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Background Low income, high-tuberculosis burden, countries are considering selective deployment of Xpert MTB/RIF assay (Xpert) due to high cost per test. We compared the diagnostic gain of the Xpert add-on strategy with Xpert replacement strategy for pulmonary tuberculosis diagnosis among HIV-infected adults to inform its implementation. Methods The first diagnostic sputum sample of 424 HIV-infected adults (67% with CD4 counts ≤200/mm3) suspected for tuberculosis was tested by direct Ziehl-Neelsen (DZN) and direct fluorescent microscopy (DFM); concentrated fluorescent microscopy (CFM); Lowenstein-Jensen (LJ) and Mycobacterial Growth Indicator Tube (MGIT) culture; and Xpert. Overall diagnostic yield and sensitivity were calculated using MGIT as reference comparator. The sensitivity of Xpert in an add-on strategy was calculated as the number of smear negative but Xpert positive participants among MGIT positive participants. Results A total of 123 (29.0%) participants were MGIT culture positive for Mycobacterium tuberculosis. The sensitivity (95% confidence interval) was 31.7% (23.6–40.7%) for DZN, 35.0% (26.5–44.0%) for DFM, 43.9% (34.9–53.1%) for CFM, 76.4% (67.9–83.6) for Xpert and 81.3% (73.2–87.7%) for LJ culture. Add-on strategy Xpert showed an incremental sensitivity of 44.7% (35.7–53.9%) when added to DZN, 42.3% (33.4–51.5%) to DFM and 35.0% (26.5–44.0%) to CFM. This translated to an overall sensitivity of 76.4%, 77.3% and 79.0% for add-on strategies based on DZN, DFM and CFM, respectively, compared to 76.4% for Xpert done independently. From replacement to add-on strategy, the number of Xpert cartridges needed was reduced by approximately 10%. Conclusions Among HIV-infected TB suspects, doing smear microscopy prior to Xpert assay in add-on fashion only identifies a few additional TB cases.
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Clinical Utility of a Novel Molecular Assay in Various
Combination Strategies with Existing Methods for
Diagnosis of HIV-Related Tuberculosis in Uganda
Willy Ssengooba
*, Lydia Nakiyingi
, Derek T. Armstrong
, Frank G. Cobelens
, David Alland
Yukari C. Manabe
, Susan E. Dorman
, Jerrold J. Ellner
, Moses L. Joloba
1Department of Medical Microbiology, College of Health Sciences, Makerere University, Kampala, Uganda, 2Infectious Diseases Institute, College of Health Sciences,
Makerere University, Kampala, Uganda, 3Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 4Department of Global Health and
Amsterdam Institute of Global Health and Development, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands, 5KNCV Tuberculosis Foundation,
The Hague, Netherlands, 6Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts, United States of America
Low income, high-tuberculosis burden, countries are considering selective deployment of Xpert MTB/RIF assay
(Xpert) due to high cost per test. We compared the diagnostic gain of the Xpert add-on strategy with Xpert replacement
strategy for pulmonary tuberculosis diagnosis among HIV-infected adults to inform its implementation.
The first diagnostic sputum sample of 424 HIV-infected adults (67% with CD4 counts #200/mm
) suspected for
tuberculosis was tested by direct Ziehl-Neelsen (DZN) and direct fluorescent microscopy (DFM); concentrated fluorescent
microscopy (CFM); Lowenstein-Jensen (LJ) and Mycobacterial Growth Indicator Tube (MGIT) culture; and Xpert. Overall
diagnostic yield and sensitivity were calculated using MGIT as reference comparator. The sensitivity of Xpert in an add-on
strategy was calculated as the number of smear negative but Xpert positive participants among MGIT positive participants.
A total of 123 (29.0%) participants were MGIT culture positive for Mycobacterium tuberculosis. The sensitivity (95%
confidence interval) was 31.7% (23.6–40.7%) for DZN, 35.0% (26.5–44.0%) for DFM, 43.9% (34.9–53.1%) for CFM, 76.4%
(67.9–83.6) for Xpert and 81.3% (73.2–87.7%) for LJ culture. Add-on strategy Xpert showed an incremental sensitivity of
44.7% (35.7–53.9%) when added to DZN, 42.3% (33.4–51.5%) to DFM and 35.0% (26.5–44.0%) to CFM. This translated to an
overall sensitivity of 76.4%, 77.3% and 79.0% for add-on strategies based on DZN, DFM and CFM, respectively, compared to
76.4% for Xpert done independently. From replacement to add-on strategy, the number of Xpert cartridges needed was
reduced by approximately 10%.
Among HIV-infected TB suspects, doing smear microscopy prior to Xpert assay in add-on fashion only
identifies a few additional TB cases.
Citation: Ssengooba W, Nakiyingi L, Armstrong DT, Cobelens FG, Alland D, et al. (2014) Clinical Utility of a Novel Molecular Assay in Various Combination
Strategies with Existing Methods for Diagnosis of HIV-Related Tuberculosis in Uganda. PLoS ONE 9(9): e107595. doi:10.1371/journal.pone.0107595
Editor: Yoshihiko Hoshino, National Institute of Infectious Diseases, Japan
Received April 11, 2014; Accepted August 14, 2014; Published September 15, 2014
Copyright: ß2014 Ssengooba et al. 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.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: This project was funded with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Departmentof
Health and Human Services, under Contract no. HHSN2722000900050C ‘‘TB Clinical Diagnostics Research Consortium’’. Additional support was provided by
Erasmus Mundus Joint Doctorate Program of the European Union to WS. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: WS, FGC, NL, DA, YM, SED, JJE and MJ have declared that no competing interests exists. David Alland (DA) and DA’s laboratory receive
royalties from licensing fees for the use of molecular beacons in the Cepheid XpertMTB/RIF assay. DA’s personal loyalties with the assay are capped at $4,999 per
year, and royalties payable to DA’s laboratory are at $50,000 per year. DA also received a research grant from Cepheid. This does not alter the authors’ adherence
to all the PLOS ONE policies on sharing data and materials.
* Email:
Tuberculosis (TB) remains the most important opportunistic
infection causing death among HIV-infected individuals in Sub-
Saharan Africa [1]. Microscopic examination of Ziehl-Neelsen
(ZN)-stained sputum smears, the most commonly available
diagnostic in resource-limited settings, has low sensitivity for
TB detection, especially among HIV-infected individuals.
Therefore, at least two sputum specimens need to be tested.
However, in practice, the second smear is difficult to obtain for
logistical reasons [2]. Fluorescence microscopy (FM) is 7–10%
more sensitive for TB detection than conventional light
microscopy [3–5], but the increase in sensitivity among HIV-
infected individuals is often lower than in HIV-uninfected
individuals [6–9].
Sputum culture, generally considered the gold standard for TB
diagnosis, takes several weeks to yield results and may not be
available before individuals are lost to follow-up or even dead [10].
The use of culture for diagnosing TB was recommended by the
PLOS ONE | 1 September 2014 | Volume 9 | Issue 9 | e107595
World Health Organization (WHO) among HIV-infected indi-
viduals [11]. However, it has not been widely scaled-up due to
infrastructure and other resource requirements, shortages of
qualified laboratory personnel, limited access to training for
specific tests and logistical limitations, as well as longer time to
detection [12].
The WHO recently endorsed use of a molecular-based
diagnostic, the Xpert MTB/RIF assay (Xpert; Cepheid, Sunny-
vale, CA, USA) as a frontline diagnostic for TB among HIV-
infected individuals [13]. An all-in-one-cartridge real-time PCR, it
offers a total run time of 2 hours, higher sensitivity, and
simultaneous susceptibility results for rifampicin in one test run.
However, this test is still under-utilized in high TB burden
countries due to the investment cost of the equipment, mainte-
nance costs and the individual cartridge cost [13,14]. Further-
more, its sensitivity for diagnosing TB among smear-negative
individuals ranges from 56–88% [15–18]; in some cases, TB
culture is still required for diagnosis.
Although WHO now recommends that Xpert be used as the
initial test in adults and children presumed to have HIV-
associated TB [19], several low-income countries are adopting
an add-on strategy in which smear microscopy is used first in
individuals clinically suspected to have TB, and if the smear is
negative and the individual is HIV-positive then the sputum is
subsequently tested by Xpert [14,20,21]. This is partly based on
considerations of affordability. Indeed, an economic analysis
suggested that for a low-income country such as Uganda an
add-on strategy had lower diagnostic and treatment costs than a
replacement strategy in which Xpert was used as the first-line
test [14]. However, this analysis did not take into account
possible differences between HIV-positive and HIV-negative
individuals, nor possible dropouts from the diagnostic process,
which may occur if individuals first have to wait for smear
results or come back. Furthermore, previous studies did not
address the effects of testing only one sputum sample, or of
using more sensitive smear microscopy methods [14,21]. In
Uganda, there were 26 health facilities with functional Xpert
services by the end of June 2012 [22]. In financial year 2012/
2013 the number of Xpert machines in Uganda increased to
45, of which 42 (93%) were fully functional and three were
awaiting installation. This was against a National Tuberculosis
Control Program target of 200 Xpert machines by the year
2015. At this Xpert roll-out rate, there will be about 38 Xpert
machines in the coming two years leading to 83 operational
Xpert centers by 2015. The national guidance is to utilize
Xpert among smear negative HIV-infected individuals and for
screening for rifampicin resistance among retreatment TB
patients, due to reasons of affordability and challenges of
optimal procurement of cartridges, in contrast to WHO
recommendation [19]. There thus remains a need to assess
the most effective diagnostic strategy that would be relevant for
routine practice using systematically collected evidence for
effectiveness before implementation [20,23].
In this study, we compared the diagnostic gain of various Xpert
add-on strategies with that of an Xpert replacement strategy for
diagnosis of pulmonary TB among HIV-infected adults to inform
its implementation in Uganda.
Study participants
This was a secondary data analysis from a prospective TB
diagnostics study among HIV-infected participants suspected of
TB disease [24]. It consisted of both inpatients and outpatients of
Mulago National referral Hospital and the Infectious Diseases
Institute (IDI) of Makerere University in Kampala, Uganda. The
participants were HIV-infected TB suspects with at least one sign
or symptom of TB. The enrollment period was January 2011
through November 2011. For the accuracy study, two sputum
samples were collected for direct ZN, direct FM, concentrated FM
and culture on both solid and liquid media. Blood samples for
CD4 cell count and for culture were collected at study enrollment.
For the present analysis, we considered a single sputum sample
only, namely the sample submitted by the participant on the first
diagnostic encounter.
Figure 1. Hypothetical Xpert implementation in either a replacement or an add-on strategy. DZN = Direct Ziehl Neelsen, DFM = Direct
Fluorescent Microscopy, CFM = Concentrated Fluorescent Microscopy, Xpert = Xpert MTB/RIF test.
Utility of Xpert MTB/RIF Assay in Various Combination Strategies
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Laboratory procedures
Mycobacteriological laboratory procedures were done at the
Mycobacteriology (BSL-3) laboratory which is under the
Medical Microbiology department of Makerere University. All
examinations were according to standard procedures for culture,
direct and concentrated smears for microscopy [25]. Briefly
smears were made from unprocessed sputum samples, stained
using standard reagents and examined by light for direct ZN
microscopy (DZN) or direct auramine O-stained FM (DFM) at
6100 and 640 objectives respectively (Olympus CX31 with
LED attachment, Olympus Corporation, Tokyo, Japan). Smear
results were reported as scanty, 1+,2+and 3+using the WHO
grading system [26]. Positive results were issued within 24 hours
to the requesting physicians to initiate TB treatment. The
remaining sputum samples were processed by digestion and
decontamination with NALC/NAOH 2% for 15 min, followed
by dilution with phosphate buffer (PH 6.8) up to 45 ml. The
homogenized sample was then centrifuged at 3000gfor 15 min.
The sediment was re-suspended with phosphate buffer (pH 6.8)
to a volume of 2 ml, of which 0.5 ml was inoculated in MGIT
and two LJ tubes per sample for mycobacterial culture. At this
point, a smear from the suspension was made for concentrated
FM (CFM). Residual aliquots were stored frozen at 280uC.
Cultures on LJ were incubated at 37uc for up to 8 weeks and
the MGIT in a machine for up to 6 weeks.
All cultures with growth were sub-cultured on blood agar to
exclude contamination and a smear was examined by ZN smear
microscopy. Specimen positive for acid-fact bacilli (AFB) under-
went Capillia Neo TB (TAUN, Numazu, Japan) testing. Capillia
positive specimens were classified as Mycobacterium tuberculosis
complex (MTB) and those negative as Non-tuberculous Mycobac-
teria (NTM). Those that were AFB-negative and had growth on
blood agar were classified as contaminated and those without
growth throughout were classified as negative.
The Xpert assay was performed on the leftover sputum pellets
from culture. Samples were thawed from 280uC to room
temperature. Procedures for Xpert were done using a 1:3 (sample:
sample reagent) dilution. This was vigorously mixed and incubated
at room temperature for 15 minutes and one mL of the mixture
was transferred to the Xpert cartridge. The cartridge was then
inserted into the Xpert machine; processing and result interpre-
tation is automated, using software version 4.0.
Statistical analysis
Data were double entered in an electronic database (MS-Access,
Microsoft Corp, Seattle WA, USA); discrepancies were solved by
checking the entries against the raw data. Data were exported to
Table 1. Characteristics and CD4 cell count categories of participants enrolled (N = 424).
Parameter Number (%)
Female 269 (63.4)
Median age(IQR) 32 (32–34)
MGIT culture positive TB cases 123 (29.0)
CD4 cell cou nt among MGIT confirmed TB cases (N = 419)
Total (N) 121 (28.9)
Median CD4/mm
(IQR) 67 (43–92)
CD4,50 (n = 164) 52 (31.7)
CD4 51–200 (n = 117) 45 (38.4)
CD4.200 (138) 24 (17.4)
Grew NTM on MGIT culture 19 (4.5)
CD4,50 (n = 164) 12 (7.3)
CD4 51–200 (n = 117) 3 (2.6)
CD4.200 (138) 4 (2.9)
Key: IQR = Inter Quartile Range, MGIT = Mycobacterial Growth Indicator Tube, NTM = Non Tuberculous Mycobacteria.
Table 2. Comparison of Xpert MTB/RIF method with sputum culture.
Xpert MTB/RIF results by MGIT status of corresponding sputum specimen
MTB positive
NTM positive but
MTB negative Negative for NTM and MTB
Xpert MTB/RIF positive 94 3 7
Xpert MTB/RIF negative 29 16 275
Xpert MTB/RIF results by LJ status of corresponding sputum specimen
Xpert MTB/RIF positive 89 1 14
Xpert MTB/RIF negative 15 2 303
Key: LJ = Lowenstein Jensen, MGIT = Mycobacterial Growth Indicator Tube, Xpert = Xpert MTB/RIF test, NTM = Non Tuberculous Mycobacteria.
Utility of Xpert MTB/RIF Assay in Various Combination Strategies
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Stata v11 (Stata Corp, College Station TX, USA) for analysis. We
used the exact binomial method for calculating 95% confidence
intervals and the 2-sided Fisher’s exact test for comparing
Diagnostic yield was defined as the observed number of TB
cases detected by each test employed. Sensitivity per test was
calculated as the proportion positive using MGIT culture as the
reference comparator. The incremental sensitivity for an add-on
strategy of Xpert to smear microscopy was calculated as the
number of participants positive by Xpert but negative on the
smear microscopy method divided by the total number of TB cases
detected by MGIT culture (Figure 1).
Ethics statement
The study protocol was approved by Makerere University
School of Public Health Institutional Review Board (MUSPHIRB)
and the Uganda National Council of Science and Technology
(UNCST). All participants gave written informed consent.
Characteristics of participants
A total of 523 HIV-infected participants were screened for the
study and 498 had the first sputum sample on the first diagnostic
encounter. A total of 74 participants were excluded from this
analysis: 19 (3.8%) due to LJ contamination, 46 (9.2%) due to
MGIT contamination, and 9 (1.8%) due to contamination on both
LJ and MGIT culture. There were no missing or invalid Xpert
results, leaving 424 participants for the analysis. Table 1 shows the
characteristics of participants included in this analysis.
Comparative performance of the methods used for
diagnosis of HIV-related tuberculosis
Overall 123 (29.0%) of the participants were microbiologically
confirmed TB cases by MGIT culture. Xpert was negative for two
specimens with DZN positive smears (one graded scanty and one
graded 1+) of which one was culture negative, for three specimens
with DFM positive smears (all graded scanty) of which two were
culture positive for M. tuberculosis, and for four specimens with
CFM positive smears (one graded scanty, two 1+and one 2+)of
which three were M. tuberculosis culture positive. The total
number of culture positive TB cases who were smear positive but
Xpert negative were four of which DZN detected one, DFM
detected two and CFM detected all those detected by DZN and
DFM with additional two TB cases (Table S1).
Of the Xpert-positive specimens, four were resistant to
rifampicin, also confirmed by phenotypic drug susceptibility
testing using MGIT. These related to two participants with CD4
cell count between 51 and 200, and two participants with CD4.
200 cells/mm
Of the five participants that were smear-positive but MGIT
culture-negative for M. tuberculosis, three grew NTM. Table 2
shows Xpert results compared with LJ and MGIT culture.
Diagnostic yield and sensitivity of the methods used for
diagnosis of HIV-related tuberculosis
Among MGIT culture positive TB patients, sputum culture by
LJ had the same yield as Xpert 104 (24.5%), however, LJ culture
had a slightly higher sensitivity of 100 (81.3%) compared to Xpert
test, 94 (76.4%) (Table 3). Xpert test detected slightly more TB
Table 3. Overall yield and sensitivity using MGIT as the reference comparator, for each of the test methods.
overall (n = 424) MGIT culture positive (n = 123)
Test Yield n (%) 95% CI Sensitivity n (%) 95% CI
DZN 42 (9.9) 7.2–13.1 39 (31.7) 23.6–40.7
DFM 46 (10.8) 8.0–14.2 43 (35.0) 26.5–44.0
CFM 58 (13.7) 10.5–17.3 54 (43.9) 34.9–53.1
Xpert 104 (24.5) 20.5–28.9 94 (76.4) 67.9–83.6
LJ 104 (24.5) 20.5–28.9 100 (81.3) 73.2–87.7
MGIT 123 (29.0) 24.7–33.5 N/A N/A
Key: Direct Ziehl Neelsen, DFM = Direct Fluorescent Microscopy, CFM = Concentrated Fluorescent Microscopy, LJ = Lowenstein Jensen, MGIT = Mycobacterial
Growth Indicator Tube, Xpert = Xpert MTB/RIF test, CI = Confidence Interval.
Table 4. Overall yield of tests, by CD4 cell count at study enrollment (n = 419).
.200 n (%)
N = 138 95% CI
51–200 n (%)
N = 117 95% CI
,/ = 50 n (%)
N = 164 95% CI
DZN 11 (8.0) 4.0–13.8 13 (11.1) 6.0–18.2 18 (11.0) 6.6–16.7
DFM 11 (8.0) 4.0–13.8 14 (12.0) 6.6–19.2 21 (12.8) 8.1–18.9
CFM 15 (10.9) 6.2–17.2 18 (15.4) 9.3–23.2 25 (15.2) 10.1–21.6
Xpert 25 (18.1) 12.1–25.5 34 (29.1) 21.0–38.1 44 (26.8) 20.2–34.2
LJ 19 (13.8) 8.4–20.6 37 (31.6) 23.3–40.8 46 (28.0) 21.3–35.5
MGIT 24 (17.4) 11.4–24.7 45 (38.5) 29.6–47.9 52 (31.7) 24.6–39.4
Key: Direct Ziehl Neelsen, DFM = Direct Fluorescent Microscopy, CFM = Concentrated Fluorescent Microscopy, LJ = Lowenstein Jensen, MGIT = Mycobacterial
Growth Indicator Tube, Xpert = Xpert MTB/RIF test, CI = Confidence Interval.
Utility of Xpert MTB/RIF Assay in Various Combination Strategies
PLOS ONE | 4 September 2014 | Volume 9 | Issue 9 | e107595
cases than LJ, only among participants with CD4 cell count .200
(Table 4).The sensitivity of Xpert decreased near-
significantly from 91.7% (95% CI 73.0–98.9%) among partici-
pants with CD4 cell count .200 cells/mm
to 73.2% (95% CI
63.2–81.7%) among participants with CD4 cell count #200 cells/
(p = 0.062). There was almost no difference in sensitivity of
Xpert between participants with CD4 cell count 50–200 cell/mm
and participants with cell count ,50 CD4 cell/mm
(68.9% vs
76.9%, p = 0.491) (Table 5).
Performance of Xpert test in an add-on strategy for TB
diagnosis among HIV-infected participants
Xpert resulted in an incremental sensitivity (IS) of 55/123
(44.7%; 95% CI 35.7–53.9%) when added to DZN, 52/
123(42.3%; 33.4–51.5%) when added to DFM and 43/123
(35.0%; 26.5–44.0%) when added to CFM. This translates into
a combined sensitivity of 76.4%, 77.3% and 78.9% for Xpert done
after a positive DZN, DFM and CFM smear examination,
respectively, compared to the sensitivity of 76.4% for Xpert done
independently (Figure 2).
In this study population one would, for the add-on strategies,
need to do 424 smear examinations, plus 385 Xperts for DZN, 381
Xperts for DFM, and 370 Xperts for CFM, whereas for Xpert
done independently one would do 424 Xpert tests. Using an add-
on strategy one would thus require 9.2% (95% CI 6.6–12.3%)
fewer Xpert tests when combined with DZN, 10.1% (7.4–13.4%)
fewer Xpert tests when combined with DFM and 12.7% (9.7–
16.2%) fewer Xpert tests when combined with CFM.
We have documented that among HIV-infected PTB suspects,
an add-on strategy in which smear examination is done first and
if negative followed by Xpert only identifies a few additional TB
cases compared to a strategy in which Xpert is used as the first-
line test. There is only 0–2.6% additional sensitivity for an add-
on strategy and the proportion of Xpert tests saved by an add-on
strategy is only around 10% (Figure 2). The smear examination
method having the most sensitivity in an add-on strategy was FM
after concentration by centrifugation, an elaborate method that
generally cannot be performed in peripheral laboratories
(Figure 2).
We found an increase in diagnostic gain from direct ZN to
fluorescent microscopy that agrees with previous studies [6,7,27]
(Table 3). Xpert detected more TB cases than any of the smear
microscopy methods irrespective of concentration; moreover,
smear microscopy is less specific as we have documented smear-
positive participants with NTM in culture that were negative by
Xpert. In agreement with previous studies [15,16], this may be
because for a positive result, Xpert requires fewer bacilli per mL of
sputum sample leading to detection of TB cases that are likely to
be missed by the smear examination methods that require more
bacilli per mL, and Xpert targets gene sequences that are specific
to M. tuberculosis[15,28]. However, Xpert did give apparent false-
positive results, including in 3 of 19 specimens that grew NTM but
no M. tuberculosis on MGIT (Table 2). Although this seems to
suggest that the specificity of Xpert for M. tuberculosis is below
100%, it may also be that the sensitivity of MGIT to detect M.
tuberculosis in these patients was incomplete, or that the NTM-
positive participants also had M. tuberculosis, which might have
been outcompeted in MGIT culture [29,30].
Sputum culture by LJ detected fewer cases than MGIT, as
previously documented [31,32]. LJ had the same yield as Xpert
although it had slightly higher sensitivity (Table 3). The high
diagnostic yield of Xpert obviates the need to do LJ culture, given
the costly infrastructure and specialized staff requirements for the
latter. Despite MGIT culture having a higher yield than the other
methods used, it is more costly with more requirements for its use
in routine TB diagnosis. Xpert assay also rapidly detected four
rifampicin resistant participants. Using Xpert assay could thereby
increase the number of individuals who are started timely on
appropriate second-line treatment. Culture using MGIT detected
more TB cases when compared with Xpert partly due to lower
detection threshold in terms of number of bacilli per mL that is
required for the MGIT culture to be positive as compared to a
molecular assay like Xpert [33]. However, the long time to
detection by culture that delays treatment initiation may not make
Table 5. Sensitivity per test by CD4 cell count among MGIT confirmed TB cases (n = 121).
.200 n (%)
N = 24 95% CI
51–200 n (%)
N = 45 95% CI
,/ = 50 n (%)
N = 52 95% CI
DZN 11 (45.8) 25.5–67.1 11 (24.4) 12.8–39.5 17 (32.7) 20.3–47.1
DFM 11 (45.8) 25.5–67.1 12 (26.7) 14.6–41.9 20 (38.5) 25.3–52.9
CFM 14 (58.3) 36.6–77.8 16 (35.6) 21.8–51.2 24 (46.2) 32.2–60.5
Xpert 22 (91.7) 73.0–98.9 31 (68.9) 53.3–81.8 40 (76.9) 63.1–87.4
LJ 19 (79.2) 57.8–92.8 36 (80.0) 65.4–90.4 43 (82.7) 69.6–91.7
Key: Direct Ziehl Neelsen, DFM = Direct Fluorescent Microscopy, CFM = Concentrated Fluorescent Microscopy, LJ = Lowenstein Jensen, MGIT = Mycobacterial
Growth Indicator Tube, Xpert = Xpert MTB/RIF test, CI = Confidence Interval.
Figure 2. Sensitivity and incremental sensitivity of Xpert
among smear negative MGIT culture confirmed TB cases
(n = 123). DZN = Direct Zielh Neelsen, DFM = Direct Fluorescent
Microscopy, CFM = Concentrated Fluorescent Microscopy, Xpert =
Xpert MTB/RIF test, IS = Incremental sensitivity.
Utility of Xpert MTB/RIF Assay in Various Combination Strategies
PLOS ONE | 5 September 2014 | Volume 9 | Issue 9 | e107595
it a better test for tuberculosis in individuals with HIV infection,
who have high mortality if left untreated [34].
On the other hand and as previously documented [15], the
sensitivity of Xpert, as well as of all smear examination methods
per test decreased when CD4 cell counts decreased below 200
, while there was no further decrease in sensitivity when
the CD4 cell count group of 50–200 cell/mm
was compared to
that of .50 cell/mm
(Table 4 and Table 5). This trend
emphasizes the need to use tests that are more sensitive in HIV-
infected individuals with CD4 cell counts below 200 cell/mm
such as molecular-based test and culture.
When we analyzed our findings in terms of incremental
sensitivity after smear microscopy, the added benefit of combining
Xpert with smear examination in an add-on strategy was small
(Figure 2). In addition, there were only limited savings in terms of
fewer Xpert tests done, whereas there will be additional costs and
potential diagnostic delays due to the smear examinations.
The limited benefit of an add-on strategy could be related to the
specific nature of this study population of HIV-infected individuals
with on average low CD4 counts (two-thirds #200 cells/mm
This reflects the likely operational cohort of HIV-infected smear-
negative TB cases in Uganda and other low income countries
where an add-on strategy is considered [21]. The sensitivity of
smear examination of a single sputum sample from an HIV-
infected individual is rather low (32–44%), whereas that of Xpert is
relatively high. Moreover, one would not want to delay diagnosis
of smear-negative TB in an HIV-infected individual because of the
high mortality [34].
Our study had limitations. We performed Xpert on previously
frozen processed sputum samples. However, previous studies
showed that this is unlikely to produce different results from testing
unprocessed samples [15]. Furthermore, we considered only one
sputum samples in our analysis, which will have limited the
sensitivity compared to testing two or more samples, including an
early morning sample or in a frontloading system as generally
recommended [31,35,36]. In practice however, often, a single
sputum sample is available and we intended to evaluate the
diagnostic value of add-on versus replacement strategies for that
situation. We calculated sensitivity relative to MGIT culture,
which is not 100% sensitive itself. Xpert positive and MGIT
culture with NTM are likely to be true positives, underestimating
the incremental sensitivity of Xpert. Moreover, NTM positive
cultures were considered negative, among which Xpert identified
additional TB cases. Finally, we used a rapid identification assay to
confirm culture positive samples for MTB complex, however,
previous studies have documented cases of false-negative results,
which could have affected the discriminatory power of culture
methods for NTM [37,38]. Future studies with more robust
identification methods such as 16s sequence based identification
on smear positive antigen test negative cultures are recommended.
Our findings support a replacement strategy (i.e. Xpert only) for
implementing Xpert for diagnosing TB among HIV-infected
individuals if Xpert is available on-site. If Xpert is not available on-
site, an add-on strategy may be helpful since, as in our study, 32–
44% of HIV-infected individuals with TB may already be detected
upon smear examination of a first sputum sample. These
individuals, or their sputum specimens, then do not need to be
referred to a clinic where Xpert tests can be performed, with
obvious savings in terms of transport cost, delays and potentially
dropout of the diagnostic process and related mortality. Indeed,
despite WHO recommendations and subsidization of cartridges
the availability at point-of-care level of Xpert will likely increase
only slowly, given the cost of the equipment and importation of
cartridges, and the difficulties in resource-constrained settings with
machine calibration and maintenance. An add-on strategy in
which smear negative samples are referred for Xpert testing will
only be economically efficient (transport, sample packaging, time-
wise) if Xpert machines are decentralized to testing hubs within
the district or to district reference laboratories. Further studies are
needed to evaluate the cost-effectiveness of various strategies for
implementing Xpert under routine, programmatic conditions
before being scaled-up in resource-limited settings.
Supporting Information
Table S1 Comparative performance of Xpert, LJ and
MGIT by smear microscopy status for diagnosis of HIV-
related tuberculosis.
The Authors wish to thank the Infectious Disease Institute Makerere
University for co-ordination support as well as the Mycobacteriology (BSL-
3) laboratory in department of medical microbiology Makerere University
for specimen analysis. Data analysis support from Ms. Sandra Armakovitch
of Boston Medical center USA. We also acknowledge the comprehensive
review of the previous versions by Prof. Dr. Bouke de Jong head of the Unit
of Mycobacteriology Institute of Tropical Medicine (ITM), Antwerp. We
also acknowledge the support from Erasmus Mundus Joint Doctorate
Program of the European Union that supported WS as a doctoral
candidate during data analysis and manuscript writing.
Author Contributions
Conceived and designed the experiments: WS. Performed the experiments:
WS LN MLJ DTA DA. Analyzed the data: WS FGC. Contributed
reagents/materials/analysis tools: MLJ JJE SED YCM. Contributed to the
writing of the manuscript: WS LN DTA FGC DA YCM SED JJE MLJ.
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... In TB/HIV endemic regions with limited resources, sputum microscopy is usually the only method available for diagnosis 4,32,34,42 . The use of only this technique to screen for TB in the population co-infected with HIV restricts its potential diagnosis because approximately 10,000 organisms per milliliter are needed in the sputum to perform the test, an uncommon amount among the population living with HIV/AIDS 34 . ...
... GeneXpert test is a sensitive, specific 43 , simple, and innovative method whose objective, since its implementation, has been to detect the presence of M. tuberculosis and resistance to rifampicin within 2 hours 6,[22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]44 . The method has an increased screening capacity 43 , besides requiring minimal bacillary concentrations in the samples for the examination 32 . ...
... Other studies do not address the simultaneous performance of both tests but point out that the use of GeneXpert substantially increased the detection of TB cases among the population co-infected with HIV/AIDS 24 The WHO recommendation to use GeneXpert, instead of sputum smear microscopy as the initial test of choice in HIV-infected individuals susceptible to TB 6 , corroborates the findings of studies that have shown that GeneXpert's sensitivity in diagnosing TB in people living with HIV ranged from 68% 31 to 100% 34 (average 81.1%), being greater than the sensitivity of sputum smear microscopy 6,23,[25][26][27][30][31][32]34,37 , which ranged from 23.0% 26 to 66,7% 37 (average 43.3%). Given the above, the implementation of GeneXpert, a fast and accurate test in different clinical contexts, has led to a significant increase in the diagnosis of pulmonary TB, both when used as a first-choice test and as a complement 45 . ...
Full-text available
Objective: To identify and synthesize the scientific knowledge produced regarding the effectiveness of the GeneXpert test in the diagnosis of pulmonary tuberculosis (TB) in people living with HIV/AIDS. Methods: Integrative literature review, which was searched on Embase, Scopus, PubMed, Cinahl, Academic Search Premier, Socindex, and LILACS platforms, in December 2019. The studies surveyed went through two stages of selection: reading of titles and abstracts by two reviewers independently; using the Rayyan platform and reading. Nineteen primary studies in English, Portuguese, and Spanish that answered the study's guiding question were included: How effective is the GeneXpert test in the diagnosis of pulmonary TB in people living with HIV/AIDS? Results: The use of GeneXpert substantially increased the detection of TB cases among the population co-infected with HIV/AIDS, with sensitivity ranging from 68% to 100%, superior to sputum smear microscopy. Specificity ranged from 91.7% to 100%; the positive predictive value from 79.2% to 96.1%; and the negative predictive value from 84.6% to 99.3%. These values were considered similar to sputum smear microscopy by most studies. We also compared these results with different ways of performing culture and other molecular tests, being considered inferior only to the Xpert Ultra. Conclusion: It is possible to affirm that places with a high incidence of HIV/AIDS would benefit from the implementation of the GeneXpert test, entailing effectiveness in diagnosing pulmonary TB in this population when compared to sputum smear microscopy, a widely used test for detection of cases.
... 9,[12][13][14][15] The performance of GeneXpert MTB/RIF ® has been shown to be better compared to LED FM in both smear positive and negative people living with HIV (PLWH), though variability exists depending on the population involved. [16][17][18] The rifampin resistance has been shown to vary in different countries from 0% in Mbeya (Tanzania), 10% in Harare (Zimbabwe) to as high as 35.1% in Moldova. 16,19 In response to WHO call to scale up the utilization of this new diagnostic, the Ministry of Health in the United Republic of Tanzania, through the National Tuberculosis and Leprosy Control Program (NTLP) 3,9 and other developmental partners, has cordially rolled out the GeneXpert MTB/RIF ® machines to various regions. ...
... But the cost-related challenges for the universal introduction of GeneXpert MTB/RIF ® in many health facilities in developing countries reiterate the need to continue strengthening the pre-existing microscopy-based TB diagnostic methods, so that the newer technique remains reserved to risky groups like smear negative PLWH, PTB patients who recently contacted MDR, and children. 3 The incremental detection of TB among smear negative PLWH in this and other studies 17,18,21,22 further justifies its utility in this risky group as recommended by the new NTLP guidelines. 3 The use of GeneXpert MTB/RIF ® to detect RIF resistance as a surrogate marker of MDR has been suggested in many studies, with concordance ranging from 88 to 100%. ...
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Laboratory confirmation among presump-tive tuberculosis (PTB) patients is pivotal in ensuring prompt management. Limited information exists in Tanzania regarding the performance of GeneXpert MTB/RIF ® in comparison with conventional methods. An operational study was conducted involving 806 PTB patients at Sekou Toure Hospital in Mwanza, Tanzania from June to November 2013. Patients' information was obtained and their respective sputum samples analyzed by light-emitting diode fluorescent microscopy (LED FM) and GeneXpert MTB/RIF ®. The mean age of study participants was 39.6±16.0 years, with males accounting for 50.5%. The majority of patients (97.5%) were new cases. The proportions of PTB patients confirmed by LED FM and GeneXpert MTB/RIF ® were 14.1% (114/806) and 23.7% (191/806) respectively, resulting into a 9.6% incremental detection rate by GeneXpert MTB/RIF ® over LED FM. The detection rate among HIV positive individuals was also higher [23.6% (63/267) vs 14.2% (38/267), respectively], with an incremental detection of 9.4%. The incremental detection of PTB by GeneXpert MTB/RIF ® over LED FM calls for expansion of its use to increase detection of smear negative PTB among people living with HIV.
... The demand for rapid TB diagnostics led to the development and introduction of commercial molecular tests into practice. Two commonly used tests in Uganda are the "Xpert MTB/RIF" (GeneXpert, Cepheid Inc.), which diagnoses TB and detects rifampicin resistance [7][8][9][10][11], and the GenoType MTBDRplus (line probe assay [LPA] from Hain Life Sciences Nehren, Germany), which detects Mycobacterium tuberculosis and its resistance to both rifampicin and isoniazid [12][13][14]. Rifampicin and isoniazid are the most important anti-tuberculosis drugs, and resistance to both drugs is commonly referred to as "multidrug resistance" (MDR-TB) [1]. ...
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Background Accurate diagnosis of tuberculosis, especially by using rapid molecular assays, can reduce transmission of drug resistant tuberculosis in communities. However, the frequency of resistance conferring mutations varies with geographic location of Mycobacterium tuberculosis, and this affects the efficiency of rapid molecular assays in detecting resistance. This has created need for characterizing drug resistant isolates from different settings to investigate frequencies of resistance conferring mutations. Here, we describe the prevalence and patterns of rifampicin- and isoniazid- resistance conferring mutations in isolates from Uganda, which could be useful in the management of MDR-TB patients in Uganda and other countries in sub-Saharan Africa. Results Ninety seven M. tuberculosis isolates were characterized, of which 38 were MDR, seven rifampicin-resistant, 12 isoniazid-mono-resistant, and 40 susceptible to rifampicin and isoniazid. Sequence analysis of the rpoB rifampicin-resistance determining region (rpoB/RRDR) revealed mutations in six codons: 588, 531, 526, 516, 513, and 511, of which Ser531Leu was the most frequent (40%, 18/45). Overall, the three mutations (Ser531Leu, His526Tyr, Asp516Tyr) frequently associated with rifampicin-resistance occurred in 76% of the rifampicin resistant isolates while 18% (8/45) of the rifampicin-resistant isolates lacked mutations in rpoB/RRDR. Furthermore, sequence analysis of katG and inhA gene promoter revealed mainly the Ser315Thr (76%, 38/50) and C(-15)T (8%, 4/50) mutations, respectively. These two mutations combined, which are frequently associated with isoniazid-resistance, occurred in 88% of the isoniazid resistant isolates. However, 20% (10/50) of the isoniazid-resistant isolates lacked mutations both in katG and inhA gene promoter. The sensitivity of sequence analysis of rpoB/RRDR for rifampicin-resistance via detection of high confidence mutations (Ser531Leu, His526Tyr, Asp516Tyr) was 81%, while it was 77% for analysis of katG and inhA gene promoter to detect isoniazid-resistance via detection of high confidence mutations (Ser315Thr, C(-15)T, T(-8)C). Furthermore, considering the circulating TB genotypes in Uganda, the isoniazid-resistance conferring mutations were more frequent in M. tuberculosis lineage 4/sub-lineage Uganda, perhaps explaining why this genotype is weakly associated with MDR-TB. Conclusion Sequence analysis of rpoB/RRDR, katG and inhA gene promoter is useful in detecting rifampicin/isoniazid resistant M. tuberculosis isolates in Uganda however, about ≤20% of the resistant isolates lack known resistance-conferring mutations hence rapid molecular assays may not detect them as resistant.
... It ignores the dropout rates associated with testing patients being asked to provide more than one sample on different days. Despite the WHO recommendations and the reduced cost of cartridges, the availability and deployment of the Xpert MTB/RIF as a point of care test will be slow due to the initial cost associated with procurement of the equipment and cartridges and difficulties resource limited settings experience in equipment calibration and maintenance [15]. ...
... It ignores the dropout rates associated with testing patients being asked to provide more than one sample on different days. Despite the WHO recommendations and the reduced cost of cartridges, the availability and deployment of the Xpert MTB/RIF as a point of care test will be slow due to the initial cost associated with procurement of the equipment and cartridges and difficulties resource limited settings experience in equipment calibration and maintenance [15]. ...
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Background Xpert MTB/RIF assay is a highly sensitive test for TB diagnosis, but still costly to most low-income countries. Several implementation strategies instead of frontline have been suggested; however with scarce data. We assessed accuracy of different Xpert MTB/RIF implementation strategies to inform national roll-out. Methods This was a cross-sectional study of 1,924 adult presumptive TB patients in five regional referral hospitals of Uganda. Two sputum samples were collected, one for fluorescent microscopy (FM) and Xpert MTB/RIF examined at the study site laboratories. The second sample was sent to the Uganda Supra National TB reference laboratory for culture using both Lowenstein Jensen (LJ) and liquid culture (MGIT). We compared the sensitivities of FM, Xpert MTB/RIF and the incremental sensitivity of Xpert MTB/RIF among patients negative on FM using LJ and/or MGIT as a reference standard. Results A total 1924 patients were enrolled of which 1596 (83%) patients had at least one laboratory result and 1083 respondents had a complete set of all the laboratory results. A total of 328 (30%) were TB positive on LJ and /or MGIT culture. The sensitivity of FM was n (%; 95% confidence interval) 246 (63.5%; 57.9–68.7) overall compared to 52 (55.4%; 44.1–66.3) among HIV positive individuals, while the sensitivity of Xpert MTB/RIF was 300 (76.2%; 71.7–80.7) and 69 (71.6%; 60.5–81.1) overall and among HIV positive individuals respectively. Overall incremental sensitivity of Xpert MTB/RIF was 60 (36.5%; 27.7–46.0) and 20 (41.7%; 25.5–59.2) among HIV positive individuals. Conclusion Xpert MTB/RIF has a higher sensitivity than FM both in general population and HIV positive population. Xpert MTB/RIF offers a significant increase in terms of diagnostic sensitivity even when it is deployed selectively i.e. among smear negative presumptive TB patients. Our results support frontline use of Xpert MTB/RIF assay in high HIV/TB prevalent countries. In settings with limited access, mechanisms to refer smear negative sputum samples to Xpert MTB/RIF hubs are recommended.
... Secondary, the low samples size obtained from ILs which probably masks the obvious significant incremental detection of Xpert; among the 600 participants of this study, only 210 (35 %) participants and 20 (20.8 %) pulmonary tuberculosis confirmed cases were from ILs. The few numbers of HIV-infected patients in this study could not allow for meaningful comparison of incremental sensitivity by HIV-status, however, significant Xpert IS among HIV-infected patients was previously documented [22]. Lastly, excluding samples which became contaminated or positive for an NTM from the analysis may have led to an under-estimation of the sensitivity of Xpert and smear microscopy, as tuberculosis cannot be definitively excluded for these patients. ...
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Background: Tuberculosis control program of Rwanda is currently phasing in light emitting diode-fluorescent microscopy (LED-FM) as an alternative to Ziehl-Neelsen (ZN) smear microscopy. This, alongside the newly introduced Xpert (Cepheid, Sunnyvale, CA, USA) is expected to improve diagnosis of tuberculosis and detection of rifampicin resistance in patients at health facilities. We assessed the accuracy of smear microscopy and the incremental sensitivity of Xpert at tuberculosis laboratories in Rwanda. Methods: This was a cross-sectional study involving four laboratories performing ZN and four laboratories performing LED-FM microscopy. The laboratories include four intermediate (ILs) and four peripheral (PLs) laboratories. After smear microscopy, the left-over of samples, of a single early-morning sputum from 648 participants, were tested using Xpert and mycobacterial culture as a reference standard. Sensitivity of each test was compared and the incremental sensitivity of Xpert after a negative smear was assessed. Results: A total of 96 presumptive pulmonary tuberculosis participants were culture positive for M. tuberculosis. The overall sensitivity in PL of ZN was 55.1 % (40.2-69.3 %), LED-FM was 37 % (19.4-57.6 %) and Xpert was 77.6 % (66.6-86.4 %) whereas in ILs the same value for ZN was 58.3 % (27.7-84.8 %), LED-FM was 62.5 % (24.5-91.5 %) and Xpert was 90 (68.3-98.8 %). The sensitivity for all tests was significantly higher among HIV-negative individuals (all test p <0.05). The overall incremental sensitivity of Xpert over smear microscopy was 32.3 %; p < 0.0001. The incremental sensitivity of Xpert was statistically significant for both smear methods at PL (32.9 %; p = 0.001) but not at the ILs (30 %; p = 0.125) for both smear methods. Conclusions: Our study findings of the early implementation of the LED-FM did not reveal significant increment in sensitivity compared to the method being phased out (ZN). This study showed a significant incremental sensitivity for Xpert from both smear methods at peripheral centers where majority of TB patients are diagnosed. Overall our findings support the recommendation for Xpert as an initial diagnostic test in adults and children presumed to have TB.
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Background: To estimate the diagnostic accuracy of Xpert MTB/RIF for rifampicin resistance in different regions, a meta-analysis was carried out. Methods: Several databases were searched for relevant studies up to March 3, 2019. A bivariate random-effects model was used to estimate the diagnostic accuracy. Results: We identified 97 studies involving 26,037 samples for the diagnosis of rifampicin resistance. The pooled sensitivity, specificity and AUC of Xpert MTB/RIF for rifampicin resistance detection were 0.93 (95% CI 0.90-0.95), 0.98 (95% CI 0.96-0.98) and 0.99 (95% CI 0.97-0.99), respectively. For different regions, the pooled sensitivity were 0.94(95% CI 0.89-0.97) and 0.92 (95% CI 0.88-0.94), the pooled specificity were 0.98 (95% CI 0.94-1.00) and 0.98 (95% CI 0.96-0.99), and the AUC were 0.99 (95% CI 0.98-1.00) and 0.99 (95% CI 0.97-0.99) in high and middle/low income countries, respectively. The pooled sensitivity were 0.91 (95% CI 0.87-0.94) and 0.91 (95% CI 0.86-0.94), the pooled specificity were 0.98 (95% CI 0.96-0.99) and 0.98 (95% CI 0.96-0.99), and the AUC were 0.98 (95% CI 0.97-0.99) and 0.99 (95% CI 0.97-0.99) in high TB burden and middle/low prevalence countries, respectively. Conclusions: The diagnostic accuracy of Xpert MTB/RIF for rifampicin resistance detection was excellent.
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Background: Xpert MTB/RIF (Xpert MTB/RIF) and Xpert MTB/RIF Ultra (Xpert Ultra), the newest version, are the only World Health Organization (WHO)-recommended rapid tests that simultaneously detect tuberculosis and rifampicin resistance in persons with signs and symptoms of tuberculosis, at lower health system levels. A previous Cochrane Review found Xpert MTB/RIF sensitive and specific for tuberculosis (Steingart 2014). Since the previous review, new studies have been published. We performed a review update for an upcoming WHO policy review. Objectives: To determine diagnostic accuracy of Xpert MTB/RIF and Xpert Ultra for tuberculosis in adults with presumptive pulmonary tuberculosis (PTB) and for rifampicin resistance in adults with presumptive rifampicin-resistant tuberculosis. Search methods: We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, Web of Science, Latin American Caribbean Health Sciences Literature, Scopus, the WHO International Clinical Trials Registry Platform, the International Standard Randomized Controlled Trial Number Registry, and ProQuest, to 11 October 2018, without language restriction. Selection criteria: Randomized trials, cross-sectional, and cohort studies using respiratory specimens that evaluated Xpert MTB/RIF, Xpert Ultra, or both against the reference standard, culture for tuberculosis and culture-based drug susceptibility testing or MTBDRplus for rifampicin resistance. Data collection and analysis: Four review authors independently extracted data using a standardized form. When possible, we also extracted data by smear and HIV status. We assessed study quality using QUADAS-2 and performed meta-analyses to estimate pooled sensitivity and specificity separately for tuberculosis and rifampicin resistance. We investigated potential sources of heterogeneity. Most analyses used a bivariate random-effects model. For tuberculosis detection, we first estimated accuracy using all included studies and then only the subset of studies where participants were unselected, i.e. not selected based on prior microscopy testing. Main results: We identified in total 95 studies (77 new studies since the previous review): 86 studies (42,091 participants) evaluated Xpert MTB/RIF for tuberculosis and 57 studies (8287 participants) for rifampicin resistance. One study compared Xpert MTB/RIF and Xpert Ultra on the same participant specimen.Tuberculosis detectionOf the total 86 studies, 45 took place in high tuberculosis burden and 50 in high TB/HIV burden countries. Most studies had low risk of bias.Xpert MTB/RIF pooled sensitivity and specificity (95% credible Interval (CrI)) were 85% (82% to 88%) and 98% (97% to 98%), (70 studies, 37,237 unselected participants; high-certainty evidence). We found similar accuracy when we included all studies.For a population of 1000 people where 100 have tuberculosis on culture, 103 would be Xpert MTB/RIF-positive and 18 (17%) would not have tuberculosis (false-positives); 897 would be Xpert MTB/RIF-negative and 15 (2%) would have tuberculosis (false-negatives).Xpert Ultra sensitivity (95% confidence interval (CI)) was 88% (85% to 91%) versus Xpert MTB/RIF 83% (79% to 86%); Xpert Ultra specificity was 96% (94% to 97%) versus Xpert MTB/RIF 98% (97% to 99%), (1 study, 1439 participants; moderate-certainty evidence).Xpert MTB/RIF pooled sensitivity was 98% (97% to 98%) in smear-positive and 67% (62% to 72%) in smear-negative, culture-positive participants, (45 studies). Xpert MTB/RIF pooled sensitivity was 88% (83% to 92%) in HIV-negative and 81% (75% to 86%) in HIV-positive participants; specificities were similar 98% (97% to 99%), (14 studies).Rifampicin resistance detectionXpert MTB/RIF pooled sensitivity and specificity (95% Crl) were 96% (94% to 97%) and 98% (98% to 99%), (48 studies, 8020 participants; high-certainty evidence).For a population of 1000 people where 100 have rifampicin-resistant tuberculosis, 114 would be positive for rifampicin-resistant tuberculosis and 18 (16%) would not have rifampicin resistance (false-positives); 886 would be would be negative for rifampicin-resistant tuberculosis and four (0.4%) would have rifampicin resistance (false-negatives).Xpert Ultra sensitivity (95% CI) was 95% (90% to 98%) versus Xpert MTB/RIF 95% (91% to 98%); Xpert Ultra specificity was 98% (97% to 99%) versus Xpert MTB/RIF 98% (96% to 99%), (1 study, 551 participants; moderate-certainty evidence). Authors' conclusions: We found Xpert MTB/RIF to be sensitive and specific for diagnosing PTB and rifampicin resistance, consistent with findings reported previously. Xpert MTB/RIF was more sensitive for tuberculosis in smear-positive than smear-negative participants and HIV-negative than HIV-positive participants. Compared with Xpert MTB/RIF, Xpert Ultra had higher sensitivity and lower specificity for tuberculosis and similar sensitivity and specificity for rifampicin resistance (1 study). Xpert MTB/RIF and Xpert Ultra provide accurate results and can allow rapid initiation of treatment for multidrug-resistant tuberculosis.
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Global tuberculosis (TB) control and eradication is hampered by the unavailability of simple, rapid and affordable diagnostic tests deployable at low infrastructure microscopy centres. We have developed and evaluated the performance of a nucleic acid amplification test for detection of Mycobacterium tuberculosis (MTB), the NWU-TB test, in clinical sputum specimens from 306 patients with suspected pulmonary tuberculosis. The test involves sputum sample processing using a lyser device within 7minutes, followed by rapid multiplex-PCR on a fast thermal cycler within 25minutes, and amplicon resolution on agarose gel electrophoresis. Samples were also examined for presence of MTB using smear microscopy, GeneXpert and MGIT culture. Results were assessed in comparison to a MGIT culture as gold standard. Of the 306 patients, 174 had a previous TB history or already on treatment, and 132 were TB naïve cases. The NWU-TB system was found to have an overall sensitivity and specificity of 80.8% (95% CI: 75-85.7) and 75.6% (95% CI: 64.9-84.4) respectively, in comparison to 85.3% (95% CI: 79.9-89.6) and 73.2% (95% CI: 62.2-82.4) respectively for GeneXpert; and 62.1% (95% CI: 55.3-68.4) and 56.1% (95% CI: 44.7-67) respectively for smear microscopy. The study has shown that the NWU-TB system allows detection of TB in less than two hours and can be utilized at low infrastructure sites to provide quick and accurate diagnosis at a very low cost. Copyright © 2015. Published by Elsevier B.V.
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The Xpert MTB/RIF assay has garnered significant interest as a sensitive and rapid diagnostic tool to improve detection of sensitive and drug resistant tuberculosis. However, most existing literature has described the performance of MTB/RIF testing only in study conditions; little information is available on its use in routine case finding. TB REACH is a multi-country initiative focusing on innovative ways to improve case notification. We selected a convenience sample of nine TB REACH projects for inclusion to cover a range of implementers, regions and approaches. Standard quarterly reports and machine data from the first 12 months of MTB/RIF implementation in each project were utilized to analyze patient yields, rifampicin resistance, and failed tests. Data was collected from September 2011 to March 2013. A questionnaire was implemented and semi-structured interviews with project staff were conducted to gather information on user experiences and challenges. All projects used MTB/RIF testing for people with suspected TB, as opposed to testing for drug resistance among already diagnosed patients. The projects placed 65 machines (196 modules) in a variety of facilities and employed numerous case-finding strategies and testing algorithms. The projects consumed 47,973 MTB/RIF tests. Of valid tests, 7,195 (16.6%) were positive for MTB. A total of 982 rifampicin resistant results were found (13.6% of positive tests). Of all tests conducted, 10.6% failed. The need for continuous power supply was noted by all projects and most used locally procured solutions. There was considerable heterogeneity in how results were reported and recorded, reflecting the lack of standardized guidance in some countries. The findings of this study begin to fill the gaps among guidelines, research findings, and real-world implementation of MTB/RIF testing. Testing with Xpert MTB/RIF detected a large number of people with TB that routine services failed to detect. The study demonstrates the versatility and impact of the technology, but also outlines various surmountable barriers to implementation. The study is not representative of all early implementer experiences with MTB/RIF testing but rather provides an overview of the shared issues as well as the many different approaches to programmatic MTB/RIF implementation.
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Light emitting diode (LED) fluorescence microscopy (FM) is an affordable, technology targeted for use in resource-limited settings and recommended for widespread roll-out by the World Health Organization (WHO). We sought to compare the operational performance of three LED FM methods compared to light microscopy in a cohort of HIV-positive tuberculosis (TB) suspects at an urban clinic in a high TB burden country. Two spot specimens collected from TB suspects were included in the study. Smears were stained using auramine O method and read after blinding by three LED-based FM methods by trained laboratory technicians in the Infectious Diseases Institutelaboratory. Leftover portions of the refrigerated sputum specimens were transported to the FIND Tuberculosis Research Laboratory for Ziehl Neelsen (ZN) smear preparation and reading by experienced technologist as well as liquid and solid culture. 174 of 627 (27.8%) specimens collected yielded one or more positive mycobacterial cultures. 94.3% (164/174) were M. tuberculosis complex. LED FM was between 7.3-11.0% more sensitive compared to ZN microscopy. Of the 592 specimens examined by all microscopy methods, there was no significant difference in sensitivity between the three LED FM methods. The specificity of the LED FM methods was between 6.1% and 7.7% lower than ZN microscopy (P<0.001), although exclusion of the single poor reader resulted in over 98% specificity for all FM methods. Laboratory technicians in routine settings can be trained to use FM which is more sensitive than ZN microscopy. Despite rigorous proficiency testing, there were operator-dependent accuracy issues which highlight the critical need for intensive quality assurance procedures during LED FM implementation. The low sensitivity of FM for HIV-positive individuals particularly those with low CD4 T cell counts, will limit the number of additional patients found by LED FM in countries with high rates of HIV co-infection.
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Sputum culture is the gold standard for diagnosis of pulmonary tuberculosis (PTB). Although mostly used for research, culture is recommended by the World Health Organization for TB diagnosis among HIV infected smear negative PTB suspects. Even then, the number of sputum samples required remains unspecified. Here, we determined the Incremental Yield (IY) and number of samples required to diagnose an additional PTB case upon second and third serial sputum culture. This was a cross sectional study done between January and March 2011. Serial sputum samples were provided by participants within two days and cultured using Lowenstein Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) methods. A PTB case was defined as a positive culture on either one or both methods. The IY from the second and third serial cultures was determined and the reciprocal of the product of the fractions of IY provided the number of samples required for an additional PTB case. Of the 170 smear negative PTB suspects, 62 (36.5%) met the case definition. The IY of the second sample culture was 12.7%, 23.6% and 12.6% and for the third sample culture was 6.8%, 7.5% and 7.3% with LJ, MGIT and LJ or MGIT, respectively. The number of samples required for an additional PTB case and 95% CI upon the second sample culture were 29.9 (16.6, 156.5), 11.3 (7.6, 21.9) and 20.8 (12.5, 62.7); while for the third sample culture were 55.6 (26.4, 500.4), 35.7 (19.0, 313.8) and 36.1 (19.1, 330.9) by LJ, MGIT and LJ or MGIT respectively. Among HIV infected smear negative PTB suspects in Kampala, 93% of PTB cases are diagnosed upon the second serial sputum culture. The number of cultures needed to diagnose an additional PTB case, ranges from 11-30 and 35-56 by the second and third sputum samples, respectively.
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RATIONALE: Xpert MTB/RIF is a novel automated molecular diagnostic recently endorsed by the World Health Organization. However, performance-related data from high HIV prevalence settings are limited. OBJECTIVES: The impact of sample-related factors on performance and the significance of Xpert MTB/RIF-positive culture-negative discordance remain unclear. METHODS: Xpert MTB/RIF was evaluated using single archived spot-sputum samples from 496 South African patients with suspected TB. Mycobacterium tuberculosis culture positivity and phenotypic resistance to rifampicin served as reference standards. MEASUREMENTS AND MAIN RESULTS: Overall, Xpert MTB/RIF detected 95% (95% confidence interval [CI], 88-98%; 89 of 94) of smear-positive culture-positive cases and the specificity was 94% (91-96%; 320 of 339). The sensitivity in smear-negative cases was 55% (35-73%; 12 of 22) when the analysis was restricted to 1 ml of unprocessed sputum and culture time-to-positivity of less than or equal to 28 days. Compared with smear microscopy (n=94), Xpert MTB/RIF detected an additional 17 cases (n=111) representing an 18% (11-27%; 111 vs. 94) relative increase in the rapid TB case detection rate. Moreover, compared with smear microscopy, the inclusion of Xpert MTB/RIF-positive culture-negative TB cases (ruled-in by an alternative diagnostic method) resulted in the detection of a further 16 cases (n=127), thus significantly increasing the rapid TB case detection rate to 35% (95% CI, 26-45%; 94 to 111 vs. 94 to 127; P<0.01), the overall specificity to 99.1% (97-100%; 320 of 323; P<0.001), and sensitivity in smear-negative TB to 60% (P=0.12). Performance strongly correlated with smear status and culture time-to-positivity. In patients infected with HIV compared with patients uninfected with HIV Xpert MTB/RIF showed a trend to reduced sensitivity (P=0.09) and significantly reduced negative predictive value (P=0.01). The negative predictive value for rifampicin resistance was 99.4%. CONCLUSIONS: XpertMTB/RIF outperformed smear microscopy, established a diagnosis in a significant proportion of patients with smear-negative TB, detected many highly likely TB cases missed by culture, and accurately ruled out rifampicin-resistant TB. Sample-specific factors had limited impact on performance. Performance in patients infected with HIV, especially those with advanced immunosuppression, warrants further study.
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Recently, new diagnostic tools for tuberculosis detection and resistance testing have become available. The World Health Organization endorses new tuberculosis diagnostics by using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) process. This endorsement process takes place when limited evidence beyond test accuracy is available. There is a need to provide guidance to tuberculosis programs about which new diagnostics to scale up and how best to position them in diagnostic algorithms. To speed adoption of new diagnostics for tuberculosis, the policy recommendation process should be revised to consist of 2 steps: technical recommendation and programmatic recommendation. Technical recommendation would follow the GRADE process and be based on accuracy with limited cost and feasibility data, while programmatic recommendation would include patient-important outcomes, cost-effectiveness when implemented under routine conditions, and factors critical to successful scale-up. The evidence for both steps should be systematically collected, but each requires different study designs.
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Although nontuberculous mycobacteria (NTM) are widely documented as a cause of illness among HIV-infected people in the developed world, studies describing the prevalence of NTM disease among HIV-infected people in most resource-limited settings are rare. To evaluate the prevalence of mycobacterial disease in HIV-infected patients in Southeast Asia. We enrolled people with HIV from three countries in Southeast Asia and collected pulmonary and extrapulmonary specimens to evaluate the prevalence of mycobacterial disease. We adapted American Thoracic Society/Infectious Disease Society of America guidelines to classify patients into NTM pulmonary disease, NTM pulmonary disease suspects, NTM disseminated disease, and no NTM categories. In Cambodia, where solid media alone was used, NTM was rare. Of 1,060 patients enrolled in Thailand and Vietnam, where liquid culture was performed, 124 (12%) had tuberculosis and 218 (21%) had NTM. Of 218 patients with NTM, 66 (30%) were classified as NTM pulmonary disease suspects, 9 (4%) with NTM pulmonary disease, and 10 (5%) with NTM disseminated disease. The prevalence of NTM disease was 2% (19 of 1,060). Of 51 patients receiving antiretroviral therapy (ART), none had NTM disease compared with 19 (2%) of 1,009 not receiving ART. Although people with HIV frequently have sputum cultures positive for NTM, few meet a strict case definition for NTM disease. Consistent with previous studies, ART was associated with lower odds of having NTM disease. Further studies of NTM in HIV-infected individuals in tuberculosis-endemic countries are needed to develop and validate case definitions.
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Capilia TB is a simple immunochromatographic assay based on the detection of MPB64 antigen specifically secreted by the Mycobacterium tuberculosis complex (MTC). Capilia TB was evaluated for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 systems in Kampala, Uganda. Since most studies have mainly dealt with respiratory samples, the performance of Capilia TB on blood culture samples was also evaluated. One thousand samples from pulmonary and disseminated tuberculosis (TB) suspects admitted to the JCRC clinic and the TB wards at Old Mulago hospital in Kampala, Uganda, were cultured in automated BACTEC MGIT 960 and BACTEC 9120 blood culture systems. BACTEC-positive samples were screened for purity by sub-culturing on blood agar plates. Two hundred and fifty three (253) samples with Acid fast bacilli (AFB, 174 BACTEC MGIT 960 and 79 BACTEC 9120 blood cultures) were analyzed for presence of MTC using Capilia TB and in-house PCR assays. The overall Sensitivity, Specificity, Positive and Negative Predictive values, and Kappa statistic for Capilia TB assay for identification of MTC were 98.4%, 97.6%, 97.7%, 98.4% and 0.96, respectively. Initially, the performance of in-house PCR on BACTEC 9120 blood cultures was poor (Sensitivity, Specificity, PPV, NPV and Kappa statistic of 100%, 29.3%,7%, 100% and 0.04, respectively) but improved upon sub-culturing on solid medium (Middlebrook 7H10) to 100%, 95.6%, 98.2%, 100% and 0.98, respectively. In contrast, the Sensitivity and Specificity of Capilia TB assay was 98.4% and 97.9%, respectively, both with BACTEC blood cultures and Middlebrook 7H10 cultured samples, revealing that Capilia was better than in-house PCR for identification of MTC in blood cultures. Additionally, Capilia TB was cheaper than in-house PCR for individual samples ($2.03 vs. $12.59, respectively), and was easier to perform with a shorter turnaround time (20 min vs. 480 min, respectively). Capilia TB assay is faster and cheaper than in-house PCR for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 culture systems in real-time testing of AFB positive cultures.
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Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens. We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [(3)H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard. All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×10(3) organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p=0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p=0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ~100 organisms. Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold.
In settings of high HIV prevalence, tuberculosis control and patient management are hindered by lack of accurate, rapid tuberculosis diagnostic tests that can be performed at point-of-care. The Determine TB LAM Ag ('TB LAM') test is a lateral flow immunochromatographic test for detection of mycobacterial lipoarabinomannan (LAM) in urine. Our objective was to determine sensitivity and specificity of the TB LAM test for tuberculosis diagnosis. Prospective diagnostic accuracy study. Hospital and outpatient settings in Uganda and South Africa. HIV-infected adults with tuberculosis symptoms and/or signs. Participants provided a fresh urine specimen for TB LAM testing, blood for mycobacterial culture, and two respiratory specimens for smear microscopy and mycobacterial culture. For the TB LAM test, sensitivity in participants with culture-positive tuberculosis and specificity in participants without tuberculosis. 1013 participants were enrolled. Among culture-positive tuberculosis patients, the TB LAM test identified 136/367 (37.1%) overall and 116/196 (59.2%) in the group with CD4≤100 cells/mm. The test was specific in 559/573 (97.6%) of patients without tuberculosis. Sensitivity of the urine TB LAM test plus sputum smear microscopy was 197/367 (53.7%) overall and 133/196 (67.9%) among those with CD4≤100. CD4≤50 (adjusted odds ratio [AOR] 6.2, P<0.001) or 51-100 (AOR 7.1, P<0.001), mycobacteremia (AOR 6.1; P<0.01) and hospitalization (AOR 2.6, P=0.03) were independently associated with a positive TB LAM test. In HIV-positive adults with CD4≤100, the TB LAM urine test detected over half of culture-positive tuberculosis patients, in less than 30 minutes and without the need for equipment or reagents.