Evaluation of the XpertMTB / RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal

Tuberculosis diagnosis and monitoring rely in Sputum microscopy of National Tuberculosis Programme, Nepal because of its low cost and easier to perform. Direct sputum microscopy is popular worldwide. Currently, there are 533 microscopy centres catering for sputum microscopy examination throughout the country. Most of the microscopy centres are established within government jurisdiction and remaining are established as nongovernmental organization as well as private sectors. A cross-sectional study was conducted from July 2013 to January 2015. A total of 2091 patients were enrolled in the study who were attending the DOTS Centre in Parsa District of Public Health Office, Nepal. Smears stained with ZN stain methods were examined microscopically followed by the GeneXpert MTB/RIF assay. Out of 2091 suspected pulmonary TB patients enrolled for sputum microscopy and GeneXpert MTB/RIF for the confirmation of TB, the 1301(62.21%) were male and 790 (37.78%) were female. The maximum TB cases were from Parsa district (555, 26.5%). The comparative study of different diagnostic tools reveals the sensitivity of MTB/RIF was 95.50% (91.87, 97.82) and significantly higher than smear microscopy performed on the same fluid, which had a sensitivity of 61.97% (55.41, 68.21). Five of 127 smear-negative cases had MTB/RIF-positive uncentrifuged sputum, resulting in a specificity of 81.23% (75.95, 85.78), which was similar to smear microscopy 98.29 % (97.34, 98.97; p=0.121). The positive predictive value (PPV) and negative predictive value (NPV) of MTB/RIF were 96.85% (93.61, 98.72) and 94.95 % (93.52, 96.14), respectively. HIV co-infection did not impact sensitivity, specificity or liquid culture time to positivity (TTP). When MTB/RIF accuracy was evaluated using composite reference standard culture positivity from sputum, the sensitivity and specificity were similar to those obtained in the primary analysis using either definite TB versus possible and non-TB combined; definite and possible TB combined versus non-TB.


Introduction
Tuberculosis (TB) is a leading cause of death worldwide.In the early 1990s, the government of Nepal brought up a program to control TB at national scale called DOTS (Directly Observed Treatment Short Course) [1].DOTS program was initiated in April 2001 with a goal to diagnose and cure TB patients which has treated more than 20 thousand patients till date [2].Until December of 2014 there are 554 microscopy centres and 22 GenXpert location in the country which specializes in diagnosis of TB [3].The expansion of DOTS program has proven its efficacy in reducing the mortality and morbidity in Nepal, however, despite of available diagnosis and treatment regimes, approximately 3000-5000 people are still dying per year of TB [4].In a DOTS centre in Kathmandu, prevalence of Multi Drug Resistant (MDR) case of Tuberculosis was found to be 3.6% [5], which could have been mitigated by early diagnosis and rapid identification of anti-TB agents resistance.Recent data from East Nepal suggests that 3.3% of all patients, who were smear microscopy negative, have RMP resistant TB [6].Despite of DOTS and available diagnosis, complications due to MDR are emerging and also there hasn't been overall increment in number of people on anti tuberculosis treatment [6].Sputum smear light microscopy, patients symptoms combined with CXR results is the preferred algorithm for TB diagnosis in Nepal.Smear test is neither relevant nor sensitive enough for the diagnosis of TB alone and needs additional diagnosis as well clinical suspecting to decide either patients should be enrolled on antibiotic or not [7].
Patients with suspected TB (≥18 ye of age) were enrolled between April 2014 and May 2015.WHO algorithm was basis for the classification of suspects of TB.Based on WHO algorithm, two subgroups were formed to classify TB suspects; 1) patients with cough for ≥ 2 weeks and 2) patients without cough for ≥ 2 weeks but having symptoms like weight loss accompanied with night fever T>37.5°C, pleural effusion or pericarditis induced breathlessness, chronic headache, swelled armpit (>2cm) or abnormal chest radiogram, thus referred as, "with cough or without cough''.Patients with WHO defined danger sign with respiratory rate >30/minute, fever >39°C, pulse rate >120/minute and unambulatory patients were excluded from the study.Smear positive were not enrolled for GenXpert thus were directly enrolled for anti TB regime.
Remaining patients with smear negative were enrolled in GenXpert and further confirmation was done by liquid culture for MTB positive and RIF resistant samples.Those cultures were further screened for MDR against first line and second line drugs.Patients with sputum negative for TB were classified as possible TB (if they are on anti-TB treatment program) or as non-TB (if they are not on anti-TB treatment program).Prospective patients (≥20 years of age) with suspected pulmonary TB (n=2091) were enrolled in the study who were attending the DOTS Centre in Parsa District of Public Health Office, Nepal from Jul 2013 to Jan 2015.Patients had at least one symptom of TB including chest radiograph with infiltrates and smear microscopy negative results in duplicate.

Specimen collection and laboratory procedures
Sputum specimens were processed using standardized protocols.The length of time between sample collection and results being issued to the clinic was also recorded.Smears stained with ZN stain methods were examined microscopically following the standard protocol.At least 200 fields were evaluated before reporting negative.Bacillary density was graded as scanty, 1+, 2+, and 3+, and all such smears were defined as ''smear-positive'.Following decontamination with N-acetyl-Lcysteine and sodium hydroxide, centrifuged sputum deposits underwent microscopy, and following re-suspension in phosphate buffer, equal volumes were tested by the GeneXpert MTB/RIF assay.The results of all tests were read by technologists blinded to the outcomes of the other assays.Sputum pellets were also tested by trained technologists using the GeneXpert MTB/RIF assay.

GeneXpert MTB/RIF and patient management
The collected sputum sample was aliquoted in two equal volumes; first half for immediate primary clinical works and other for reference for future works at -20°C.From the first aliquot, the specimen was used for smear tests followed by GeneXpert MTB/RIF assay.The diagnostic assays were followed by a single specialized clinical research associate blind to the patient's diagnosis in reference standard and index test, throughout the study period.

Statistical analysis
Collected data were analyzed and interpreted statistically using graphPad prism version 6.0 and SPSS 17.0.All the values are expressed as mean±SD and are analyzed using Student's t test which is parametric as well Mann-Whitney test wherever applicable.P value (P<0.05), was considered significant unless stated otherwise.The suspected pulmonary TB patients (n= 2091) were enrolled for sputum microscopy and GeneXpert MTB/RIF for the confirmation of TB.Among them, 1301(62.21%)were male and 790 (37.78%) were female.All the patients were referred to this centre by local doctors and physicians after having tuberculosis symptoms and abnormal chest radiograph for sputum microscopy and GeneXpert MTB/RIF for the confirmation of Makwanpur, Mahottari, Panchthar, Saptari, Siraha, Morang, Sindhuli, Rasuwa and Chitwan (Figure 1).Since the study conducted at Parsa district, the actual number of case presented here does not highlight the prevalence of TB in the respective district.As Bara is the adjacent district to Parsa, the number of patients visited is more than other district.The prevalence of TB in Parsa district is 3 times higher than the prevalence in the Bara district.TB.The clinical symptoms showed most of the patients have fever, cough, and weight loss and night sweat (Table 4).The maximum TB cases were from Parsa district (555, 26.5%) followed by Bara, Rautahat, Sarlahi,

Diagnostic accuracy of different method and MTB/RIF performed on uncentrifuged sputum
Direct sputum microscopy is popular worldwide.In Nepal, the Tuberculosis diagnosis and monitoring rely in Sputum microscopy because of its low cost and easier to perform.At present there are 533 microscopy centres catering the sputum microscopy examination throughout the country.The region wise distribution is given in Table 1.Most of the microscopy centres are established in government setting and few are established in nongovernmental organization and private sectors.NTC-National   2 and Figure 3 respectively.6) or TTP.When MTB/RIF accuracy was evaluated using composite reference standard culture positivity from sputum, the sensitivity and specificity were similar to those obtained in the primary analysis using either definite TB versus possible and non-TB combined; definite and possible TB combined versus non-TB.

Discussion
In Nepal, the estimated incidence of TB is 163 per 100,000 with a prevalence rate of 241 per 100,000 populations.In 2014-15, the Nepal Tuberculosis Programme (NTP) registered 17,788 sputum smearpositive cases and 8,367 sputum smear negative cases [6,8].A total of 2091 individuals from thirteen districts of Nepal were included in the present study and the prevalence of smear positive pulmonary tuberculosis was found to be 788 out of 2091(37.06 %).We found the most of the patients were passively participated in the study and almost all patients are visited Government hospital for the treatment.Approximately 66.76% of the patient had the cough duration for last 2 days.Although, only 37.09 % of patients had AFB positive on smear, out of which 97.8 % were new  Although the GeneXpert has several limitations, including requirement for stable electricity supply, limited temperature range, availability of maintenance, and bulky consumables, wider availability of the accurate GeneXpert assay may counter the use of these serological tests by providing a viable alternative to the patient and healthcare provider.It is highly probable that a small number of cases of TB were missed in this study as GeneXpert does not have as high sensitivity as culture.There is a danger that clinicians will "exclude" a TB diagnosis on the basis of a negative GeneXpert test and it is important that education is carried out to ensure clinicians are aware of the test limitations prior to the test being implemented.However, it is not sustainable to implement TB culture facilities at general hospitals in South Asia and the long turnaround of results means loss to follow-up in the diagnostic pathway is high.This study was not an assessment of GeneXpert sensitivity and specificity, as this has been comprehensively evaluated in comparison with culture by others.

Conclusion
The prevalence of TB is high in Parsa DOTS centre in comparable to other districts of Nepal.However, the relatively higher rate of RIF resistance observed in our study signals the danger of increasing MDRTB in the study areas in the future in Nepal.
On the other hand, MTB RIF positivity among sputum negative, RIF resistant (DR TB) among sputum negative and RIF resistant among retreatment cases is large in this study.The higher level of MDR-TB among previously treated patients suggests the need to strengthen the DOTS strategy and the capacity of laboratories to undertake effective treatment especially among previously treated patients in Nepal.The GeneXpet is more specific and sensitive that smear AFB detection.The Government of Nepal Ministry of Health and population should implement GeneXpert in each DOTS centre in Nepal for better diagnosis.

Figure 1 :
Figure 1: District wise number of Tuberculosis Patients visited at GeneXpert Centre in Parsa

Figure 2 :
Figure 2: MDR Pattern in TB Patients

Table 4 :
Clinical Feature of Tuberculosis

Table 1 :
Number of DOTS and Microscopy Centres by Region * Showed the number of DOTS and Microscopy centres in which number of sub-centres are more followed by number of treatment centres and microscopy centres.There is few DR TB treatment centres.(EDR: Eastern Development Region.CDR: Central Development Region; WDR: Western Development Region; MWDR: Mid Western Development Region; FWDR: Far Western Development Region)

Table 3 :
Comparison of different test for diagnosis of

Table 5 :
Pattern of sputum culture with active TB

Table 6 :
Accuracy of Xpert MTB/RIF for the detection of Smear positive TB in sputum