A comparative study of AFB smear and geneXpert MTB / riF assay in pulmonary and extrapulmonary specimens and detection of rifampicin resistance in a tertiary care hospital

Vol. 5 | Issue 4 | October December 2017 Abstract Tuberculosis is a major public health problem worldwide including India causing increased morbidity and mortality. There is a need for rapid identification for early diagnosis and treatment. In this study, GeneXpert MTB/RIF assay is used for rapid identification and was compared with AFB staining (by Zeihl Neelsen method). GeneXpert was used for Mycobacterium tuberculosis (MTB) complex identification and rifampicin detection from smear positive and smear negative patients. Out of 320 patients, 19 patients were acid-fast bacilli (AFB) smear positive and 67 patients detected for MTB by MTB/RIF assay. Rifampicin resistance (RR) was detected in 2 cases out of 62 positive cases by MTB/RIF assay. The test is considered more sensitive for AFB smear positive and showed high level of detection of MTB but less sensitive for AFB smear negative and showed low and medium level of detection of MTB. The GeneXpert is considered as an effective diagnostic tool for rapid diagnosis of MTB and RR in smear negative and positive specimens.

introduction In 2016, globally there were an estimated 10.4 million incident cases of tuberculosis (ranging from 8.8 million to 12.2 million), equivalent to 140 cases per 100,000 population.Most of the estimated number of cases in 2016 occurred in the WHO South-East Asia Region (45%).The five countries that stood out, as having the largest number of incident cases were India, Indonesia, China, the Philippines and Pakistan, which together accounted for 56% of the global total.Of these, China, India and Indonesia alone accounted for 45% of global cases.Nigeria and South Africa each accounted for 4% of the global total [1].India stands highest, in burden of tuberculosis (TB).The World Health Organisation (WHO) TB statistics for India for 2016, estimated 2.79 million cases for India.Incidence of multi-drug resistance tuberculosis (MDR TB) with rifampicin resistance is 11 per 100,000 population [2].
GeneXpert MTB/RIF assay (Cepheid, USA) is a rapid molecular test for diagnosis of TB recommended by WHO.It was initially recommended (in 2010) for diagnosis of pulmonary TB in adults.Since 2013, it has also been recommended for use in children and to diagnose specific forms of extrapulmonary TB (EPTB).The test has much better accuracy than sputum smear microscopy [1].GeneXpert MB/RIF assay is currently used to detect Mycobacterium tuberculosis (MTB) complex DNA and its susceptibility to rifampicin (RMP) in a single reaction.It is based on seminested real-time PCR (rT-PCR) that targets the rpo-B gene hot spot region [3,4].Monoresistance to RMP is rare, however, >90% of RMP resistant isolates also exhibit resistance to isoniazid (INH).Therefore, the detection of RMP resistance may serve as surrogate marker of MDR Mycobacterium tuberculosis [5,6].The test is carried out within 2 hours in a disposable cartridge [7].
Early diagnosis is helpful for early patient management and successful patient outcomes.False-negative results and misdiagnosis of TB suspects are common in developing nations, as most TB control programmes use Ziehl-Neelsen (ZN) smear microscopy, which has poor sensitivity and multiple visits are required which leads to higher default.Mycobacterial culture, although considered as the gold standard but is slow and usually takes 2-6 weeks time to yield a final result and requires proper infrastructure and technical expertise [8][9][10].Diagnosis of EPTB is difficult to establish due to the low number of bacteria in clinical specimens.Rapid and accurate diagnosis of pulmonary and EPTB is still a great challenge [11].
In the present study, aim is to study pulmonary and extrapulmonary specimens by AFB staining (Zeihl Neelsen method) and molecular method of detection by cartridge based nucleic acid amplification test (CB-NAAT) or GeneXpert MTB/RIF assay (Cepheid, USA) in patients attending to a tertiary care hospital.
AIM: 1.To detect MTB by GeneXpert MTB/RIF in inpatient (IP) and outpatient (OP) and to find out the incidence rate, 2. To find out predominant gender and age group for MTB in IP and OP, 3. To compare pulmonary and extrapulmonary AFB smear report with GeneXpert RIF/MTB assay, along with detection of rifampicin resistance.

Materials and method
The study was conducted in the Department of Microbiology at Krishna Institute of Medical Sciences Ltd., Secunderabad, Telangana.The study duration was one year (1 August 2016-31 July 2017).It is a laboratory based prospective study.A total of 320 various clinical specimens of tuberculosis suspected patients from inpatients and outpatients were studied.A total of 198 pulmonary specimens (bronchial wash, pleural fluid, sputum, ET secretion and tracheal secretion) and a total of 122 extrapulmonary specimens (tissue biopsy, lymph nodes, ultrasound guided aspiration, FNAC, pus, CSF, drain fluid, ascitic fluid, urine) were included in the study.
Inclusion criteria: All pulmonary and extrapulmonary specimens from IP and OP were received with request of two tests: 1. AFB smear examination by Ziehl Neelsen method and 2. GeneXpert MTB/RIF assay, which were included in this study.
Exclusion criteria: Those specimens which had only one method, either GeneXpert or AFB smear by Ziehl Neelsen staining method were excluded from the study.

Ziehl neelsen staining method for detection of MTB
The smear preparation was done in biosafety cabinet level 2B.For sputum specimens, a direct smear and a concentration method smear (by Petroff's method) were prepared and heat fixed.Acid fast bacilli (AFB) staining was done by Ziehl Neelsen staining method and then visualised under 40x and 100x light microscope [12,13].Reporting of sputum is done as per RNTCP guidelines [13,14].

geneXpert MTB/riF assay (Figure 1a,b)
It detects MTB DNA in under 2hrs and detects mutations that cause resistance to rifampicin (Figure 1).Sample reagent was added at a 2:1 ratio to clinical specimens.The closed specimen container was manually agitated twice during a 15-min period at room temperature, before 2 ml of the inactivated material was transferred to the test cartridge.The last result was used for the analysis [15].
A total of 320 specimens data were collected in excel format and analysed by using SPSS 20.  detected by GeneXpert and Out of 124 OP, 42 MTB were detected by GeneXpert MTB/RIF assay (Table 1).Among 196 IP patients, male was predominant gender (n=133) and among 124 OP patients, female was predominant gender (n=70) (Table 2).Among different age groups, 41-60years was most commonly affected age group followed by 21-40 years (Table 3).Among all IP patients, 8 were AFB smear positive and 188 were AFB smear negative.Among all OP patients, 11 were AFB smear positive and 113 were AFB smear negative.All smear positive and negative patients were subjected to GeneXpert MTB/ RIF assay (Table 4).Among 8 smear positive IP patients, 7 patients were GeneXpert positive and one was negative.Among 188 smear negative IP patients, 18 patients were GeneXpert positive and 170 were negative.Among 11 smear positive OP patients, 11 patients were GeneXpert positive.Among 113 smear negative IP patients, 31 patients were GeneXpert positive and 82 were negative (Table 5).In this study, it was observed that patients can be categorized according to their AFB staining report and GeneXpert MTB/RIF assay report in both inpatients and outpatients (Table 7).

Discussion
In the prospective study, the diagnostic yield of GeneXpert to detect MTB in pulmonary and extrapulmonary specimens was evaluated and compared with AFB smear staining by Zeihl Neelsen staining.GeneXpert is a simple bench top point of care diagnostic assay that can be performed with minimal training.The results are available within 2 hours, much earlier than the culture which usually takes days to come positive [16][17][18].
Steingart et al. showed a systematic review of 27 studies, in which GeneXpert assay of respiratory specimens had a pooled sensitivity of 89% (95% CI: 85%-92%) and specificity of 99% (95% CI: 98%-99%) in the diagnosis of pulmonary TB [19].Zeka et al. [20] showed that due to low number of bacilli in extrapulmonary samples, they are not detected by AFB staining frequently.They are detected by GeneXpert MTB/RIF assay, which were the limits of detection of the test, as comparable to the present study as shown in Table 6.

citation:
Rani P, Bilolikar AK, Sarma VC, Udayasri B, Reddy SG, Kakarla PL, Shukla R, Poonam AR.A comparative study of AFB smear and GeneXpert MTB/RIF assay in pulmonary and extrapulmonary specimens and detection of rifampicin resistance in a tertiary care hospital.J Med Sci Res.2017; 5(4):115-120.DOI: http://dx.doi.org/10.17727/JMSR.2017/5-22copyright: © 2017 Rani P, et al.Published by KIMS Foundation and Research Center.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
of 320 clinical samples, 196 IP and 124 OP specimens were collected.Out of 196 IP, 25 MTB

Table 1 :
Mycobacterium tuberculosis detected by GeneXpert MTB/RIF assay in inpatient and outpatient specimens.

Table 2 :
Mycobacterium tuberculosis detected by GeneXpert MTB/RIF assay in Males and Females.

Table 3 :
Mycobacterium tuberculosis detected by GeneXpert MTB/RIF assay age group wise.

Table 4 :
Mycobacterium tuberculosis detected by AFB staining by Zeihl Neelsen method.

Table 5 :
GeneXpert MTB/RIF assay result among AFB smear positive and AFB smear negative patients.

Table 6 :
AFB smear report, GeneXpert report along with rifampicin resistance in pulmonary and extrapulmonary specimens.

Table 7 :
Observation of AFB staining report & GeneXpert MTB/RIF assay report in both inpatients and outpatients.
[21] level detection of MTB is observed in pulmonary specimens [sputum(5)followed by bronchial wash (4) and pleural fluid(1)].In extrapulmonary specimens, low level detection of MTB is observed.High level detection was observed in AFB smears positive cases.Rifampicin resistance was seen in only two cases of extrapulmonary specimens (one in tissue biopsy and one in pus).However, GeneXpert does not eliminate the need of conventional microscopy, culture and anti-tubercular drug sensitivity that are required to monitor the progression of treatment and to detect resistance to drugs other than Rifampicin[21].conclusionTheGeneXpert MTB/RIF assay is a rapid and easy-toperform fully automated Nucleic acid amplification test, which is extremely helpful in early diagnosis and to initiate the treatment of tuberculosis in extrapulmonary and pulmonary tuberculosis.It has a high capability to detect MTB complex DNA in AFB microscopy negative samples of pulmonary and extrapulmonary origin.The assay correctly detects the information from the rpoB hot spot region regarding rifampicin resistance.The early diagnosis by MTB/RIF assay and to initiate correct regimen with regular follow up would be helpful in management of tuberculosis patients.