Background. T-SPOT.TB is an interferon gamma release assay for detecting Mycobacterium tuberculosis infection. The requirement to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T Cell Xtend reagent may allow delayed processing, but has not been extensively field tested. Design. Consecutive AFB smear positive adult tuberculosis patients were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1–3 sputum samples for culture and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage, 25 μL of T Cell Xtend reagent was added per mL of blood, and the sample was tested using T-SPOT.TB. Results. 143 patients were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement between T-SPOT.TB and culture was 95.4% (95%CI; 87.1–99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TB when using T Cell Xtend reagent was 96.8% (95%CI; 88.8–99.6%). Conclusions. When T Cell Xtend reagent is added to specimens held overnight at recommended temperatures, T-SPOT.TB is as sensitive as the standard assay in patients with tuberculosis

Adams, Lisa V.

Ferguson, Katherine

Maro, Isaac

Matee, Mecky

Mtei, Lillian

Talbot, Elizabeth A.

von Reyn, C. Fordham

English

PubMed

Elizabeth A. Talbot

Isaac Maro

Katherine Ferguson

Lisa V. Adams

Lillian Mtei

Mecky Matee

C. Fordham von Reyn

Directory of Open Access Journals

Tuberculosis Research and Treatment

Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania

Background. T-SPOT.TB is an interferon gamma release assay for detecting Mycobacterium tuberculosis infection. The requirement to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T Cell Xtend reagent may allow delayed processing, but has not been extensively field tested. Design. Consecutive AFB smear positive adult tuberculosis patients were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1-3 sputum samples for culture and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage, 25 μL of T Cell Xtend reagent was added per mL of blood, and the sample was tested using T-SPOT.TB. Results. 143 patients were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement between T-SPOT.TB and culture was 95.4% (95%CI; 87.1-99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TB when using T Cell Xtend reagent was 96.8% (95%CI; 88.8-99.6%). Conclusions. When T Cell Xtend reagent is added to specimens held overnight at recommended temperatures, T-SPOT.TB is as sensitive as the standard assay in patients with tuberculosis

Talbot, Elizabeth A

Adams, Lisa V

von Reyn, C Fordham

Digital Library of the Tanzania Health Community

Hindawi Publishing Corporation
Tuberculosis Research and Treatment
Volume 2012, Article ID 345290, 4 pages
doi:10.1155/2012/345290
Clinical Study
Maintenance of Sensitivity of the
T-SPOT.TB Assay after Overnight Storage of
BloodSamples,Dar esSalaam, Tanzania
ElizabethA. Talbot,1,2 IsaacMaro,3 Katherine Ferguson,4 LisaV.Adams,1 LillianMtei,3
Mecky Matee,3 andC. Fordham vonReyn1
1Dartmouth Medical School, Hanover, NH 03755, USA
2Infectious Diseases and International Health Section, 1 Medical Center Drive, Lebanon, NH 03756, USA
3Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania
4Dartmouth College, Hanover, NH 03755, USA
Correspondence should be addressed to Elizabeth A. Talbot, elizabeth.talbot@dartmouth.edu
Received 25 November 2011; Accepted 15 January 2012
Academic Editor: Catharina Boehme
Copyright © 2012 Elizabeth A. Talbot et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Background.T - S P O T . TB is an interferon gamma release assay for detecting Mycobacterium tuberculosis infection. The requirement
to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T Cell Xtend reagent may allow
delayed processing, but has not been extensively ﬁeld tested. Design. Consecutive AFB smear positive adult tuberculosis patients
were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1–3 sputum samples for culture
and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage,
25μLo fTC e l lXtend reagent was added per mL of blood, and the sample was tested using T-SPOT.TB. Results. 143 patients
were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red
blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement
between T-SPOT.TB and culture was 95.4% (95%CI; 87.1–99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TB when using T
Cell Xtend reagent was 96.8% (95%CI; 88.8–99.6%). Conclusions.W h e nTC e llXtend reagent is added to specimens held overnight
at recommended temperatures, T-SPOT.TB is as sensitive as the standard assay in patients with tuberculosis.
1.Introduction
The T-SPOT.TB (Oxford Immunotec Ltd, Abingdon, UK)
is an interferon gamma release assay (IGRA) for detection
of latent Mycobacterium tuberculosis infection (LTBI). This
assay detects T-lymphocytes speciﬁc for M. tuberculosis
antigens that are absent from M. bovis BCG and most envi-
ronmental mycobacteria [1]. Currently, T-SPOT.TB must be
performed within 8 hours of sample collection. Logistically,
this requires that patients have blood drawn early enough
to transport the sample to the laboratory, which must assay
samples on the day of receipt. This precludes batching
samples, which would save resources and testing supplies.
Moreover, in many countries, laboratories are centralized
and are not in close proximity to where the blood sample
is drawn. Therefore, it is not possible to transport samples to
arrive at the receiving laboratory for processing on the same
day.
To allow greater test processing ﬂexibility, the manufac-
turer of T-SPOT.TB developed a proprietary reagent called T
Cell Xtend, which they report can be added to whole blood
samples stored at 10–25◦C to increase this timeframe up to
32 hours [2].
2.MaterialsandMethods
The ethical review boards of Dartmouth Medical School
(Hanover NH), the National Institute for Medical Research2 Tuberculosis Research and Treatment
(Dar es Salaam, Tanzania) and Muhimbili University of
Health and Allied Sciences (MUHAS, Dar es Salaam, Tanza-
nia) approved the study. The study was conducted according
to the principles of good clinical practice and under the
International Conference on Harmonization guidelines.
2.1. Participant Enrolment. All AFB smear positive indi-
viduals referred for initiation of TB treatment to one of
two National Tuberculosis and Leprosy Programme (NTLP)
TB treatment clinics in Dar es Salaam, Tanzania, were
approached by a trained study nurse to determine their
interest in being included in the study. The study nurse
explained the study and answered any questions in the
patient’s native language. The participants then signed the
approved informed consent or, in the presence of a witness,
indicated their signature with a ﬁngerprint. All participants
provided a medical history, up to three sputum samples and
a blood sample for testing by IGRA and were oﬀered HIV
testing according to the NTLP guidelines.
2.2. IGRA Testing. A single tube of blood was drawn from
each participant. During the initial enrolment period, blood
samples were transported to the laboratory under ambient
temperature conditions (maximum average temperatures
during the enrolment months were 28◦Cf o rS e p t e m b e ra n d
29◦C for October) and, in the latter enrolment period, under
temperatures ensured as <25◦Cb yu s eo fat e m p e r a t u r e
control shipping box (GreenBox Thermal Management
System (Thermosafe Brands, Arlington Heights, IL, USA)).
The blood samples were then stored overnight in an air
conditionedlaboratory(<25◦C)andtestedusingT-SPOT.TB
according to the manufacturer’s instructions for use [3].
TC e l lXtend reagent was added to each blood sample
at 25μL/mL blood, mixed and incubated for 20 minutes
a tr o o mt e m p e r a t u r eb e f o r ep r o c e s s i n g .P e r i p h e r a lb l o o d
mononuclear cells (PBMC) were harvested by Ficoll den-
sity gradient centrifugation using Leucosep tubes (Oxford
Immunotec, Abingdon, UK). The PBMCs were washed,
counted using a hemocytometer, and plated at 2.5 × 105
cells per well into a membrane-bottomed plate, coated with
anti-interferon gamma antibody. PBMCs were incubated
overnight in the presence of the provided TB antigens
(ESAT-6 and CFP10) along with controls (positive mitogen
control and a nil control). The PBMCs producing interferon
gamma were revealed as spots by incubation with an enzyme
conjugated secondary antibody for interferon gamma and
a colour producing enzyme substrate. Spots were counted,
and clinical result was recorded according to the approved
algorithm where, compared to the nil control, 6 spots and
above are positive and 5 spots and below are negative.
Results with a low-mitogen response (<20 spots) or a high-
nil control response (>10 spots) were recorded as invalid.
2.3. Mycobacterial Studies. AFB smears were done according
to the Ziehl-Neelsen method by trained research personnel.
SputumwasculturedformycobacteriaonLowensteinJensen
slants.
2.4. Statistical Analyses. Sensitivity was calculated as the
number of T-SPOT.TB positive samples divided by the
number of culture positive samples multiplied by 100. 95%
conﬁdence limits were calculated. The Kappa statistic was
used to measure overall agreement between culture and T-
SPOT.TB results. Analyses were conducted using Excel.
3. Results
A total of 143 participants were enrolled into the study
(Figure 1). The initial 57 blood samples drawn at one clinic
were transported and stored at ambient temperatures prior
to processing. After processing, it was recognized that these
specimens were not reliably kept at temperatures below
25◦C (the required temperature per protocol), and therefore
these 57 samples were excluded from the ﬁnal sensitivity
analysis. Temperature control measures were instituted for
the subsequent collection of samples from an additional 86
participants. Nineteen of these 86 (22.1%) participants were
excluded because the laboratorian observed red blood cell
contamination in the PBMC layer so the PBMCs could not
be accurately counted. One of 86 (1.2%) participants failed
to provide a sputum sample for culture and was excluded.
Therefore, 66 results were available for analysis. The
participants were all black African and ranged in age from
18–60 years. The majority were male and BCG vaccinated
(71%and77%,resp.);17(26%)wereHIV-positive,24(36%)
were HIV-negative, and 25 (38%) declined testing.
Of the 66 T-SPOT.TB results, 61 (92.4%) were positive,
4 (6.1%) were negative, and 1 (1.5%) was invalid. The
overall agreement between the 65 valid T-SPOT.TB and
cultureresultswas95.4%(62/65:95%CI;87.1–99.0%)witha
Kappa value of 0.548. The sensitivity of the T-SPOT.TB assay
when run after delayed processing using the T Cell Xtend
reagent was 96.8% (60/62: 95%CI; 88.8–99.6%). Among 41
T-SPOT.TBresultsfromparticipantswithknownHIVstatus,
T-SPOT.TB results were positive in 14 (82.4%) of those who
were HIV-positive and 23 (95.8%) of those who were HIV-
negative (P>0.05). However, limiting to valid results from
culture positive patients, the sensitivity of the assay among
samples from HIV-positive participants was 92.9% (13/14).
In spite of exclusion because of protocol noncompliance,
results from the 57 samples transported at ambient tempera-
ture were also analyzed. Of the 57, 40 (70.2%) were positive,
11 (19.3%) were negative, and 6 (10.5%) was invalid. Com-
pared with the results from the 66 samples with temperature
control, false negative results among these 57 samples were
more common (P = 0.05). Results from 19 samples were
excluded because of red blood cell contamination showed
14 (73.7%) positive and 5 (26.3%) negative. Compared with
the results from 66 samples with temperature control, false
negativeresultsamongthese19werestatisticallysigniﬁcantly
more common (P = 0.04).
4. Discussion
T-SPOT.TB with delayed processing using T Cell Xtend
among patients with culture-conﬁrmed tuberculosis showed
high sensitivity, comparable to that reported for immediateTuberculosis Research and Treatment 3
Screened
143
Evaluable
143 (100%)
Samples at
ambient
temperature
57
Samples at
controlled
temperature
86
Pos
40
(70.2%)
Neg
11
(19.3%)
Invalid
6
(10.5%)
Analyzed
66
Failed
to give
sputum
1
RBC
contamination
19
Pos
61
(92.4%)
Neg
4
(6.1%)
Invalid
1
(1.5%)
Pos
14
(73.7%)
Neg
5 (26.3%)
Figure 1: Patient ﬂow and testing results using T-SPOT.TB and T Cell Xtend, Dar es Salaam, Tanzania.
processing. In a recent meta-analysis of 14 studies of T-
SPOT.TB, (without Xtend) in low- and middle-income
countries, Metcalfe et al. reported sensitivity of 83% (95%
CI, 63–94%) among HIV-positive and -negative tuberculosis
patients [4].
This high sensitivity we observed is also consistent with
pilotstudieswiththeTCellXtend reagentcompletedatthree
clinical sites including the UK and South Africa [5]. Lenders
et al. studied the utility of T Cell Xtend, conducting T-
SPOT.TBassaysthesamedayascollectionandapproximately
one and two days after collection [6]. Spot counts from
66 specimens T-SPOT.TB assayed without the T Cell Xtend
reagent one day after collection were higher than those
processed on the day of collection. In contrast, spot counts
from 215 specimens assayed two days after collection with
the T Cell Xtend reagent were similar to those processed on
the day of collection. The T Cell Xtend reagent reduced the
proportionofsamplesthatchangedfrompositivetonegative
and vice versa (4.54% to 2.83%), but this was not statistically
signiﬁcant [6]. Wang et al. had similar high agreement
(98.2%) among 108 specimens of results of immediate T-
SPOT.TB and T-SPOT.TB with the T Cell Xtend reagent
processed 23–32 hours after collection [7].
In one of our patients, T-SPOT.TB was positive and
culture was negative (Table 1). This patient was an HIV-
infectedmale,whosubmittedthreesputumspecimenswhich
were 3+, 4+ and 1+ AFB smear positive, but all cultures were
negative. The T-SPOT.TB result was interpreted as positive,
given that the Nil control well showed 6 spots, Panel A 6, and
Panel B 15. Per the package insert, the test result is positive if
Panel A minus Nil control and/or Panel B minus Nil control
is 6 spots [3]. It may be that the T-SPOT.TB result was falsely
positive, or that the culture was falsely negative, perhaps
becausethepatienthadreceivedantituberculosistherapy.We
do not have follow-up data on the patient to know if they
Table 1: Overall agreement between TB culture and T-SPOT.TB.
T-SPOT.TB
Positive Negative Invalid
Culture Positive 60 2 1∗
N e g a t i v e 120
∗1/66 (1.5%) of T-SPOT.TB results was invalid due to a high nil control
result.
demonstrated a typical response to antituberculosis therapy,
or there was an alternative diagnosis made.
Our samples transported using a validated system for
temperature control at or below 25◦C showed sensitivity that
is equivalent to that reported for the same day T-SPOT.TB
assay [1, 3]. However, our study clearly illustrates the need to
controltemperature.Insub-SaharaAfricansettings,asimple
solution for shipment over long distances would be required,
such as the use of an insulated package system.
Red blood cell contamination also interferes with assay
reliability because the presence of large numbers of red
blood cells makes accurate counting of the PBMC fraction
diﬃcult, and therefore the required number of PBMCs
cannot be reliably determined. Red blood cell contamination
is more common when a hemocytometer is used (as in this
study), and the use of automated instruments that provide
ad i ﬀerential count of white blood cells would eliminate this
problem.
5. Conclusions
T-SPOT.TB can be run on blood samples not contaminated
with red blood cells and maintained overnight by the use
of T Cell Xtend reagent without aﬀecting the sensitivity of
the assay. Storage and transport of samples at temperatures4 Tuberculosis Research and Treatment
<25◦C are critical to obtaining optimum sensitivity, and use
of an automated cell counting instrument may correct the
reduced sensitivity observed with red blood cell contamina-
tion.
Authors’ Contributions
E .A .T a l b o t ,L .V .A d a m s ,L .M t e i ,a n dC .F .v o nR e y n
designed the study. E. A. Talbot, I. Maro, K. Ferguson, L. V.
Adams, L. Mtei, M. Matee, and C. F. von Reyn participated
in the implementation of the trial. E. A. Talbot, L. Mtei, M.
Matee, and C. F. von Reyn participated in administration.
I. Maro, K. Ferguson, and L. Mtei participated in data
collection. E. A. Talbot analyzed the data and wrote the ﬁrst
d r a f t .E .A .T a l b o t ,L .V .A d a m s ,L .M t e ia n dC .F .v o nR e y n
contributed to the interpretation of the data. All authors
reviewed and approved the ﬁnal paper.
Acknowledgments
The authors would like to express their gratitude to the
volunteers who participated in this study. They appreci-
ate the eﬀorts of the clinical research staﬀ: Dr. Ibrahim
Mteza, Esther Kayichile, Joyce Wamsele, and Emmanuel
Balandya. They also thank Betty Mchaki (DarDar Project)
and Wendy Wieland-Alter (Dartmouth Medical School) for
their generous laboratory assistance and Miriam Zayumba
(DarDar Project) and Susan Tvaroha (Dartmouth College)
for assistance with data management. The authors disclose
thatOxfordImmunotec,themanufactureroftheT-SPOT.TB
and the T Cell Xtend reagent, funded this study and also
contributedinon-sitestudymonitoring.OxfordImmunotec
did not interpret the ﬁndings or write this paper. C. F. von
Reyn has been a one-day consultant for Oxford Immunotec
on two occasions, and C. F. von Reyn, L. V. Adams, and E.
A. Talbot received additional research funding from Oxford
Immunotecmorethanoneyearpriortothissubmission.The
other authors have no potential conﬂicts to declare.
References
[1] CDC, “Updated guidelines for using interferon gamma release
assays to detect Mycobacterium tuberculosis infection—United
States, 2010,” Morbidity and Mortality Weekly Report, vol. 59,
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[2] http://www.oxfordimmunotec.com/XtendpackInsert-english,
2010.
[3] http://www.oxfordimmunotec.com/96-UK, 2010.
[4] J. Z. Metcalfe, C. K. Everett, K. R. Steingart et al., “Interferon-
γ release assays for active pulmonary tuberculosis diagnosis in
adults in low-and middle-income countries: systematic review
and meta-analysis,” Journal of Infectious Diseases, vol. 204, no.
4, pp. S1120–S1129, 2011.
[ 5 ]I .D u r r a n t ,J .R a d c l i ﬀe, and T. Day, “TB testing using T-
SPOT.TB after overnight sample storage,” Clinical Microbiology
and Infection, vol. 15, p. pS393, 2009.
[ 6 ]L .M .L e n d e r s ,R .M e l d a u ,R .N .v a nZ y l - S m i te ta l . ,“ C o m p a r -
ison of same day versus delayed enumeration of TB-speciﬁc T
cell responses,” Journal of Infection, vol. 60, no. 5, pp. 344–350,
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[7] S. Wang, D. A. Powell, H. N. Nagaraja, J. D. Morris, L. S.
Schlesinger,andJ.Turner,“Evaluationofamodiﬁedinterferon-
gamma release assay for the diagnosis of latent tuberculosis
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Interferonγ release assays for active pulmonary tuberculosis diagnosis in adults in low-and middle-income countries: systematic review and meta-analysis,”

L e n d e r s ,R .M e l d a u ,R .N .v a nZ y l - S m i te ta l . ,“ C o m p a r -ison of same day versus delayed enumeration of TB-speciﬁc T cell responses,”

Schlesinger,andJ.Turner,“Evaluationofamodiﬁedinterferongamma release assay for the diagnosis of latent tuberculosis infection in adult and paediatric populations that enables delayed processing,”

u r r a n t ,J .R a d c l i ﬀe, and

Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosis infection—United States,

Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania.

Talbot, E.A.

Maro, I.

Ferguson, K.

Adams, L.V.

Mtei, L.

Matee, M.

von Reyn, C.F.

MUHAS Institutional Repository

Background. T-SPOT.TBis an interferon gamma release assay for detectingMycobacterium tuberculosisinfection. The requirement to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T CellXtendreagent may allow delayed processing, but has not been extensively field tested.Design. Consecutive AFB smear positive adult tuberculosis patients were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1–3 sputum samples for culture and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage, 25 μL of T CellXtendreagent was added per mL of blood, and the sample was tested using T-SPOT.TB.Results. 143 patients were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement between T-SPOT.TBand culture was 95.4% (95%CI; 87.1–99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TBwhen using T CellXtendreagent was 96.8% (95%CI; 88.8–99.6%).Conclusions. When T CellXtendreagent is added to specimens held overnight at recommended temperatures, T-SPOT.TBis as sensitive as the standard assay in patients with tuberculosis.</jats:p

Crossref

Hindawi Publishing CorporationTuberculosis Research and TreatmentVolume 2012, Article ID 345290, 4 pagesdoi:10.1155/2012/345290Clinical StudyMaintenance of Sensitivity of theT-SPOT.TB Assay after Overnight Storage ofBlood Samples, Dar es Salaam, TanzaniaElizabeth A. Talbot,1, 2 Isaac Maro,3 Katherine Ferguson,4 Lisa V. Adams,1 Lillian Mtei,3Mecky Matee,3 and C. Fordham von Reyn11 Dartmouth Medical School, Hanover, NH 03755, USA2 Infectious Diseases and International Health Section, 1 Medical Center Drive, Lebanon, NH 03756, USA3 Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania4 Dartmouth College, Hanover, NH 03755, USACorrespondence should be addressed to Elizabeth A. Talbot, elizabeth.talbot@dartmouth.eduReceived 25 November 2011; Accepted 15 January 2012Academic Editor: Catharina BoehmeCopyright © 2012 Elizabeth A. Talbot et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.Background. T-SPOT.TB is an interferon gamma release assay for detecting Mycobacterium tuberculosis infection. The requirementto process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T Cell Xtend reagent may allowdelayed processing, but has not been extensively field tested. Design. Consecutive AFB smear positive adult tuberculosis patientswere prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1–3 sputum samples for cultureand 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage,25 μL of T Cell Xtend reagent was added per mL of blood, and the sample was tested using T-SPOT.TB. Results. 143 patientswere enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to redblood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreementbetween T-SPOT.TB and culture was 95.4% (95%CI; 87.1–99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TB when using TCell Xtend reagent was 96.8% (95%CI; 88.8–99.6%). Conclusions. When T Cell Xtend reagent is added to specimens held overnightat recommended temperatures, T-SPOT.TB is as sensitive as the standard assay in patients with tuberculosis.1. IntroductionThe T-SPOT.TB (Oxford Immunotec Ltd, Abingdon, UK)is an interferon gamma release assay (IGRA) for detectionof latent Mycobacterium tuberculosis infection (LTBI). Thisassay detects T-lymphocytes specific for M. tuberculosisantigens that are absent from M. bovis BCG and most envi-ronmental mycobacteria [1]. Currently, T-SPOT.TB must beperformed within 8 hours of sample collection. Logistically,this requires that patients have blood drawn early enoughto transport the sample to the laboratory, which must assaysamples on the day of receipt. This precludes batchingsamples, which would save resources and testing supplies.Moreover, in many countries, laboratories are centralizedand are not in close proximity to where the blood sampleis drawn. Therefore, it is not possible to transport samples toarrive at the receiving laboratory for processing on the sameday.To allow greater test processing flexibility, the manufac-turer of T-SPOT.TB developed a proprietary reagent called TCell Xtend, which they report can be added to whole bloodsamples stored at 10–25◦C to increase this timeframe up to32 hours [2].2. Materials and MethodsThe ethical review boards of Dartmouth Medical School(Hanover NH), the National Institute for Medical Research2 Tuberculosis Research and Treatment(Dar es Salaam, Tanzania) and Muhimbili University ofHealth and Allied Sciences (MUHAS, Dar es Salaam, Tanza-nia) approved the study. The study was conducted accordingto the principles of good clinical practice and under theInternational Conference on Harmonization guidelines.2.1. Participant Enrolment. All AFB smear positive indi-viduals referred for initiation of TB treatment to one oftwo National Tuberculosis and Leprosy Programme (NTLP)TB treatment clinics in Dar es Salaam, Tanzania, wereapproached by a trained study nurse to determine theirinterest in being included in the study. The study nurseexplained the study and answered any questions in thepatient’s native language. The participants then signed theapproved informed consent or, in the presence of a witness,indicated their signature with a fingerprint. All participantsprovided a medical history, up to three sputum samples anda blood sample for testing by IGRA and were oﬀered HIVtesting according to the NTLP guidelines.2.2. IGRA Testing. A single tube of blood was drawn fromeach participant. During the initial enrolment period, bloodsamples were transported to the laboratory under ambienttemperature conditions (maximum average temperaturesduring the enrolment months were 28◦C for September and29◦C for October) and, in the latter enrolment period, undertemperatures ensured as <25◦C by use of a temperaturecontrol shipping box (GreenBox Thermal ManagementSystem (Thermosafe Brands, Arlington Heights, IL, USA)).The blood samples were then stored overnight in an airconditioned laboratory (<25◦C) and tested using T-SPOT.TBaccording to the manufacturer’s instructions for use [3].T Cell Xtend reagent was added to each blood sampleat 25 μL/mL blood, mixed and incubated for 20 minutesat room temperature before processing. Peripheral bloodmononuclear cells (PBMC) were harvested by Ficoll den-sity gradient centrifugation using Leucosep tubes (OxfordImmunotec, Abingdon, UK). The PBMCs were washed,counted using a hemocytometer, and plated at 2.5 × 105cells per well into a membrane-bottomed plate, coated withanti-interferon gamma antibody. PBMCs were incubatedovernight in the presence of the provided TB antigens(ESAT-6 and CFP10) along with controls (positive mitogencontrol and a nil control). The PBMCs producing interferongamma were revealed as spots by incubation with an enzymeconjugated secondary antibody for interferon gamma anda colour producing enzyme substrate. Spots were counted,and clinical result was recorded according to the approvedalgorithm where, compared to the nil control, 6 spots andabove are positive and 5 spots and below are negative.Results with a low-mitogen response (<20 spots) or a high-nil control response (>10 spots) were recorded as invalid.2.3. Mycobacterial Studies. AFB smears were done accordingto the Ziehl-Neelsen method by trained research personnel.Sputumwas cultured for mycobacteria on Lowenstein Jensenslants.2.4. Statistical Analyses. Sensitivity was calculated as thenumber of T-SPOT.TB positive samples divided by thenumber of culture positive samples multiplied by 100. 95%confidence limits were calculated. The Kappa statistic wasused to measure overall agreement between culture and T-SPOT.TB results. Analyses were conducted using Excel.3. ResultsA total of 143 participants were enrolled into the study(Figure 1). The initial 57 blood samples drawn at one clinicwere transported and stored at ambient temperatures priorto processing. After processing, it was recognized that thesespecimens were not reliably kept at temperatures below25◦C (the required temperature per protocol), and thereforethese 57 samples were excluded from the final sensitivityanalysis. Temperature control measures were instituted forthe subsequent collection of samples from an additional 86participants. Nineteen of these 86 (22.1%) participants wereexcluded because the laboratorian observed red blood cellcontamination in the PBMC layer so the PBMCs could notbe accurately counted. One of 86 (1.2%) participants failedto provide a sputum sample for culture and was excluded.Therefore, 66 results were available for analysis. Theparticipants were all black African and ranged in age from18–60 years. The majority were male and BCG vaccinated(71% and 77%, resp.); 17 (26%) wereHIV-positive, 24 (36%)were HIV-negative, and 25 (38%) declined testing.Of the 66 T-SPOT.TB results, 61 (92.4%) were positive,4 (6.1%) were negative, and 1 (1.5%) was invalid. Theoverall agreement between the 65 valid T-SPOT.TB andculture results was 95.4% (62/65: 95%CI; 87.1–99.0%) with aKappa value of 0.548. The sensitivity of the T-SPOT.TB assaywhen run after delayed processing using the T Cell Xtendreagent was 96.8% (60/62: 95%CI; 88.8–99.6%). Among 41T-SPOT.TB results from participants with knownHIV status,T-SPOT.TB results were positive in 14 (82.4%) of those whowere HIV-positive and 23 (95.8%) of those who were HIV-negative (P > 0.05). However, limiting to valid results fromculture positive patients, the sensitivity of the assay amongsamples from HIV-positive participants was 92.9% (13/14).In spite of exclusion because of protocol noncompliance,results from the 57 samples transported at ambient tempera-ture were also analyzed. Of the 57, 40 (70.2%) were positive,11 (19.3%) were negative, and 6 (10.5%) was invalid. Com-pared with the results from the 66 samples with temperaturecontrol, false negative results among these 57 samples weremore common (P = 0.05). Results from 19 samples wereexcluded because of red blood cell contamination showed14 (73.7%) positive and 5 (26.3%) negative. Compared withthe results from 66 samples with temperature control, falsenegative results among these 19 were statistically significantlymore common (P = 0.04).4. DiscussionT-SPOT.TB with delayed processing using T Cell Xtendamong patients with culture-confirmed tuberculosis showedhigh sensitivity, comparable to that reported for immediateTuberculosis Research and Treatment 3Screened143Evaluable143 (100%)Samples atambienttemperature57Samples atcontrolledtemperature86Pos40(70.2%)Neg11(19.3%)Invalid6(10.5%)Analyzed66Failedto givesputum1RBCcontamination19Pos61(92.4%)Neg4(6.1%)Invalid1(1.5%)Pos14(73.7%)Neg5 (26.3%)Figure 1: Patient flow and testing results using T-SPOT.TB and T Cell Xtend, Dar es Salaam, Tanzania.processing. In a recent meta-analysis of 14 studies of T-SPOT.TB, (without Xtend) in low- and middle-incomecountries, Metcalfe et al. reported sensitivity of 83% (95%CI, 63–94%) among HIV-positive and -negative tuberculosispatients [4].This high sensitivity we observed is also consistent withpilot studies with the T Cell Xtend reagent completed at threeclinical sites including the UK and South Africa [5]. Lenderset al. studied the utility of T Cell Xtend, conducting T-SPOT.TB assays the same day as collection and approximatelyone and two days after collection [6]. Spot counts from66 specimens T-SPOT.TB assayed without the T Cell Xtendreagent one day after collection were higher than thoseprocessed on the day of collection. In contrast, spot countsfrom 215 specimens assayed two days after collection withthe T Cell Xtend reagent were similar to those processed onthe day of collection. The T Cell Xtend reagent reduced theproportion of samples that changed from positive to negativeand vice versa (4.54% to 2.83%), but this was not statisticallysignificant [6]. Wang et al. had similar high agreement(98.2%) among 108 specimens of results of immediate T-SPOT.TB and T-SPOT.TB with the T Cell Xtend reagentprocessed 23–32 hours after collection [7].In one of our patients, T-SPOT.TB was positive andculture was negative (Table 1). This patient was an HIV-infectedmale, who submitted three sputum specimens whichwere 3+, 4+ and 1+ AFB smear positive, but all cultures werenegative. The T-SPOT.TB result was interpreted as positive,given that the Nil control well showed 6 spots, Panel A 6, andPanel B 15. Per the package insert, the test result is positive ifPanel A minus Nil control and/or Panel B minus Nil controlis 6 spots [3]. It may be that the T-SPOT.TB result was falselypositive, or that the culture was falsely negative, perhapsbecause the patient had received antituberculosis therapy.Wedo not have follow-up data on the patient to know if theyTable 1: Overall agreement between TB culture and T-SPOT.TB.T-SPOT.TBPositive Negative InvalidCulturePositive 60 2 1∗Negative 1 2 0∗1/66 (1.5%) of T-SPOT.TB results was invalid due to a high nil controlresult.demonstrated a typical response to antituberculosis therapy,or there was an alternative diagnosis made.Our samples transported using a validated system fortemperature control at or below 25◦C showed sensitivity thatis equivalent to that reported for the same day T-SPOT.TBassay [1, 3]. However, our study clearly illustrates the need tocontrol temperature. In sub-Sahara African settings, a simplesolution for shipment over long distances would be required,such as the use of an insulated package system.Red blood cell contamination also interferes with assayreliability because the presence of large numbers of redblood cells makes accurate counting of the PBMC fractiondiﬃcult, and therefore the required number of PBMCscannot be reliably determined. Red blood cell contaminationis more common when a hemocytometer is used (as in thisstudy), and the use of automated instruments that providea diﬀerential count of white blood cells would eliminate thisproblem.5. ConclusionsT-SPOT.TB can be run on blood samples not contaminatedwith red blood cells and maintained overnight by the useof T Cell Xtend reagent without aﬀecting the sensitivity ofthe assay. Storage and transport of samples at temperatures4 Tuberculosis Research and Treatment<25◦C are critical to obtaining optimum sensitivity, and useof an automated cell counting instrument may correct thereduced sensitivity observed with red blood cell contamina-tion.Authors’ ContributionsE. A. Talbot, L. V. Adams, L. Mtei, and C. F. von Reyndesigned the study. E. A. Talbot, I. Maro, K. Ferguson, L. V.Adams, L. Mtei, M. Matee, and C. F. von Reyn participatedin the implementation of the trial. E. A. Talbot, L. Mtei, M.Matee, and C. F. von Reyn participated in administration.I. Maro, K. Ferguson, and L. Mtei participated in datacollection. E. A. Talbot analyzed the data and wrote the firstdraft. E. A. Talbot, L. V. Adams, L. Mtei and C. F. von Reyncontributed to the interpretation of the data. All authorsreviewed and approved the final paper.AcknowledgmentsThe authors would like to express their gratitude to thevolunteers who participated in this study. They appreci-ate the eﬀorts of the clinical research staﬀ: Dr. IbrahimMteza, Esther Kayichile, Joyce Wamsele, and EmmanuelBalandya. They also thank Betty Mchaki (DarDar Project)and Wendy Wieland-Alter (Dartmouth Medical School) fortheir generous laboratory assistance and Miriam Zayumba(DarDar Project) and Susan Tvaroha (Dartmouth College)for assistance with data management. The authors disclosethat Oxford Immunotec, themanufacturer of the T-SPOT.TBand the T Cell Xtend reagent, funded this study and alsocontributed in on-site study monitoring. Oxford Immunotecdid not interpret the findings or write this paper. C. F. vonReyn has been a one-day consultant for Oxford Immunotecon two occasions, and C. F. von Reyn, L. V. Adams, and E.A. Talbot received additional research funding from OxfordImmunotecmore than one year prior to this submission. Theother authors have no potential conflicts to declare.References[1] CDC, “Updated guidelines for using interferon gamma releaseassays to detect Mycobacterium tuberculosis infection—UnitedStates, 2010,” Morbidity and Mortality Weekly Report, vol. 59,no. RR-5, pp. 2–9, 2010.[2] http://www.oxfordimmunotec.com/XtendpackInsert-english,2010.[3] http://www.oxfordimmunotec.com/96-UK, 2010.[4] J. Z. Metcalfe, C. K. Everett, K. R. Steingart et al., “Interferon-γ release assays for active pulmonary tuberculosis diagnosis inadults in low-and middle-income countries: systematic reviewand meta-analysis,” Journal of Infectious Diseases, vol. 204, no.4, pp. S1120–S1129, 2011.[5] I. Durrant, J. Radcliﬀe, and T. Day, “TB testing using T-SPOT.TB after overnight sample storage,” Clinical Microbiologyand Infection, vol. 15, p. pS393, 2009.[6] L. M. Lenders, R. Meldau, R. N. van Zyl-Smit et al., “Compar-ison of same day versus delayed enumeration of TB-specific Tcell responses,” Journal of Infection, vol. 60, no. 5, pp. 344–350,2010.[7] S. Wang, D. A. Powell, H. N. Nagaraja, J. D. Morris, L. S.Schlesinger, and J. Turner, “Evaluation of amodified interferon-gamma release assay for the diagnosis of latent tuberculosisinfection in adult and paediatric populations that enablesdelayed processing,” Scandinavian Journal of Infectious Diseases,vol. 42, no. 11-12, pp. 845–850, 2010.Submit your manuscripts athttp://www.hindawi.comStem CellsInternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014MEDIATORSINFLAMMATIONofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Behavioural NeurologyEndocrinologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Disease MarkersHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioMed Research InternationalOncologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Oxidative Medicine and Cellular LongevityHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014PPAR ResearchThe Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofObesityJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Computational and  Mathematical Methods in MedicineOphthalmologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Diabetes ResearchJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Research and TreatmentAIDSHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Gastroenterology Research and PracticeHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Parkinson’s DiseaseEvidence-Based Complementary and Alternative MedicineVolume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

Maintenance of Sensitivity of the T-SPOT.<i>TB</i>Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania

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Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania

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