was conducted to:
1. Show the agreement of conventional and new methods in detecting Mycobacterium in sputum and other body fluids.
2. To detect rifampin resistance among smear positive cases.

A cross sectional study recruited 72 patients who presented to Chest and Respiratory Disease Clinic in Kirkuk for the period between 4.12.2013-4.2.2014. Sputum samples were obtained from each patient for direct smear microscopy and GeneExpert for detection of Mycobacterium tuberculosis, then agreement in results of both investigations was assessed.

Regarding sputum sampling each TB suspect was requested to submit two sputum specimens, one for conventional smear plus Xpert MTB/RIF and the second for the conventional smear alone. When the results of Xpert were positive the Rifampin sensitive patients were registered as Cat 1 or Cat 2 according to drug history. If the specimen proved to be RIF/ resistant, it would be subjected to culture and drug sensitivity test (DST).

Table 1 shows that during the period of the study; a total of 958 sputum samples were examined for direct smear microscopy to detect the presence of mycobacterium tuberculosis.

Regarding GeneXpert (PCR), a total of 321 were examined; all smear positive cases, highly suspected smear negative TB cases and other body fluids referred by public and private sectors were included in the study.

Regarding gender difference, the ratio of male to female was 1.9:1. The percentage of positive cases by DSM was 3.03%, while in PCR it was 11.52%.

It was clear that there was significant statistical difference between the two tests (P<0.05).

Table 1: Distribution of cases in DSM & PCR according to Gender


It is clear that PCR has detected higher positive cases than the conventional method AFB; also it is obvious that males are affected more than females.

It was found that the proportion of positive cases in GeneXpert was greater than that with AFB. There was a statistically significant difference between the two methods (P< 0.007), as indicated in Table 2.

Table 2: Agreement between DSM and GeneXpert for study sample


Table 3 illustrates the distribution of positive cases detected by PCR. Out of 321 samples tested for detection of MTB by GeneXpert, 37 cases were positive, distributed as 35 pulmonary TB and 2 extra pulmonary (pus); apart from these 2 extra pulmonary samples , all other samples were negative.

Among the 37 positive cases only 4 (10.81%) cases were proved to be resistant to Rifadin by the new test (GeneXpert). The percentage of Rifadin resistance was 11.42% among pulmonary cases.

Table 3: Distribution of types of TB cases according to gender and Rifadin resistance detected by PCR

RFS=Rifadin sensitive; RFR=Rifadin resistant

It is obvious from table 3 that PCR has advantage to detect Rifampin resistance cases within less than 2 hours.

Regarding other body fluids, all were negative when tested by PCR.

WHO have started the largest roll-out of GeneXpert, as detecting TB rapidly and identifying drug resistance on the spot is an essential step to improve care of the infected people and avoid transmission in the community (5).
The higher percentage of positive cases among males than females detected by PCR, is in agreement with WHO report as males have much higher rates than females.

In a study conducted in Vantanilla, Lima, in 2005 showed that tuberculosis globally kills more women than any other single infectious disease. It is noted that 70% of the poor are women and they face obstacles in seeking medical advice and care (6).

Detection of Mycobacterium TB by PCR was increased in comparison with direct sputum smear; this result is in agreement with a study done in Brazil and published as Clinical trials during 2011.

In a survey conducted among prisoners, in Malaysia, it was documented that a single GeneXpert assay proved to get 53% sensitivity, 100% specificity and concluded that single test yields low screening sensitivity (7).

WHO technical report has concluded that PCR is the most common method of amplification, in spite of many other methods for rapid test for diagnosing MTB which are commercially available as in Roche (Amplicor PCR-FDA-approved), Becton Dickson CBD probe Tec, Standard Displacement amplification (SDA), Gene probe (Amplified Mycobacterium tuberculosis Direct (AMTD),Transcription machinated amplification (TMA) in addition to the assay used in the current study under the commercial name Cepheid (GeneXpert MTB/RIF) nested real time PCR (8).
In addition to excellent sensitivity with smear positive specimens, the device can identify 72.5% of smear negative culture positive samples from single tests. WHO recommended that the GeneXpert MTB/RIF is suitable for use at district and sub -district levels (9&10).

Detection of drug resistance in comparing the conventional (AFB) test and new PCR, showed that males had higher resistance than females although the number of cases is small as tuberculosis is a rare disease and among all notified cases resistance occupies 3% of the TB cases (11).

In MTB, mono resistance to (RIF) is rare and almost 90% of resistant cases to RIF are also resistant to INH so a positive result of RIF is a strong surrogate of MDR-TB, and in resistant isolates it has been shown that from to 95% to 98% RIF resistance is caused by mutations in the proB gene encoding the RNA polymerase B-unit (12&13).

1-Colledge, N. R.; Walker, B.R. & Ralston, S.H. (2010). Davidson's Principles & Practice of Medicine. 21 st Edition. Churchill Livingstone Elsevier, Edinburgh.
2-Maiga, M; Mohoudan, A.; William, R. & Bishai, et al (2012). Current tuberculosis diagnostic tools role of urea breath test.
Indian J. Med. Res., 135: 731-736.
3-Virella, G. (2004). Microbiology and Infectious Diseases. 3rd Edit. Williams & Wilkins PP. 168-174.
4-Piatek, S.A.; Cleff, M.V.; Alexander, H. et al. (2013). Gene expert for tuberculosis diagnosis planned and purposeful implementation.
Global Health Sci. Pract., 1, 18-23. WWW. Ghsc Journal.org.
5-Onifade, D.A.; Bayer, A.M.; Montaya, R. et al. (2010). Gender related factors influencing tuberculosis control in Shanty towns. A qualitative study.
BMC Public Health, 10, 381.
6-Keertan, D. (2013) Trial of point-of-treatment Xpert MTB/RIF Assay.
Clin. Trials. Gov. Identifier. University of Cape Town.
7-AbduRazzak, H.; Al-darragi, A.; Razak, H.A., et al. (2013). The diagnostic performance of a single Genexpert MTB/RIF Assay in an intensified tuberculosis case finding among HIV infected prisoners in Malaysia.
PlosONE 8(9).
8-WHO (2011) Automated Real-time Nucleic acid Amplification Technology and Rapid and Simultaneous Detection of Tuberculosis and Rifampin Resistance. Xpert MTB/RIF system. Policy statement.
9-UNITAID (2012) Tuberculosis, Diagnostic Technology Landscape Semi-annual update.
10-Merza, M.A.; Famia, P.; Tabars, P. et al. (2011) Anti Tuberculosis drug resistance and associated risk factors in a tertiary level TB Center in Iran.
A retrospective analysis. J. Infect. Dev. Ctries, 5(70: 511-519.
11-Zeka, N.A.; Tasbakan, S.; Cavasogli, V.C. (2011). Evaluation of the Gene Xpert MTB/RIF. Assay for rapid diagnosis of tuberculosis and detection of Rifampin Resistance in Pulmonary and Extra-pulmonary specimens.
J. Clin. Microbiol, 49(12): 4138-4141.
12-Prachi Pinglay (2012) GeneXpert test for drug resistant TB detect 28 cases during month. Hindustan times, Mumbai, April 28.
13-Osama, M.; Simpsa, J.A.; Galdwell,j. et al. (2014) GeneExpert MTB/RIF Version G4 for identification of Rifampin-Resistance Tuberculosis in a programmatic setting. J. Clin. Microbiol., 52(2): 635-637.