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Tuberculosis Diagnostics in the New Millennium: Role in TB Identification and ControlGuest Editors: Soumitesh Chakravorty, Catharina Boehme, and Jongseok Lee

Tuberculosis Research and Treatment

Tuberculosis Diagnostics in the New Millennium:Role in TB Identification and Control

Tuberculosis Research and Treatment

Tuberculosis Diagnostics in the New Millennium:Role in TB Identification and Control

Guest Editors: Soumitesh Chakravorty, Catharina Boehme,and Jongseok Lee

Copyright © 2012 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Tuberculosis Research and Treatment.” All articles are open access articles distributed under theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

Editorial Board

J. K. Actor, USAA. S. Apt, RussiaM. S. Baird, UKM. R. Barer, UKChristos Chouaid, FranceLuis E Cuevas, UKKarl Drlica, USABrian Eley, South AfricaCarlo Garzelli, ItalyE. A. Graviss, USAJacques Grosset, USA

A. S. Hammond, MaliS. E. Hasnain, IndiaR. L. Hunter, USAJuraj Ivanyi, UKVincent Jarlier, FranceP. R. Klatser, The NetherlandsJos R. Lapa e Silva, BrazilRoy Mugerwa, UgandaT. Ottenhoff, The NetherlandsDavid C. Perlman, USANalin Rastogi, France

Giovanna Riccardi, ItalyMenico Rizzi, ItalyW. N. Rom, USAEuzenir N. Sarno, BrazilSarman Singh, IndiaJeffrey R. Starke, USAIsamu Sugawara, JapanT. E. Tupasi, PhilippinesRobert S. Wallis, USA

Contents

Tuberculosis Diagnostics in the New Millennium: Role in TB Identification and Control,Soumitesh Chakravorty, Catharina Boehme, and Jongseok LeeVolume 2012, Article ID 768603, 2 pages

Pouched Rats’ Detection of Tuberculosis in Human Sputum: Comparison to Culturing and PolymeraseChain Reaction, Amanda Mahoney, Bart J. Weetjens, Christophe Cox, Negussie Beyene, Klaus Reither,George Makingi, Maureen Jubitana, Rudovick Kazwala, Godfrey S. Mfinanga, Amos Kahwa, Amy Durgin,and Alan PolingVolume 2012, Article ID 716989, 5 pages

The PCR-Based Diagnosis of Central Nervous System Tuberculosis: Up to Date, Teruyuki Takahashi,Masato Tamura, and Toshiaki TakasuVolume 2012, Article ID 831292, 17 pages

Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples,Dar es Salaam, Tanzania, Elizabeth A. Talbot, Isaac Maro, Katherine Ferguson, Lisa V. Adams, Lillian Mtei,Mecky Matee, and C. Fordham von ReynVolume 2012, Article ID 345290, 4 pages

The Use of Interferon Gamma Release Assays in the Diagnosis of Active Tuberculosis,Silvan M. Vesenbeckh, Nicolas Schonfeld, Harald Mauch, Thorsten Bergmann, Sonja Wagner,Torsten T. Bauer, and Holger RussmannVolume 2012, Article ID 768723, 4 pages

Comparison of Overnight Pooled and Standard Sputum Collection Method for Patients with SuspectedPulmonary Tuberculosis in Northern Tanzania, Stellah G. Mpagama, Charles Mtabho,Solomon Mwaigwisya, Liberate J. Mleoh, I Marion Sumari-de Boer, Scott K. Heysell, Eric R. Houpt,and Gibson S. KibikiVolume 2012, Article ID 128057, 5 pages

Hindawi Publishing CorporationTuberculosis Research and TreatmentVolume 2012, Article ID 768603, 2 pagesdoi:10.1155/2012/768603

Editorial

Tuberculosis Diagnostics in the New Millennium: Role inTB Identification and Control

Soumitesh Chakravorty,1 Catharina Boehme,2 and Jongseok Lee3

1 Division of Infectious Diseases, Department of Medicine, New Jersey Medical School, University of Medicine & Dentistry of New Jersey,Newark, NJ 07103, USA

2 Foundation for Innovative New Diagnostics (FIND), Avenue de Bude 16, 1202 Geneva, Switzerland3 Department of Microbiology, International Tuberculosis Research Center, Gyeongsang, Changwon 631-710, Republic of Korea

Correspondence should be addressed to Soumitesh Chakravorty, [email protected]

Received 10 December 2012; Accepted 10 December 2012

Copyright © 2012 Soumitesh Chakravorty et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Tuberculosis (TB) continues to remain a significant threateven as we have moved into the second decade of the 21stcentury. As a matter of fact TB has assumed an even moreominous stance with the emergence of totally drug resistantor TDR Mycobacterium tuberculosis (M. tb) strains which arevirtually untreatable. Unfortunately, there has been no neweffective vaccine against tuberculosis, and in spite of intro-duction of several new drugs like bedaquiline, delamanid,PA824, or SQ109 and a new generation of fluoroquinoloneslike gatifloxacin and moxifloxacin, they are yet to be involvedin the current routine antituberculosis regimen, thoughclinical trials for combinatorial therapy along with currentlyused drugs are underway. The emergence of resistanceagainst these new classes of drugs is also a likely possibilitywhich will result in the same problems in the future withnewer group of drug-resistant strains, as we are facing todaywith the multidrug resistant (MDR) and extensively drug-resistant (XDR) strains. Under these circumstances, rapidand definitive molecular diagnostics for effective interven-tion and treatment of TB patients is a cornerstone forappropriate disease control and eradication. Recently, a lotof attention has been devoted towards rapid TB diagnosticsespecially those which enable rapid drug susceptibility testing(DST) to break free from the century long dependence onsmear microscopy and culture methods, which are frustrat-ingly insensitive and time consuming, respectively. WHOhas recently recommended automated liquid culture systems,line probe assays, and the Xpert MTB/RIF tests which allowfaster DST results and highly sensitive detection of M. tb fromclinical samples. These assays signal towards the emerging

era of rapid and decisive tests employing new generation ofmolecular and microbiological methods. Against this excit-ing backdrop of the emerging trends and innovations in newTB diagnostic techniques, the special edition of TuberculosisResearch and Treatment focuses on tuberculosis diagnosticsin the new millennium, role in TB identification and control,which represents a perspective on the emerging trends inthe next of generation TB diagnostics. From unconventionalapproaches like using sputum sniffing pouched rats tothe latest immunodiagnostics like interferon gamma releaseassays (IGRAs), a detailed overview on the current PCR-based techniques of diagnosis of TB meningitis, as well asimportance of the methods of sample collection for accuratediagnosis of TB, are dealt with in this issue. One elegant studyby A. Mahoney et al. examines the utility of using pouchedrats for detection of tuberculosis as an adjunct to smearmicroscopy in resource poor countries. The paper by T.Takahashi et al. explores the current PCR-based approachesto diagnose one of the most deadly forms of extrapulmonaryTB, TB meningitis, and describes a novel “wide range quanti-tative nested real-time PCR” assay. Two papers by E. A.Talbot et al. and S. M. Vesenbeckh et al. deal with one ofthe most widely used immunodiagnostics for TB, namely,IGRA and its applicability in TB diagnostics. One paperexamined its utility in overnight-stored blood samples, anda second study assessed the accuracy of the recommendedcut-off values for diagnosing active TB, in which both haveimportant implications on utility and applicability of IGRA.The paper by Mpagama et al. brings forth the importance ofusing overnight-pooled sputum in enhancing the sensitivity

2 Tuberculosis Research and Treatment

and shortening the time of detection using the BACTECMGIT system. This special edition on the current scenarioof TB diagnostics will serve as a concise but comprehensivespectrum of the different approaches aimed at faster andmore definitive detection of TB disease and its control.

Soumitesh ChakravortyCatharina Boehme

Jongseok Lee


Clinical Study

Pouched Rats’ Detection of Tuberculosis in Human Sputum:Comparison to Culturing and Polymerase Chain Reaction

Amanda Mahoney,1, 2 Bart J. Weetjens,2 Christophe Cox,2 Negussie Beyene,2

Klaus Reither,3, 4 George Makingi,5 Maureen Jubitana,2 Rudovick Kazwala,5

Godfrey S. Mfinanga,6 Amos Kahwa,6 Amy Durgin,1, 2 and Alan Poling1, 2

1 Department of Psychology, Western Michigan University, Kalamazoo, MI 49008-5200, USA2 Tuberculosis Research, Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling (APOPO), Morogoro, Tanzania3 Tuberculosis Research, Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland4 TB Research and Training Center, Ifakara Health Institute, Bagamoyo, Tanzania5 Department of Veterinary Medicine, Sokoine University of Agriculture, Morogoro, Tanzania6 Clinical Research Laboratory, National Institute for Medical Research, Dar es Salaam, Tanzania

Correspondence should be addressed to Amanda Mahoney, [email protected]

Received 22 December 2011; Revised 16 May 2012; Accepted 16 May 2012

Academic Editor: Soumitesh Chakravorty

Copyright © 2012 Amanda Mahoney 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.

Setting. Tanzania. Objective. To compare microscopy as conducted in direct observation of treatment, short course centers topouched rats as detectors of Mycobacterium tuberculosis. Design. Ten pouched rats were trained to detect tuberculosis in sputumusing operant conditioning techniques. The rats evaluated 910 samples previously evaluated by smear microscopy. All sampleswere also evaluated through culturing and multiplex polymerase chain reaction was performed on culture growths to classify thebacteria. Results. The patientwise sensitivity of microscopy was 58.0%, and the patient-wise specificity was 97.3%. Used as a groupof 10 with a cutoff (defined as the number of rat indications to classify a sample as positive for Mycobacterium tuberculosis) of 1,the rats increased new case detection by 46.8% relative to microscopy alone. The average samplewise sensitivity of the individualrats was 68.4% (range 61.1–73.8%), and the mean specificity was 87.3% (range 84.7–90.3%). Conclusion. These results suggest thatpouched rats are a valuable adjunct to, and may be a viable substitute for, sputum smear microscopy as a tuberculosis diagnosticin resource-poor countries.

1. Introduction

A major hurdle in combating tuberculosis (TB) is diagnos-ing the disease in resource-poor countries. Sputum smearmicroscopy, the technique typically used, is relatively slowand characteristically has high specificity but low sensitivity[1, 2]; therefore, the international medical community hasprioritized developing a quick, accurate, and affordable alter-native diagnostic. In an attempt to develop one, researchersrecently have investigated the use of scent-detecting pouchedrats (Cricetomys gambianus) as a TB diagnostic. An initialproof of principle investigation [3] revealed that pouchedrats trained through operant conditioning procedures coulddetect TB in human sputum, and three subsequent studies,involving a total of over 20,000 patients, showed that

using the rats in second-line screening of sputum samplesinitially screened by smear microscopy at direct observationof treatment—short course (DOTS) centers in Tanzaniaincreased new case detections by 31.4% [4], 44% [5], and42.8% [6].

These results are promising, but the accuracy of Crice-tomys in detecting TB has not been extensively evaluatedrelative to an established reference standard. Culturing isconsidered the “gold standard” for TB detection [2], andWeetjens et al. [3] reported the results of a study in which tworats, Mandela and Kingston, evaluated 817 sputum samplesalso evaluated by culturing, which revealed 67 TB-positivesamples. Sensitivity relative to culturing for both rats was73.1%, while specificity was 97% and 97.8% for Mandelaand Kingston, respectively. In an attempt to provide more


comprehensive information regarding pouched rats’ TB-detection accuracy relative to the best available and afford-able method, this experiment evaluated 10 rats’ performancecompared to culture in combination with Multiplex PCR.

2. Method

2.1. Subjects and Materials. Ten adult Cricetomys obtainedfrom our breeding colony, 5 males and 5 females, eval-uated all sputum samples. The animals were housed andmaintained as detailed elsewhere [3, 7]. Ethical clearanceto conduct the research was obtained from the TanzanianNational Institute for Medical Research. Some of the rats hadbeen used in previous studies and all of the rats had beenevaluating sputum samples for TB for at least one year.

Testing was conducted in a chamber 205 cm long, 55 cmwide, and 55 cm high with clear plastic walls and ceiling anda stainless steel floor. Ten holes with sliding lids 2.5 cm indiameter were spaced equidistance apart along the centerlineof the chamber floor’s long axis. Pots containing sputumwere placed beneath the holes for the rats to evaluate. Ediblereinforcers (rewards), consisting of a mixture of mashedbanana with ground rodent diet pellets, were deliveredthrough a plastic syringe through feeding holes.

2.2. Collection of Sputum Samples. Sputum samples werecollected weekly from eight DOTS centers in Dar es Salaamand Morogoro, Tanzania, using World Health Organization(WHO) recommended sputum containers. Direct smearmicroscopy after Ziehl-Neelsen staining was conducted at theDOTS centers prior to collection of the samples. Samplesof less than 2 mL were excluded to ensure that there wassufficient volume for culturing and rat evaluation. In all, 910samples from 456 patients (two from each of 454, one fromeach of two) were evaluated. Before evaluation by rats, analiquot was taken from each sample for culture purposes,and then sterile phosphate buffered saline solution (5 mL)was added to each sputum sample and microorganisms wereinactivated by heating the sample at 90◦C in a water bathfor 30 min [8]. The samples were then frozen at −20◦Cuntil the day of evaluation (up to seven days). Thoughthere is some controversy surrounding the cellular impactof freezing and thawing sputum, past research suggests thatsamples may be kept frozen without significant alteration ofcell quality or cell counts [9]. Furthermore, data collectedinternally suggest that the rats’ performance is unaffectedby the freezing procedures employed. Samples were thawedfour times for the purpose of this study: once on the day ofcollection, once to take aliquots for culture, once to evaluatethe sputum quantity and add buffer, and once on the day ofevaluation by the rats.

2.3. Rats’ Evaluation of Samples. Prior to this study, the ratswere trained to detect TB as detailed elsewhere [3, 7]. Inthe present study, each rat evaluated each sample twice, in adifferent order, across 13 sessions. In each session, 63 samplesfound negative by microscopy at DOTS centers and sevensamples found positive were presented to the rats. The seven

positive samples served as reinforcement opportunities tomaintain the rats’ indications while the remaining sampleswere categorized as “unknown”. During the sessions, theexperimenter opened each hole in the cage as the rat passedover and sniffed. When the rat paused for 5 s (i.e., emittedan indicator response), the experimenter informed a datacollector who then stated whether the sample was smearpositive according to DOTS-center microscopy. If the ratmade an indicator response above a smear-positive sample,the experimenter sounded a click and delivered food, afterwhich the rat then moved to the next hole to continueevaluations. If the rat emitted an indicator response at asmear-negative sample, which is considered an unknownsample, the experimenter closed the hole but did not sounda click or present food.

2.4. Data Analysis of Rat Results. Following evaluations, therats’ performance was assessed relative to the results ofculture with M. tuberculosis Multiplex PCR. Sensitivity andspecificity were calculated for the group of 10 rats, and thusthe criterion for counting a rat-positive indication could bean indication on either or both sample presentations by onerat, ten rats, or any number of rats in between, which arereferred to hereafter as cutoffs 1–10. At a cutoff of 3, forexample, a sample was deemed rat positive if three or morerats indicated it; samples indicated by only 2, 1, or 0 rats weredeemed negative.

2.5. Culturing and PCR. Culturing was conducted in accor-dance with an established and recommended procedure forculturing sputum samples on Lowenstein-Jensen (LJ) solidmedia (WHO Guidelines on Standard Operating Proce-dures for Microbiology, Tuberculosis, WHO Regional Officefor Southeast Asia, 2006). Decontaminated samples wereinoculated onto different tubes of Lowenstein-Jensen solidmedia, one with pyruvate and the other with glycerol. Thetubes were incubated at 37◦C and inspected weekly for eightweeks. Media on which microbial growth was observed werescraped, stained by the ZN method, and analyzed by lightmicroscopy. Each specimen which exhibited either AFB-positive culture material or characteristic bacterial growthwas further analyzed by Multiplex PCR.

In the first step of PCR [10], Multiplex PCR genustyping was conducted to identify species belonging tothe Mycobacterium genus. This genotyping distinguishedspecies belonging to the Mycobacterium tuberculosis complex(MTC), specifically M. bovis, M. africanum, M. tuberculosis,and M. microti, from nontuberculous mycobacteria (M.avium, M. intracellulare, and others). All bacterial suspen-sions or DNA extracts containing MTC were subjected toanother PCR, deletion typing. This procedure differentiatedbacteria that were M. tuberculosis, M. bovis, or M. africanum.

3. Results

All sputum samples were classified as positive or negativefor M. tuberculosis by microscopy at the DOTS centers,culturing (with PCR as appropriate), and rats’ evaluation.

Tuberculosis Research and Treatment 3

Table 1: Samples classified as M. tuberculosis and non-M. tubercu-losis by Multiplex PCR.

M. tuberculosis Non M. tuberculosis

Total PCR+ 129 13

ZN+ glycerol/pyruvate (102/87) (2/13)αRat+ 109 10

Smear+ (DOTS) 86 7αA sample was considered rat positive if at least one rat indicated.

Table 2: Sample-wise and patient-wise sensitivity and specificity atrat agreements cutoffs 1–10.

CutoffSamplewise Patientwise

Sensitivity Specificity Sensitivity Specificity

1 84.50 64.00 85.54 49.06

2 81.40 75.70 81.93 64.61

3 76.00 81.80 75.90 73.99

4 74.40 86.60 73.49 80.70

5 68.20 89.40 66.27 84.18

6 66.70 91.90 65.06 88.47

7 65.10 93.30 63.86 90.88

8 62.80 95.50 62.65 94.64

9 58.90 96.90 60.24 96.51

10 53.50 98.10 54.22 98.12αRelative to Multiplex PCR.

Culture-positive samples were those in which character-istic growth was stained with a Ziehl-Neelsen stain andthe presence of acid-fast bacilli confirmed. A sample wasfurther considered PCR-positive if, following amplification,the amplified nucleotide sequence for M. tuberculosis wasdetected (see Table 1 for these results). DOTS centers’microscopy identified 96 positive samples and 49 positivepatients, and culture identified 162 positive samples and 104positive patients. PCR identified 129 positive samples and 81positive patients. DOTS-centers’ microscopy found 86 of thePCR positive samples and 771 of the PCR negative samples.Thus, relative to PCR, sputum smear microscopy conductedat the DOTS centers yielded a sample-wise sensitivity of66.7% and a specificity of 98.7%.

Table 2 displays rat results relative to PCR at rat cutoffs1–10. At a cutoff of 1, sample-wise sensitivity was 84.5%and specificity was 64%. As the cutoff increased, specificitycharacteristically improved while sensitivity worsened. At thelargest cutoff, 10 (meaning all rats must indicate upon thesample to score it as rat-positive), sensitivity was 53.5% andspecificity was 98.1%. At a cutoff of 7 sample-wise sensitivitywas 65.1% and specificity was 93.3%.

Although it is tenable to use 10 rats, using fewer animalswould render the evaluation process faster and less expensive.To ascertain the effects of using fewer animals, 36 combina-tions of randomly selected rats were created from the presentdata set, 12 combinations of 4 rats, 12 combinations of 3rats, and 12 combinations of 2 rats. Sample-wise sensitivityand specificity relative to culture/PCR were calculated foreach rat combination (Table 3). Using four rats at a cutoff

of one rat, average sensitivity was 79.9% (range 76.2–82.5%)and average specificity was 73.8% (range 71.2–75.4%). Whenonly two rats were used, average sensitivity decreased to74.9% (range 69–88.1%) while average specificity increasedto 81.6% (range 78.1–85.6%). As the cutoff selected isincreased, meaning more rat indications are required tocount a sample as rat-positive, sensitivity decreases andspecificity increases. For example, using four rats at a cutoffof four, sensitivity dropped to 57.8% (from 79.9% at a cutoffof 1) while specificity improved to 96.2% (from 73.8% at acutoff of 1).

Table 4 shows patient-wise data comparing the sensitivityand specificity of DOTS centers’ microscopy to the averagefor the 10 individual rats. Relative to combined MultiplexPCR and MTB/RIF results, DOTS centers’ microscopyidentified 47 of 81 positive patients and 407 of 409 negativepatients. Therefore, at the patient-wise level, the sensitivityof microscopy at DOTS centers was 58% and the specificitywas 99.5%. The rats correctly classified, on average, 57.1positive and 329.1 negative patients and 104.2 positive and675 negative samples. For the rats, the mean patient-wisesensitivity was 70.5% (99% confidence interval [CI] .68–.73)and mean specificity was 80.5% (95% CI .78–.83). Predictivevalues were calculated for patient-wise results of DOTSmicroscopy and the rats (Table 4). The positive predictivevalue (i.e., the probability that a patient with a positive testresult really does have the condition of interest) was .96(95% confidence interval [CI] .84–.99) for microscopy and.42 (95% CI .33–.50) for the rats. The negative predictivevalue (i.e., the probability that a patient with a negative testresult really is free of the disease) was .92 (95% CI .89–.95)for microscopy and .93 (95% CI .89–.96) for the rats.

4. Discussion

In this study, 10 adult Cricetomys evaluated 910 sputumsamples collected from patients suspected for tuberculosis.Weetjens et al. [3] previously reported that each of twopouched rats yielded a sensitivity of 73.1% relative toculturing, and their specificities were 97% and 97.8%. Inthe present study, which included Multiplex PCR, somewhatlower values were obtained where the mean individualsample-wise sensitivity of 10 rats relative to culture/PCR was68.4%, and the mean specificity was 87.3%. Nonetheless,each rat’s sensitivity exceeded that of ZN smear microscopyperformed as part of routine TB screening at DOTS centers,although their specificity was lower. Because the rats canevaluate samples quickly, it is tenable to have several ofthem evaluate each sputum sample, and this has been donein studies examining their use in second-line screening ofsamples initially evaluated by ZN microscopy [4–6]. Forexample, Poling et al. [5] used a cutoff of 2 of 10 rats foridentifying a sample as TB-positive. The present data suggestthat this is a reasonable criterion in terms of balancingsensitivity and specificity, which were 81.4% and 75.7%when it was used in the present study. Similar values wereobtained with cutoffs of 3 of 10 and 4 of 10. With a cutoffof 2, the rats as a group detected 66 of 81 patients found


Table 3: Average sensitivity and specificity for 12 groups of 4, 3, and 2 rats.

Cutoff4 Rats 3 Rats 2 Rats

Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity

1 79.9 73.8 77.2 77.4 74.9 81.6

2 71.5 86.4 68.8 89.7 63.4 93.4

3 64.8 92.0 60.6 95.6

4 57.8 96.2αRelative to culture/PCR.

Table 4: Patient-wise smear microscopy and average rat reference results.

Sensitivity Specificity Pos predictive value Neg predictive value

Culture/PCR No TB Culture/PCR No TBαSmear

Correct/total samples (%) 47/81 (58.0) 407/409 (99.5) .96 .92

95% confidence interval (CI) 46.6–68.7 98–99.9 .84–.99 .89–.95

Rat (average of 10)

Correct/total samples (%) 57.1/81 (70.5) 329.1/409 (80.5) .42 .93

95% CI 68.2–72.8 78.4–82.6 .33–.50 .89–.96αSmear microscopy conducted at DOTS centers.

to be TB-positive by culturing/PCR, whereas DOTS centers’microscopy detected 47 of these patients. Therefore, had therats been used in second-line screening as in prior studies[5, 6] and their results verified, they would have increasednew-case detections by 40.4%.

Results revealed 57 culture-negative and three culture-positive, but PCR-negative samples indicated by six or morerats. To clarify the status of these samples, an internal testwas done on all of these plus 100 randomly-selected rat-negative samples using the GeneXpert MTB/RIF (Cephid,Sunnyvale, CA, USA) [11]. Analysis by the GeneXpertrevealed 25 positive samples and 23 positive patients that hadpreviously been classified as negative by culture or MultiplexPCR, bringing the combined Multiplex PCR and MTB/RIFpositive samples to 154 and positive patients to 98. The testreclassified 18 of the 60 rat-positive samples and 7 of the 100rat-negative samples. An analysis was conducted includingthese reclassified participants and revealed a patient-wisesensitivity of microscopy at DOTS centers of 48% andspecificity of 98.3%. For the rats, patient-wise sensitivity was67% (range 62.2–72.5%) while their mean specificity was93.5% (range 91.1–95.3%). Due to the costs of the cartridgesrequired for the GeneXpert, it was not possible to evaluateall samples and, to avoid a possible bias, these data werenot incorporated into the main results. These additional datasuggest that the specificity of the rats may be higher thanthat suggested by the comparison to culture and, to testthis possibility these findings, a study is underway that willthoroughly evaluate the rats relative to MTB/RIF.

In prior studies, a second microscopy was used to verifythe status of DOTS-negative, rat-positive samples, but suchconfirmation is weak. The rats identify as positive a relativelyhigh number of TB-negative samples; however, relying onmicroscopy alone allows a substantial number of patientswith TB to go undetected. A better procedure would be to use

the GeneXpert to confirm the status of rat-positive, smear-negative samples. Confirmation of samples in this way islikely to reveal a substantial number of TB-positive patientsmissed with the present procedure and thus slow the spreadof transmission. This benefit would seemingly justify thefinancial cost of using the GeneXpert.

In addition to finding TB-positive patients overlooked bymicroscopy, the rats may potentially yield savings in time andcost. The rats are faster in evaluation than a lab technician,but require that the samples be transported and processed.These steps are completed with large batches of samples andit is important that future studies clearly demonstrate thecost-efficiency of these procedures relative to microscopy andinvestigate potential savings in costs and time. Prospectively,should the rats be called upon as a first-line screening tool,research on the MTB/RIF assay indicates that its high costmay limit its global utility [12], and the rats seem particularlywell suited to work in conjunction with this technology toreduce costs. The rats screen samples quickly and, if usedin areas with prevalence similar to that in which they havebeen tested, will reduce the number of patients in need offollowup. The outcome of such a setup depends largely uponthe number of rats used. Extrapolating from the currentresults, one could expect to recheck about 10% of samplesif one rat is used and recheck about 45% of samples if 10rats are used. The ideal number, as illustrated in Table 4, isprobably between 2 and 4 rats as sensitivity remains relativelyhigh while the false alarm rate improves with fewer rats.

A significant limitation of the present study is thatno clinical data were available for comparison. TB-positivepatients evaluated by TB specialists are more likely to beidentified than those diagnosed by smear results alone[13], and so a study is underway at APOPO that willincorporate clinical data. A second limitation is that theHIV status of patients was not available to us; therefore, the


sensitivity and specificity of DOTS microscopy and the ratscould not be compared in HIV-positive and HIV-negativepatients. Further research in this area is planned to make thecomparison and to evaluate the value of the rats in detectingTB in children.

5. Conclusions

In this study, DOTS microscopy found 58% of the cul-ture/Multiplex PCR positive patients, which is similar toresults found in past studies [2, 11], compared to an averageof 70.5% of positive patients found by individual rats.The results presented herein, combined with previouslypublished operational data, demonstrate that the rats arefaster than smear microscopy as commonly practiced andcan identify more TB-positive patients. There is now sub-stantial evidence that when used for second-line screening,Cricetomys can have a large positive impact on TB detectionand public health in high-incidence areas, such as sub-Saharan Africa, although future research is necessary torefine training techniques to identify the applications forwhich the rats are best suited and to ascertain their per-case-detected cost relative to alternative diagnostics.

Authors’ Contribution

This work was carried out in collaboration among all authorslisted. C. Cox, B. J. Weetjens, A. Poling, G. Makingi, and A.Mahoney conceptualized this research and provided criticalintellectual content. R. Kazwala, G. S. Mfinanga, and K.Reither provided expertise on the laboratory operationsnecessary for carrying out this experiment, contributed tothe conceptualization of this research, and participated in thediscussion on presentation of the results. A. M. Mahoney, N.Beyene, M. Jubitana, D. Kuipers, and A. Durgin oversaw thelaboratory experiments, analyzed the data, and interpretedthe results. A. Poling, A. Mahoney, and N. Beyene wrote thepaper. All authors have contributed to, seen, and approvedthis paper.

Acknowledgments

The authors wish to acknowledge the trainers, secretaries,and laboratory technicians in the TB laboratory at APOPOfor their hard work throughout this experiment. Thiswork would not have been possible without their support.Financial support for this work was provided by the UBSOptimus Foundation. The authors declare that they haveno competing interests, and specifically have no competinginterests with respect to the use of the Cepheid GeneXpert.

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[11] M. D. Perkins, G. Roscigno, and A. Zumla, “Progress towardsimproved tuberculosis diagnostics for developing countries,”The Lancet, vol. 367, no. 9514, pp. 942–943, 2006.

[12] P. M. Small and M. Pai, “Tuberculosis diagnosis-time for agame change,” The New England Journal of Medicine, vol. 363,no. 11, pp. 1070–1071, 2010.

[13] P. K. Lam, P. A. Lobue, and A. Catanzaro, “Clinical diagnosis oftuberculosis by specialists and non-specialists,” InternationalJournal of Tuberculosis and Lung Disease, vol. 13, no. 5, pp.659–661, 2009.


Review Article

The PCR-Based Diagnosis of Central Nervous SystemTuberculosis: Up to Date

Teruyuki Takahashi,1 Masato Tamura,1, 2 and Toshiaki Takasu1, 2

1 Department of Neurology, Nagaoka-Nishi Hospital Mitsugohya-machi, 371-1 Nagaoka City, Niigata, Japan2 Division of Neurology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan

Correspondence should be addressed to Toshiaki Takasu, [email protected]

Received 2 December 2011; Accepted 14 February 2012


Copyright © 2012 Teruyuki Takahashi 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.

Central nervous system (CNS) tuberculosis, particularly tuberculous meningitis (TBM), is the severest form of Mycobacteriumtuberculosis (M.Tb) infection, causing death or severe neurological defects in more than half of those affected, in spite of recentadvancements in available anti-tuberculosis treatment. The definitive diagnosis of CNS tuberculosis depends upon the detectionof M.Tb bacilli in the cerebrospinal fluid (CSF). At present, the diagnosis of CNS tuberculosis remains a complex issue because themost widely used conventional “gold standard” based on bacteriological detection methods, such as direct smear and cultureidentification, cannot rapidly detect M.Tb in CSF specimens with sufficient sensitivity in the acute phase of TBM. Recently,instead of the conventional “gold standard”, the various molecular-based methods including nucleic acid amplification (NAA)assay technique, particularly polymerase chain reaction (PCR) assay, has emerged as a promising new method for the diagnosis ofCNS tuberculosis because of its rapidity, sensitivity and specificity. In addition, the innovation of nested PCR assay technique isworthy of note given its contribution to improve the diagnosis of CNS tuberculosis. In this review, an overview of recent progressof the NAA methods, mainly highlighting the PCR assay technique, was presented.

1. Introduction

Central nervous system (CNS) disease caused by Mycobac-terium tuberculosis (M.Tb), particularly tuberculous menin-gitis (TBM), is uncommon and accounts for approximately1% of all tuberculosis cases in the United States [1, 2]. CNStuberculosis is the severest form of M.Tb infection, causingdeath or severe neurological defects in more than half ofthose affected, in spite of recent advancements in availableantituberculosis treatment (ATT) [1–5]. In addition, owingto an increasing number of immunocompromised hostscaused by the prevalence of AIDS, increasing numbers ofolder people, the wider use of immunosuppressive agents,and other factors, TBM remains a serious clinical and socialproblem [1–5]. Owing to its relative rarity and the widespectrum of its neurological symptoms, CNS tuberculosisremains a formidable diagnostic challenge [1–5]. In TBM,accurate and rapid diagnosis and early treatment for tuber-culosis are the most important factors with regard to theprognosis and the prevention of long-term neurological

sequelae [1–5]. However, the conventional “gold standard”based on bacteriological detection methods of M.Tb, includ-ing the direct smear examination for acid-fast bacilli (AFB)and culture identification, is inadequate for early diagnosis,owing to the poor sensitivity or the long time required (4–8weeks) for cultures [1–5].

Recently, the detection of M.Tb DNA in the cerebrospinalfluid (CSF) through the use of various molecular-basedmethods, including nucleic acid amplification (NAA) assaytechnique, particularly polymerase chain reaction (PCR)assay, has emerged as a promising new method for thediagnosis of CNS tuberculosis because of its rapidity,sensitivity, and specificity [2–42]. Many investigators havereported on the usefulness of PCR assay for the detectionof M.Tb DNA in CSF, although the sensitivity of PCRassay has significant discrepancies (65–83%) between eachtype of measuring methods and different laboratories [2–42]. Moreover, nested PCR assay has been reported asa prominent method for detecting M.Tb DNA in CSF


[3, 4, 17, 19, 24–27, 32]. This new method drasticallyincreases the sensitivity and specificity of DNA amplificationcompared with conventional single-step PCR [3, 4, 17,19, 24–27, 32]. However, the nested PCR assay using CSFsamples has yet to be widely used in TBM diagnosis owing toits laborious and time-consuming procedure, which carriesa high risk of cross-contamination [3, 4, 17, 19, 24–27,32]. Currently, real-time PCR assay is applied in routinediagnostic laboratory testing [33, 38, 40–42]. In addition toconventional qualitative analysis, real-time PCR assay makesit possible to perform accurate quantitative analyses with ahigh degree of reproducibility [33, 38, 40–42].

In this paper, the authors highlight the recent advance-ment of NAA assay techniques, in particular PCR assay andprovide an overview of the current issues and evolution ofdiagnosis and clinical aspects of CNS tuberculosis.

2. The Global Epidemiologic Burdenof Tuberculosis

In 2007, the World Health Organization (WHO) estimatedthat 9.27 million new cases (139/100,000 population) ofactive tuberculosis occur annually, resulting in an estimated1.6 million deaths per year [1, 2]. Tuberculosis remains aworldwide burden, with a large majority of new active tuber-culosis cases occurring in underdeveloped and developingcountries [1, 2]. In fact, India, China, Indonesia, Nigeria,and South Africa rank first to fifth in the total numberof incident cases of tuberculosis [1, 2]. In 80% of newtuberculosis cases, social and demographic factors such aspoverty, overcrowding, malnutrition, and a compromisedimmune system play a major role in the worldwide epidemic,while the remaining 20% of tuberculosis cases are associatedwith HIV in sub-Saharan Africa [1, 2]. Among the 9.27million new tuberculosis cases in 2007, an estimated 1.37million (14.8%) were HIV positive [1, 2].

CNS infection is one of the severest forms of tuber-culosis [1–5]. In a large-scale epidemiological study ofextrapulmonary tuberculosis in the United States, CNSinvolvement was noted in 5 to 10% of extrapulmonarytuberculosis cases, with more recent CDC data in 2010indicating that 5.5% of extrapulmonary cases involve CNStuberculosis (=1.2% of total tuberculosis cases) [43–46].In the largest prospective epidemiological study of CNStuberculosis, the incidence of developing CNS tuberculosiswas approximately 1.0% among 82,764 tuberculosis casesfrom 1970 to 2001 in a Canadian cohort [1, 2, 43].However, despite an overall decrease in the total number oftuberculosis cases in advanced nations such as the UnitedStates, a gradual and continuous increase in the proportionof extrapulmonary tuberculosis cases has been reported [1,2, 43–46]. This increase has been mainly attributed to therecent increase of immunocompromised patients and theHIV/AIDS epidemic [1, 2, 43–46]. In addition, although theoverall population-based mortality rate from tuberculosis islow and decreasing, several studies have shown that mortalityrates are substantially higher in patients with several forms of

extrapulmonary tuberculosis, including CNS tuberculosis orTBM and disseminated disease [1, 2, 43–46].

Several risk factors for CNS tuberculosis have beenidentified. Both age (children > adults) and HIV-coinfectedpatients are at high risk for developing CNS tuberculosis[1, 2, 44]. Other risk factors include malnutrition, recentmeasles in children, alcoholism, malignancies, the use ofimmunosuppressive agents in adults, and disease prevalencein the community [1, 2]. Several studies in developedcountries have also identified that foreign-born individuals(individuals born outside of developed countries) are over-represented among CNS tuberculosis cases [1, 2, 44–46].

3. Current Issues for Diagnosis inCNS Tuberculosis

At present, the diagnosis of CNS tuberculosis remains acomplex issue because the most widely used conventionalbacteriological detection methods, such as direct smear forAFB and culture for M.Tb, cannot rapidly detect M.Tbin CSF specimens with sufficient sensitivity in the acutephase of TBM [2–42]. Rapid and accurate diagnosis in theacute phase of CNS tuberculosis and early starting ATT arethe most important factors with regard to the prognosisand the prevention of long-term neurological sequelae [2–5]. The poor sensitivity and often delayed results fromthe conventional “gold standard” based on microbiologicaltechniques in the traditional TBM diagnosis underscore theneed for a more sensitive, rapid, and accurate diagnosticmethod in clinical practice [2–5]. Several molecular-basedmethods, often drawn from successful techniques used forthe diagnosis of tuberculosis in respiratory specimens, havebeen evaluated for their applicability in the diagnosis ofCNS tuberculosis. These techniques include commerciallyavailable NAA methods and other PCR-based methods [2–42]. In addition, the use of neuroradiographic techniquessuch as magnetic resonance imaging (MRI) has prominentlyimproved the diagnostic accuracy of TBM and tubercu-lomas [2, 47–49]. Recently, the role of neuroradiographictechniques in the evaluation of CNS tuberculosis hasbeen reviewed in various reports [1–5, 47–49]. Commonlyidentified neuroradiological features of TBM include basalmeningeal enhancement, hydrocephalus, and infarctions inthe supratentorial brain parenchyma and brain stem [2, 47–49]. Moreover, tuberculomas are generally defined as low-or high-intensity, round or lobulated masses with irregularwalls and show homogeneous or ring enhancement afterthe administration of contrast [2, 47–49]. They occur assolitary or multiple nodules and are typically found inthe frontal and parietal lobes [2, 47–49]. However, thedifferential diagnosis of tuberculomas and other intracranialfocal massive lesions such as fungal granulomas is difficultwhen using only neuroradiographic techniques [2]. There-fore, the combination of molecular-based techniques andneuroradiographic techniques is regarded as a promising andpowerful diagnostic tool for TBM and tuberculomas, insteadof neuroradiographic techniques with or without classicalbacteriological detection methods [2].


4. Recent Advancement of PCR Assay Technique

The NAA methods for M.Tb are diagnostic techniques todemonstrate the presence of tubercle bacilli by the extractionand amplification of DNA or RNA of M.Tb from clinicalspecimens such as sputum or CSF. The representativeDNA amplification method is the PCR assay technique. Inthis section, an overview of the principles of PCR assaytechniques is presented, mainly with regard to their recentadvancement and evolution.

4.1. The Basic Principle of PCR Assay. A schema indicatingthe basic principle of the PCR assay is shown in Figure 1(a).Fundamentally, the PCR assay technique depends on “ther-mal cycling,” consisting of cycles with repeated heatingand cooling for the reactions of DNA denaturation andenzymatic replication of DNA. Short DNA fragments called“primers” containing sequences complementary to the targetregion along with a DNA polymerase are key componentsto enable sequence-specific DNA amplification. The thermalcycling procedure involves a first step for physical separa-tion of the two strands of DNA double helix at a hightemperature (94–98◦C for 20–30 seconds); this is called theDNA denaturation step. At a lower temperature (50–65◦Cfor 20–40 seconds), the primers that are complementaryto the target DNA region anneal to each separated single-stranded DNA as the template; this is called the annealingstep. The specificity of PCR mainly results from both theprimer sequence setting that closely matches the templatesequence and the annealing temperature setting dependingon the length of primers. The DNA polymerase binds tothe primer-template hybrid and begins DNA synthesis. TheDNA polymerase enzymatically assembles and synthesizes anew DNA strand complementary to the DNA template inthe 5′ to 3′ direction; this is called the extension/elongationstep. In general, almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzymeoriginally isolated from the bacterium Thermus aquaticus. Asa result, it is possible to repeat serially the thermal cyclingprocedure, consisting of alternate heating and cooling steps.As the thermal cycling procedure progresses, the synthesizedDNA fragment is itself used as a template for replication,setting in motion a “chain reaction” in which the DNAtemplate is exponentially amplified.

Through the use of agarose gel electrophoresis, the am-plified DNA fragments can be separated by their lengths(molecular weights). The agarose gel is then treated witha solution containing ethidium bromide (EtBr), which isthe most commonly used dye for visualizing DNA bands inagarose gel electrophoresis. Because EtBr fluoresces underUV light when intercalated into the major groove of DNA,through EtBr treatment, the amplified target DNA fragmentcan be visualized and detected as a distinctive band.

4.2. The Principle of Nested PCR Assay. A schema indicatingthe principle of nested PCR assay is shown in Figure 1(b).

Nested PCR assay is a modified version of the PCRtechnique intended to increase the amplification efficiency

markedly and to reduce the level of nonspecific PCRproducts due to the amplification of untargeted primerbinding sites. Although the specificity of the standard single-step PCR depends on the primers′ complementarity to thetarget DNA sequence, a commonly occurring problem is thatprimers bind to other similar regions of the DNA, givinguntargeted PCR products such as primer dimers, hairpins,and alternative primer target sequences. Nested PCR assayrequires two sets of primer pairs, used in two successive stepsof the PCR procedure. In particular, a second set of primerpairs is prepared to amplify a secondary target within thefirst-step PCR product; this is the source of the term “nested”.The first set of primers amplifies a fragment similarly tothe standard single-step PCR. However, the second set ofprimers binds inside the first-step PCR product to allowamplification of the second-step PCR product, which isshorter than the first one. The advantage of the nested PCRassay is that, if an untargeted or nonspecific PCR product isamplified, the probability is quite small that the region wouldbe amplified in the second-step PCR by the second set ofprimers. Thus, nested PCR assay is an excellent techniquefor obtaining a sufficient amount of target DNA througha two-step amplification procedure, and for prominentlyimproving the specificity to the target sequence by reducingthe amplification of nonspecific products.

4.3. The Principle of Real-Time Quantitative PCR Assay.Real-time PCR assay is a variation of the PCR techniqueintended to amplify and simultaneously quantify a targetedDNA molecule; it enables both detection and quantification.Although the basic procedure of real-time PCR follows thegeneral principle of classical PCR, its key feature is thatthe amplified DNA fragment is detected as the reactionprogresses in “real time.” This is a novel and revolutionaryapproach compared with conventional standard PCR, wherethe PCR product is detected at the end of the reactionprocedure. Two common methods for detection of productsin real-time PCR are as follows: (1) nonspecific fluorescentdyes such as SYBR Green that intercalate with any double-stranded DNA and (2) sequence-specific oligonucleotideprobes such as TaqMan probe that are labeled with afluorescent reporter dye, which permits detection onlyafter hybridization of the probe with its complementarytarget sequence. The former (SYBR Green) will bind to alldouble-stranded DNA PCR products including nonspecificPCR products (such as primer dimer). This is a potentialdisadvantage as it could obstruct accurate quantification ofthe intended target DNA fragment.

In contrast, fluorescent reporter probes such as TaqManprobe detect only the DNA fragment containing the com-plementary probe sequence; therefore, use of such reporterprobes significantly increases the specificity and enablesaccurate quantification, even in the presence of nonspecificamplified fragments. A schema indicating the principle ofreal-time quantitative PCR assay based on a fluorescentreporter probe (TaqMan PCR) is shown in Figure 1(c).The sequence-specific oligonucleotide probe is labeled witha fluorescent reporter dye, such as FAM or VIC, at the 5′-


DNA polymerase

(1) Denaturation

(2) Primer annealing

Forward primer

(4) PCR product detection

(3) Extention

Template: extracted DNA specimen

Reverse primer

DNA polymerase

5

5

5

3

3

3

5

5

5

3

3

3

3

5

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3

(a)


Inner forward primerInner reverse primer

DNA polymerase

Detection

2nd PCR product

5

5

5

3

3

3

3

5

DNA polymerase Outer reverse primer

Template: 1st PCR product

Outer forward primer

[First step PCR]

[Second step PCR]

(b)


Forward primerReverse primer

Specific probe (TaqMan probe)

Fluorescent reporter dye Quencher dye

DNA polymerase

DNA polymerase

5

5

53

3

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53

(c)

Figure 1: Schemata of the principles of polymerase chain reaction (PCR) assay techniques. (a) A schema indicating the basic principle ofPCR assay. (b) A schema indicating the principle of nested PCR assay. (c) A schema indicating the principle of real-time (TapMan) PCRassay.

end and conjugated with a quencher dye, such as TAMRA, atthe 3′end. The close proximity between a fluorescent reporterdye and a quencher dye prevents emission of its fluorescence.As the reaction is initiated, both probe and primers annealto the target DNA sequence during the annealing step ofPCR. As the PCR procedure progresses, breakdown of theprobe by the 5′ to 3′ exonuclease activity of Taq polymeraseseparates the reporter from the quencher and thus allowsunquenched emission of fluorescence of the reporter dye,which can be detected after excitation with a laser. Thestrength of fluorescence increases exponentially becausefluorescent reporter dyes separate from quencher dyes in amanner corresponding to the progress of PCR cycles, and itsgeometric increase is used to determine the threshold cycle(Ct) value in order to calculate the amplification rate in eachreaction. As a result, the primary amount of DNA can bequantified accurately.

5. Commercially Available NAAMethods for M.Tb

Currently, two commercially available NAA methods for thedirect detection of M.Tb complex have been approved by

the United States Food and Drug Administration (FDA),as follows: Roche Amplicor Mycobacterium tuberculosis Test(Roche Diagnostic Systems, Inc., Indianapolis, IN, USA) andGene-Probe Amplified Mycobacterium tuberculosis Direct(MTD) Test (Gene-Probe, Inc., San Diego, CA, USA) [2, 6–12]. Both tests use the 16S ribosomal (r) RNA gene of M.Tb(GenBank accession no. NC 000962.2 (1471846–1473382))as the target sequence for amplification [2, 6–12]. The 16SrRNA gene represents a stable property of microorganismsand is widely used as the target for identifying mycobacterialspecies [2, 6–12].

Roche Amplicor Test involves the conventional standardPCR-based method. In this test kit, the DNA fragmentis amplified and detected by the primer pair and probethat are specific for the 16S rRNA gene of M.Tb [2, 6, 8,10]. In addition, Roche Cobas TaqMan MTB Test is theimproved successor to the Amplicor Test and adopts a real-time (TaqMan) PCR assay technique. Meanwhile, Gen-ProbeMTD Test is the isothermal amplification method for RNA[2, 7, 9–12]. In this test kit, the 16S rRNA of M.Tb isdirectly amplified by the coupling of reverse transcriptaseand RNA polymerase under a constant temperature (43◦C),and detected by hybridization using specific oligo-RNA


probe [2, 7, 9–12]. At present, Amplicor is approved by theFDA for testing of AFB smear-positive respiratory specimensonly [2, 6–12]. Meanwhile, MTD is approved for testingof respiratory specimens, regardless of the result of smearfor AFB [2, 6–12]. Several studies have reported excellentresults for both tests (sensitivity and specificity levels of morethan 95%) in AFB smear-positive respiratory specimens,but reduced sensitivity (60 to 85%) when applied for AFBsmear-negative respiratory specimens [2, 6–12]. Neither testis approved by the FDA for testing of CSF specimens [2, 6–12].

6. Clinical Application of PCR Assay Techniquefor the Diagnosis of CNS Tuberculosis

Table 1 summarizes the performance of PCR-based assays forthe diagnosis of CNS tuberculosis [2, 6–42]. The challengesof applying NAA assay techniques to the rapid diagnosisof M.Tb in the CSF specimens stem largely from the smallnumber of bacilli typically present in TBM and the presenceof amplification inhibitors in the CSF [2–42]. In actualclinical practice, the sensitivity and specificity of PCR-basedassay methods are the most serious issues in the diagnosis ofCNS tuberculosis. In order to improve both the sensitivityand the specificity of PCR assay, the efficient extraction andpurification of DNA from a small number of M.Tb bacilliin the CSF specimens and the setting of primers to amplifyM.Tb DNA as the template specifically and efficiently are themost important factors [2–42]. Therefore, many researchershave worked intensively on these issues [2–42]. In the 1990s,a number of improved extraction and purification methodsof M.Tb DNA from CSF specimens were reported by Shankaret al. [14], Kaneko et al. [13], and Lin et al. [20], Accordingto these studies, the CSF specimen was treated with cytolysisbuffer containing proteinase-K and sodium dodecyl sulfate(SDS) as a surface-active agent, and then M.Tb DNAwas extracted and purified using phenol-chloroform andethanol precipitation from the treated CSF specimen [13,14, 20]. In order to extract a small amount of M.Tb DNAfrom a CSF specimen more efficiently, the authors used ahigh-molecular-weight carrier, Ethachinmate (Nippon Gene,Tokyo, Japan), as a coprecipitating agent for the nucleotidestogether with the previously reported phenol-chloroformextraction and ethanol precipitation [3, 4, 32, 33, 38, 40,41]. However, in recent studies, the conventional phenol-chloroform extraction and ethanol precipitation have tendedto be regarded as inadequate for routine use in clinical exam-inations because of their laborious and time-consuming pro-cedures [25–31, 34–37]. Generally, commercially availablecolumn extraction kits, such as the QIAmp Blood Kit andQIAmp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA),and the Cobas Amplicor respiratory specimen preparationkit (Roche Diagnostic Systems, Inc., Indianapolis, IN, USA),have been widely used for DNA extraction from varioussamples in previous studies [6–12, 25–31, 34–37]. However,in the current study, it was impossible to extract M.TbDNA from CSF specimens sufficiently using commercialcolumn extraction kits; therefore, these popular kits may be

inadequate for extracting a small amount of M.Tb DNA fromCSF specimens [3, 4, 32, 33, 38, 40, 41].

Currently, the four major M.Tb DNA-specific sequences,including the regions of IS6110 insertion sequence (Rv3475:GenBank accession no. NC 000962.2 (3891051–3892091)),65-kDa heat shock protein antigen (Rv0251c: NC 000962.2(302173–302652)), 16S rRNA gene and MPT64 (NC000962.2 (2223343–2224029)) are evaluated by NAA assays(Table 1) [6–42]. Of these four sequence regions, the IS6110insertion sequence, which is a repetitive element exclusivelyfound in the genome of M.Tb complex, has been most widelyused as the target sequence with superior amplificationefficiency in many studies (Table 1) [15, 16, 18, 19, 21,22, 24, 25, 28, 34, 36, 37, 39]. In these previous studies,PCR assays targeting the IS6110 insertion sequence revealedrelatively good results (an overall sensitivity of 70–98% andspecificity of 80–100%) for TBM diagnosis (Table 1). Inaddition, next to the IS6110 insertion sequence, the 16SrRNA gene has been frequently used for NAA assays, and itis the target sequence of two commercially available M.Tbdetection methods, namely, the Roche Amplicor Test andthe Gen-Probe MTD Test, which have been approved bythe FDA for testing of respiratory specimens, as describedabove [2, 6–12]. At present, no commercially available NAAassay methods have been approved for testing of CSF, butseveral studies have evaluated their performance in TBMcases (Table 1) [2, 6–12]. A recent meta-analysis concerningthe accuracy of commercially available NAA assay methodsfor TBM diagnosis revealed an overall sensitivity of 56%and specificity of 98% [10]. On the basis of these results,the commercially available NAA assay methods are evaluatedas follows: they may play a role in confirming TBM, butbecause of low sensitivity, they are not ideal for rulingout TBM [2, 10]. As the major reason for the insensitiveperformance of these two commercially available NAA assaymethods for TBM diagnosis, it is considered that, since theyhave been approved for testing of respiratory specimenscontaining relatively large numbers of M.Tb bacilli, theyare inadequate for detecting a small amount of M.Tb DNAfrom CSF specimens. Recently, in several studies, Gen-Probe MTD Tests modified to improve the performance fordetecting M.Tb DNA in CSF specimens have been reported[7, 9, 11, 12]. However, the use of these modified techniqueswas limited to a single laboratory, and they have not beenwidely used [7, 9, 11, 12]. Meanwhile, MPT64 is the genesequence encoding the MPB64 protein that is specific forM.Tb complex and exists at only one site on the M.Tbgenome. Therefore MPT64 is regarded as the most specificsequence for PCR assays and has been used as the targetsequence for PCR in many studies (Table 1) [3, 4, 13, 14, 16,17, 20, 23, 26, 27, 32, 33, 38, 40, 41]. Lee et al. [16] comparedthree sequence regions (IS6110, 65 kDa antigen, and MPT64)and reported that MPT64 was the most specific and sensitivesequence for the amplification of M.Tb DNA by PCR.

Beyond the commercially available NAA methods, theapplication of PCR-based methods to amplify M.Tb DNAhas received a lot of attention clinically (Table 1) [13–42].As described above, in order to improve the performance of


Table 1: Performance of PCR-based assays for the diagnosing TBM.

AuthorReported

yearAssay technique Specimens and cases

%Sensitivity

%Specificity

Reference

Commercially availableNAA assays

Bonington et al. 1998 Roche amplicor PCR83 CSF/69 patients (40 TBM,29 non-TBM): South Africa

60 100 [6]

Lang et al. 1998 Modified Gen-Probe MTD84 CSF and children (24 TBM,60 non-TBM): Dominica

83 100 [7]

Bonington et al. 2000 Roche Cobas Amplicor PCR83 CSF/69 patients (40 TBM,29 non-TBM): South Africa

17.5 100 [8]

Chedore and Jamieson 2002 Gen-Frobe MTD 311 CSF: Canada †93.8 †99.3 [9]

Pai et al. 2003 Review and Meta-Analysis14 studies with commercialNAA assays

56 98 [10]

Thwaites et al. 2004 Gen-Probe MTD341 CSF/152 patients (73TBM, 79 non TBM): Vietnam

38 99 [11]

Cloud et a1. 2004 Modified Gen-Probe MTD27 CSF specimens spiked withM. tuberculosis

17–100 100 [12]

Other PCR-based assays

Kaneko et al. 1990 MPT64 single PCR26 CSF and patients (6 TBM,20 non-TBM): Japan

83.3 100 [13]

Shanker et al. 1991 MFT64 single PCR85 CSF and patients (34 TBM,51 non-TBM): India

65 88 [14]

Donald et a1. 1993 IS6110 single PCR43 CSF/20 TBM chidren:South Africa

80 — [15]

Lee et al. 1994IS6110/65kDa antigen/MFT64Single FCR

27 CSF and patients (6 TBM,21 non-TBM): Singapore

100/83/83 38/67/90 [16]

Liu et al. 1994 MFT64 nested PCR100 CSF and patients (21TBM, 79 non-TBM): Taiwan

90 100 [17]

Folgueira et al. 1994 IS6110 single PCR25 AIDS patients (11 TBM, 14non-TBM): Spain

82 100 [18]

Scarpellini et al. 1995 IS6110 nested PCR68 CSF/36 AIDS patients (12TBM, 24 non-TBM): Italy

100 100 [19]

Lin et al. 1995 MFT64 single PCR47 CSF/45 patients (18 TBM,27 non-TBM): Taiwan

70 100 [20]

Kox et al. 1995 IS6110 single PCR42 patients (24 TBM, 18non-TBM): The Netherlands

48 100 [21]

Nguyen, et a1. 1996 IS6110 single PCR 136 TBM patients: Vietnam 32 100 [22]

Seth et a1. 1996 MFT64 single PCR89 CSF and patients (40 TBM,49 non-TBM): India

85 94 [23]

Wei et al. 1999IS6110/65kDa antigen/MTF40multiplex nested PCR

11 CSF and patients (5 TBM,6 non-TBM): China

60 66 [24]

Caws et al. 2000 IS6110 nested PCR131 TBM patients: UnitedKingdom

†75 †94 [25]

Martins et al. 2000 MFT64 nested FCR73 specimens (30 PF, 26 PB,17 CSF): Brazil

†70 †88 [26]

Brienza et al. 2001 MPT64 nested PCR91 patients (41 TBM, 50non-TBM): Brazil

53 100 [27]

Narayanan et al. 2001 IS6110/TRC4 single PCR96 CSF and patients (67 TBM,29 non-TBM): India

80.5/91 79/76 [28]

Desai et al. 2002 pKSIO single PCR120 CSF and patients (105TBM, 15 non-TBM): India

31 100 [29]

Rafi and Naghily 2003Single PCR (target notavailable)

36 CSF and patients (29 TBM,6 non-TBM): Iran

86.2 100 [30]

Kulkarni et a1. 2005 38 kDa protein single PCR60 CSF and patients (30 TBM,30 non-TBM): India

90 100 [31]

Takahashi et al. 2005 MPT64 nested PCR29 CSF and patients (9 TBM,20 non-TBM): Japan

100 100 [32]

Takahashi and Nakayama 2006 MPT64 QNRT-PCR29 CSF and patients (9 TBM,20 non-TBM): Japan

100 100 [33]

Quan et a1. 2006 IS61 10 single PCR74 CSF and patients (25 TBM,49 non-TBM): China

75 93.7 [34]

Desai et al. 2006Single PCR (target notavailable)

57 CSF and patients (30 TBM,27 non-TBM): India

66.7 100 [35]


Table 1: Continued.

AuthorReported

yearAssay technique Specimens and cases

%Sensitivity

%Specificity

Reference

Rafi et al. 2007IS6110 single PCR, MPT64/65kDa antigen nested PCR

176 CSF and patients (75TBM, 101 non-TBM): India

98/91/51 100/91/92 [36]

Rafi and Naghilys 2007 IS6110 uniplex (single) PCR945 CSF and patients (677TBM, 268 non-TBM): India

76.4 89.2 [37]

Takahashi et al. 2007 MPT64 QNRT-PCR63 CSF/28 patients (8 TBM,20 non-TBM): Japan

55.8 100 [38]

Deshpande et a1. 2007 IS6110 Single PCR80 CSF and patients (51 TBM,29 non-TBM): India

91.4 75.9 [39]

Takahashi et al. 2008 MPT64 WR-QNRT-PCR96 CSF/53 patients (24 TBM,29 non-TBM): Japan

95.8 100 [40, 41]

Haldar et al. 2009 devR qRT-PCR167 CSF and patients (81TBM, 86 non-TBM): India

87.6 92 [42]

NAA: nucleic acid amplification, CSF: cerebrospinal fluid, PF: pleural fluids, PB: pleural biopsies, QNRT-PCR: quantitative nested real-time PCR,WR-QNRT-PCR: wide range quantitative nested real-time PCR, pRT-PCR: quantitative real-time PCR, †: results versus culture as gold standard.

the PCR-based assays for the diagnosis of CNS tuberculosis,various ideas and modifications have already been tried [2,6–42]. Recently, as a revolutionary assay technique that con-veys drastically increased sensitivity and specificity comparedwith the conventional standard PCR assay, nested PCR assayhas been innovated for the diagnosis of CNS tuberculosis [3,4, 17, 19, 24–27, 32]. Liu et al. [17] reported that nested PCRassay targeting MPT64 was performed for CSF specimenscollected from 21 patients with clinically suspected TBMand enabled diagnosis of TBM with a sensitivity of 90%and a specificity of 100% within 24 hours. In addition,they reported that the nested PCR assay had approximately1000 times higher sensitivity than the conventional single-step PCR assay [17]. The authors performed the nestedPCR assay targeting MPT64 for the CSF specimens collectedfrom 9 patients with clinically highly suspected TBM [32].In our study, both the sensitivity and the specificity ofnested PCR assay were 100%, but the sensitivity of theconventional single-step PCR assay and culture for M.Tbwas only 22.2% [32]. Moreover, the minimum detectionsensitivity of our nested PCR assay technique was examined.This technique enabled detection at a level as low as 1–10copies/2 µL of purified M.Tb DNA and had 1000 to 10,000times higher sensitivity than the conventional single-stepPCR assay [32]. Concerning the relationship between thePCR assay results and the responses for ATT, Lin et al.[20] examined this relationship by conventional single-stepPCR assay and Scarpellini et al. [19] examined it by nestedPCR assay. In particular, Scarpellini et al. [19] performeddiachronic study by nested PCR assay targeting IS6110 forserial CSF specimens collected from 7 TBM patients duringthe clinical treatment course. In their diachronic study,the nested PCR assay results were converted from positiveto negative in 4 TBM patients whose clinical conditionsrecovered during ATT [19]. In contrast, the nested PCR assayresults remained positive throughout the clinical course in 3patients who demonstrated no ATT response and died [19].Therefore, Scarpellini et al. concluded that the nested PCRassay was useful for assessing the clinical treatment course ofTBM [19]. Similarly, in our study, 11 out of 27 serial CSFspecimens that were available from the 7 patients with highly

suspected TBM and collected during the clinical treatmentcourse of ATT revealed positive results (40.7%) of the nestedPCR assay targeting MPT64 [32]. Moreover, our nested PCRassay results were converted from positive to negative in the 6patients whose clinical conditions recovered during the ATT[32]. In contrast, the conventional methods including single-step PCR assay and culture for M.Tb all revealed negativeresults for a series of 27 CSF specimens during the clinicaltreatment course of 7 patients [32]. The nested PCR assayis a useful assay technique with superior sensitivity andspecificity. Because of the demonstration of the capacity ofthe nested PCR assay in the diagnosis of difficult cases inwhich other conventional assay methods cannot detect M.Tb,the authors speculated that, if this assay technique was widelyand appropriately used within clinical practice, it would be apowerful tool for the rapid and accurate diagnosis of CNStuberculosis.

Currently, although the wide use of nested PCR assay inclinical practice is expected, regrettably, it has rarely beenperformed for TBM diagnosis [3, 4, 17, 19, 24–27, 32]. Themain argument against the use of nested PCR assay is that,because of its complicated, laborious, and time-consumingprocedures, it is an inadequate assay technique for routineuse in clinical examination [3, 4, 17, 19, 24–27, 32]. Inaddition, owing to its markedly increased sensitivity and therequirement for an additional amplification step, in general,it is considered that cross-contamination can easily occurthrough the nested PCR assay procedure [3, 4, 17, 19, 24–27,32]. However, the possibility of cross-contamination can beminimized by good laboratory practice. Certainly, the nestedPCR assay may be inadequate for routine use in clinicalexaminations dealing with many sample specimens such asscreening examinations. However, even if any other “simple”assay methods are used for TBM diagnosis, a negativeresult cannot exclude the possibility of M.Tb infection,and clinical judgment in TBM diagnosis remains a seriousproblem with the requirement of an appropriate clinicalexamination [2]. In actual clinical practice, the diagnosis ofCNS tuberculosis requires not a screening examination butrather a definitive examination. Therefore, the nested PCRassay should become a prominent and useful assay technique


Table 2: Sequences of primers and TaqMan probes for WR-QNRT-PCR assays.

Objective Target Type SequencePCR

productsize

First stepPCR

Wild M.Tb DNA (MPT64)or W-plasmid

WFl: Outer wild forwardprimerWRl: Outer wild reverseprimer

5′-ATCCGCTGCCAGTCGTCTTCC-3′

Total 21 nucleotides, A:2, T:6, G:4, C:9 (GC% 62)5′-CTCGCGAGTCT AGGCCAGCAT-3′

Total 21 nucleotides, A:4, T:4, G:6, C:7 (GC% 62)239 bp

New internal control(NM-plasmid)

MFl: Outer mutation forwardprimerMRl: Outer mutation reverseprimer

5′-TCGATTCTGTCCCACCGCCGT-3′

Total 21 nucleotides, A:2, T:6, G:4, C:9 (GC% 62)5′-AGACTCGACGCGTAGTCCTCG-3′

Total 21 nucleotides, A:4, T:4, G:6, C:7 (GC% 62)

Wild M.Tb DNA (MPT64)or W-plasmid

TqMn-WF2: TaqMan innerwild forward primerTqMn-WR2: TaqMan innerwild reverse primer

5′-GTGAACTGAGCAAGCAGACCG-3′

Total 21 nucleotides, A:7, T:2, G:7, C:5 (GC% 57)5′-GTTCTGATAATTCACCGGGTCC-3′


Second stepPCR

New internal control(NM-plasmid)

TqMn-MF2: TaqMan innermutation forward primerTqMn-MR2: TaqMan innermutation reverse primer

5′-AGATCGGATAGCCAGCACGGA-3′

Total 21 nucleotides, A:7, T:2, G:7, C:5 (GC% 57)5′-TGCGCTGCGTCGACATATTCT A-3′

Total 22 nucleotides, A:4, T:7, G:5, C:6 (GC% 50)77 bp

Wild M. Tb DNA (MPT64)or W-plasmid

TqMn-W-VIC: TaqManprobe- wild-VIC

5′-VIC-TATCGATAGCGCCGAATGCCGG-TAMRA-3′


New internal Control(NM-plasmid)

TqMn-M-FAM: TaqManprobe-mutation-FAM

5′-FAM-ATGGGACGGCTAGCAATCCGTC-TAMRA-3′


Underline: artificial sequence.

if used on well-defined and appropriate clinical specimenscollected from “highly suspected” TBM patients. Finally, aremaining challenge of using PCR-based assay methods inthe diagnosis of CNS tuberculosis is the requirement foran appropriate laboratory infrastructure to perform thesemore sophisticated assay techniques; it is a fact that theinfrastructure is often lacking in areas where TBM is highlyendemic. This is a crucial issue that should be solved as soonas possible in the diagnosis of CNS tuberculosis.

7. Development of NovelDiagnostic Assay Technique Based onPCR for CNS Tuberculosis

Recently, the authors developed an internally controllednovel “wide-range (WR)” quantitative nested real-time(QNRT) PCR assay for M.Tb DNA, in order to rapidly diag-nose CNS tuberculosis [32, 33, 38, 40, 41]. This techniquecombines the high sensitivity of nested PCR with the accuratequantification of real-time (TaqMan) PCR (Figure 2(a))[40].

In WR-QNRT-PCR assay, two types of original plasmid,“wild” (W) and “new mutation” (NM) plasmids, wereprepared for quantitative detection of M.Tb DNA [40,41]. W-plasmid, which was inserted a 239-base-pair (bp)DNA fragment of the MPT64 gene into pCR 2.1 vector(Invitrogen Corp., San Diego, CA, USA), was constructedfor use as standard template (Figure 2(b)) [40]. NM-plasmid

was developed on the basis of the W-plasmid for useas a new internal control “calibrator” in WR-QNRT-PCRassay (Figure 2(b)) [40]. In NM-plasmid, a total of fiveregions, where two pairs of (outer and inner) forward andreverse primers and TaqMan probe annealed, were replacedwith artificial random nucleotides (Figure 2(b)) [40]. Thesequences of the artificial random nucleotides were set tohave the same nucleotide composition as MPT64 of wildM.Tb [40, 41]. For use in WR-QNRT-PCR assay, four pairsof specific primers and two types of specific TaqMan probeswere prepared. The sequences and positions of these primersand probes are shown in Table 2 and Figure 2(b) [40, 41].The two pairs of forward and reverse primers (outer primerpair WF1 and WR1 for use at the first step and inner primerpair TqMn-WF2 and TqMn-WR2 for use at the second step)and TaqMan probe (TqMn-W-VIC) were specific for MRT64of wild M.Tb or W-plasmid. In contrast, the two pairs offorward and reverse primers (outer primer pair MF1 andMR1 and inner primer pair TqMn-MF2 and TqMn-MR2)and TaqMan probe (TqMn-M-FAM) were specific for theartificial random nucleotides in NM-plasmid for use as anew internal control “calibrator.” These primers and probeswere set to have the same nucleotide composition but adifferent and random sequence (Table 2) [40, 41]. Therefore,the annealing efficiencies of these primers and probes to wildMPT64 or NM-plasmid as a template can be regarded as thesame.

WR-QNRT-PCR assay consists of two consecutive PCRamplification steps, which are conventional PCR at the


1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 1E+10


Fluorescent reporter dye

Quencher dye

53

35

5

3

3

5

Outer forward primer

Template: 1st PCR product

Outer reverse primer

Inner forward primer Inner reverse primerSpecific probe (TaqMan probe)

[First step PCR]

[Second step PCR: real-time PCR]

(a)

Inner primer pair annealing site

Outer primer pair annealing site

TaqMan probe annealing site

New mutation (NM) plasmidWR-QNRT-PCR assay

MF1 TqMn-MF2TqMn-M-FAM

TqMn-MR2 MR1

MPT64 (GenBank accession no. NC

pCR 2.1 vector

WF1WR1

TqMn-WF2 TqMn-WR2TqMn-W-VIC

The 239 bp DNA fragment of MPT64 (= first step PCR product)

35000962)

−→

Artificial random nucleotides

(b)

0

5

10

15

20

25

30

3 3 XY = − . 3 log + 43.06R2 = 0.997,F = 1.007

Starting copy number of M. Tb DNA

PCR-Eff = 99.7%

Ct

valu

e

(c)

0

5

10

15

20

25

30

3. 41.01Y = − 28 logX +R2 = 0.998,F = 1.015

= 101.8%

1E+05 1E+06 1E+07 1E+08 1E+09 1E+101E+04

Starting copy number of NM-plasmid

PCR-Eff

Ct

valu

e

(d)

−→→400

101

100

ΔRn

Cycle number

Thresholdlimit

(e)

M. Tb DNA 105 copies + NM-plasmid 103 copiesM. Tb DNA 103 copies + NM-plasmid 103 copiesM. Tb DNA 10 copies + NM-plasmid 103 copies

M. Tb DNA 104 copies + NM-plasmid 103 copiesM. Tb DNA 102 copies + NM-plasmid 103 copiesM. Tb DNA 101 copy + NM-plasmid 103 copies

36322824201612842 6 10 14 18 22 26 30 34 38

Figure 2: Continued.


0

5000

10000

15000

20000

25000

0 1 2 3 4 5 6 7 8 3 4 5

Time course

Case 3Case 8Case 9Case 10

Case 11

Case 12Case 13

Case 14Case 15Case 16

Week Month

ATT effective cases

ATT noneffective cases

DN

A c

opy

nu

mbe

r (/

mL

CSF

)

Right Left

1 2

3 4 5

(g)

(h) (i)

0

5000

10000

15000

20000

25000

Clinical stage of TBM

CSF sample from ATT noneffective casesCSF sample from ATT effective cases

Stage 0 : 0 Stage I : 1 Stage II : 2 Stage III : 3

R2 = 0.597, ∗ < 0.0001P

5

7

9

11

13

W-plasmid copy number

1E+05 1E+04 1E+03 1E+02 1E+01 1E+00

Statistically analyzed by the one-way ANOVA

NSD:F= 1.086, P= 0.774

FAM

ct

valu

e

(f)

∗P = 0.0041

Figure 2: The principle of wide-range (WR) quantitative nested real-time (QNRT) PCR assay and its results. (a) A schema indicating theprinciple of WR-QNRT-PCR assay. (b) A schema of wild (W) and new mutation (NM) plasmids NM-plasmid was developed for use as anew internal control. (c) The specific standard curve to detect Mycobacterium tuberculosis (M.Tb) DNA or W-plasmid quantitatively. (d)The specific standard curve to detect the NM-plasmid as a new internal control quantitatively. (e) Amplification curves for 103 copies ofNM-plasmids as a new internal control. (f) One-way ANOVA against Ct values for 103 copies of NM-plasmid. (g) The progress of M.TbDNA copy numbers calculated by the WR-QNRT-PCR assay during clinical time course in 10 suspected tuberculous meningitis (TBM)patients (cases 3 and 8–16). Statistical comparison between the cases in which anti-tuberculosis treatment (ATT) was effective (cases 8–14and 16) and those in which it was not effective (cases 3 and 15) was carried out by repeated-measure ANOVA. (h) Cranial MRI findings forcases 11 and 12 on admission. 1, 2: Cranial MRI findings for case 11. 1: FLAIR image (TR 9000/TE 110). High-intensity lesions of cerebralinfarction (circle), which were probably caused by tuberculous vasculitis, are noted on both sides of the frontal lobe. 2: T1-WI (TR 500/TE17). A cranial tuberculoma was noted in the right thalamus. 3, 4, 5: Cranial MRI findings (Gd T1-WI: TR 400/TE 15) for case 12. Multiplecranial tuberculomas with marked Gd enhancement (arrows) were noted (3: midbrain, 4: right cerebellum, 5: right temporal lobe). (i) Resultof simple regression analysis between M.Tb DNA copy number (y axis) and clinical stage of TBM (x axis).

first step and real-time (TaqMan) PCR at the second step(Figure 2(a)) [33, 38, 40, 41]. In this assay, the entireprocedure including extraction, amplification, and detectionfor both M.Tb DNA and NM-plasmid as a new internalcontrol is performed simultaneously under the same assayconditions by using four pairs of primers and two probesthat have equivalent annealing efficiency to the respectivetemplates. Therefore, the initial copy number of M.Tb DNAin CSF samples can be calculated by the amplification

ratio against the new internal control (NM plasmid) as a“calibrator,” using the following equation; X : W = C : M ∴X= W × C/M. (X, the initial copy number of M.Tb DNAper 1 mL of CSF sample; C, the initial copy number ofthe new internal control (=“calibrator” 103 copies of NM-plasmid); W and M, the copy numbers of M.Tb DNA andNM-plasmid, respectively, after passing through extractionand PCR amplification procedures.) [40, 41] In M.Tb, itis universally acceptable that a single copy of MPT64 gene


represents one bacterial cell. Therefore, it can be consideredthat the copy numbers calculated by WR-QNRT-PCR assaycorrespond to the bacterial cell numbers of M.Tb in CSFsamples.

For WR-QNRT-PCR assay, two specific standard curvesfor quantitative detection of M.Tb DNA and NM-plasmidas a new internal control are needed [40, 41]. The precisionof the standard curve is the principal factor for quantitativedetection in real-time (TaqMan) PCR assay [40, 41]. Thetwo specific standard curves are shown in Figures 2(c)and 2(d) [40, 41]. In simple regression analysis, both ofthese two standard curves demonstrated a significant linearrelationship (R2 > 0.99) between the threshold cycle (Ct)values (y axis) and log of the starting copy numbers foreach standard template (x axis). In both standard curves,no significant differences were found among the plots inrepeated experiments (n = 10; F = 1.007, P = 0.65and F = 1.015, P = 0.53) by two-way ANOVA. ThePCR-efficiency (Eff) of real-time PCR can be calculated bythe slope of the standard curve, in the following equation:PCR − Eff = 10(−1/slope) − 1. The PCR-Eff calculated bythis equation based on the slopes (−3.33 or −3.28) of twostandard curves was 99.7 or 101.8%, respectively [40, 41].These results indicated that the efficiencies of amplificationand detection for both M.Tb DNA and the new internalcontrol were almost equivalent in the WR-QNRT-PCR assay.

When a value of 103 copies of NM-plasmid was setas the optimal copy number of new internal control, theamplification curves of NM-plasmids revealed extremelyuniform patterns in all starting copy numbers (1–105) ofW-plasmids as a mimic of M.Tb DNA (Figure 2(e)) [40]. Inaddition, the Ct values for 103 copies of the NM-plasmid alsorevealed statistically significant uniform variance between allstarting copy numbers of W-plasmids (F = 1.086, P =0.774) by one-way ANOVA (Figure 2(f)) [40]. These resultsindicated that there was no interference between M.Tb DNAand the new internal control in the entire PCR amplificationprocedures. Therefore, NM-plasmid could be regarded asappropriate for use as a new internal control “calibrator”in WR-QNRT-PSR assay. Owing to the development ofNM-plasmid, the WR-QNRT-PCR assay enabled statisticallysignificant stable and accurate quantitative detection of M.TbDNA with a wide detection range (1–105 copies) [40, 41].

The authors examined the clinical usefulness of theWR-QNRT-PCR assay for rapid and accurate diagnosisand assessment of the clinical course of CNS tuberculosis[41]. Twenty-four patients with clinically suspected TBMand 29 non-TBM control patients were collected between1998 and 2005 [41]. A total of 67 CSF samples werecollected from these 24 patients. Of 67 CSF samples, 43were available serially from the 10 patients (cases 3 and8–16) who had followups of more than 2 weeks [41].Table 3 summarizes the clinical features of the 24 suspectedTBM patients upon admission (before ATT) [41]. All 24patients met the (A) clinical criteria and (B) supportingevidence for TBM (shown in Table 3) and were classifiedas 8 “confirmed” cases (cases 1 to 8) (CSF culture positivefor M.Tb) and 16 “highly probable” cases (cases 9 to 24)(meeting all the clinical criteria and with three positive

results for supporting evidence, but having no bacterialisolation). In clinical applications, the WR-QNRT-PCR assayrevealed sufficiently high sensitivity (95.8%) and specificity(100%) for 24 clinically suspected TBM patients [41]. Themeasured copy numbers of M.Tb DNA (per 1 mL of CSF)are shown in Table 3 [41]. In conditional logistic regressionanalysis, a copy number of M.Tb DNA (per 1 mL CSF) >8000was an independent risk factor for poor prognosis of TBM(=death) (OR = 16.142, 95%CI = 1.191–218.79, ∗P =0.0365) [41]. In the diachronic study, the copy numbersof M.Tb DNA demonstrated significant alterations duringthe clinical treatment course in 10 suspected TBM patients,in these 10 patients including 2 “confirmed” cases (cases3 and 8) and 8 “highly probable” cases (cases 9–16) [41].The classical cultures for M.Tb revealed positive results inonly three out of 43 serial CSF samples collected duringthe clinical treatment course in cases 3 and 8. In contrast,the quantitative detection of M.Tb DNA was possible in 25CSF samples (58.1%) in the WR-QNRT-PCR assay [41].In addition, the copy numbers of M.Tb DNA decreasedgradually to below the detection limit of the WR-QNRT-PCR assay in the 8 patients (cases 8–14 and 16) for whomATT was effective and who demonstrated improvement inboth clinical stages and routine CSF findings during clinicaltreatment course (Figure 2(g)) [41]. However, in cases 3 and15, in whom ATT was not effective and who died due toaggravation of TBM, the copy numbers were continually highthroughout the clinical course (Figure 2(g)) [41]. The trendin the alterations of M.Tb DNA copy numbers during clin-ical treatment course demonstrated a significant difference( ∗P = 0.0041) between the ATT effective cases (cases 8–14 and 16) and the ATT noneffective cases (cases 3 and8) by repeated-measure ANOVA (Figure 2(g)) [41]. In cases11 and 12, initial cranial MRI on admission demonstratedmultiple intracranial focal masses that were regarded astypical tuberculomas (Table 4 and Figure 2(h)) [38, 41].In these two cases with tuberculomas, after conversion tonegative results of WR-QNRT-PCR assay, transient positivitywas once again revealed without any symptoms of meningitisduring the course of ATT [38, 41]. In general, tuberculomasoften occur along with TBM, but certainly may occur inthe absence of TBM [2]. Tuberculomas in these two casesdisappeared at 5 and 3 months after the initiation of ATTduring the follow-up MRI (Table 4) [38, 41]. Interestingly,WR-QNRT-PCR assay results became completely negativealong with the disappearance of tuberculomas [38, 41]. Theauthors speculate that the transient detections of M.Tb DNAby WR-QNRT-PCR assays in these two cases during the ATTwere correlated with tuberculomas. A small amount of M.Tbmight have survived within the tuberculomas. The highsensitivity of WR-QNRT-PCR assay might have detected alittle DNA of extinct M.Tb leaking into CSF along withthe rupture of tuberculomas by ATT. These findings suggestthat the combination of molecular-based techniques andneuroradiographic techniques is a promising and powerfuldiagnostic tool for TBM and tuberculomas in actual clinicalpractice. To our knowledge, there have been no previousreports on cases such as these two cases; therefore, theymay be regarded as clinically important. Moreover, in simple


Ta

ble

3:T

he

sum

mar

yof

basa

lclin

ical

feat

ure

sin

24pa

tien

tsw

ith

susp

ecte

dT

BM

.

Pati

ent

No.

Age

/Sex

∗ Clin

ical

stag

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fin

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efor

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gle

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ure

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firm

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ses

173

/MII

I28

829

913

23.4

−+

++

2872

1M

E,H

C,

CV

D,I

FMSp

,GA

Abo

ut

3w

eeks

Non

effec

tive

276

/MII

I16

556

946

12.3

−+

++

1002

8M

E,C

VD

Sp2

days

Eff

ecti

ve

328

/MII

I60

543

425

16.3

−+

−+

2257

1M

E,H

C,

CV

D,I

FMSp

Abo

ut

1m

onth

Non

effec

tive

438

/MII

7663

718

6.5

−+

−+

7161

HC

,IFM

Sp,G

A1

day

Eff

ecti

ve5

53/F

III

344

354

3810

.3−

++

+45

47IF

M−

1da

yE

ffec

tive

672

/FII

I24

732

957

18.4

−+

−+

6340

HC

,IFM

−7

days

Non

effec

tive

734

/MII

612

320

1820

.2−

++

+12

43−

−N

otav

aila

ble

Eff

ecti

ve8

42/M

II41

845

636

22.6

−+

++

1053

2M

E,I

FMSp

Abo

ut

2w

eeks

Eff

ecti

veH

igh

lypr

obab

leca

ses

935

/FII

208

300

1316

.3−

−−

+78

92M

E,H

C,

CV

D,I

FM−

7da

ysE

ffec

tive

1065

/FI

107

7048

7.8

−−

−+

1904

−−

3da

ysE

ffec

tive

II52

/MII

1813

554

8.6

−−

−+

5858

ME

,HC

,C

VD

,IFM

Sp,G

A1

day

Eff

ecti

ve

1224

/FI

3025

304.

4−

−−

+54

36IF

M−

1da

yE

ffec

tive

1344

/FII

I60

7052

N.D

.−

−−

+96

00C

VD

−1

day

Eff

ecti

ve14

59/F

II40

359

783.

7−

−−

+51

12H

C−

Abo

ut

1m

onth

Eff

ecti

ve15

44/M

III

117

8748

3.9

−−

−+

8400

ME

Sp,G

AA

bou

t3

wee

ksN

oneff

ecti

ve16

40/M

III

800

188

6612

−−

−+

7050

CV

D−

1da

yE

ffec

tive

1730

/FII

I72

021

150

9.7

−−

−+

5596

IFM

−5

days

Eff

ecti

ve18

20/F

II44

216

446

17.6

−−

−−

Not

dete

cted

IFM

GA

Not

avai

labl

eE

ffec

tive


Ta

ble

3:C

onti

nu

ed.

Pati

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No.

Age

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Sin

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PC

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PC

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)A

DA

(IU

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AFB

smea

rM

.Tb

cult

ure

1963

/MII

I75

8447

15.9

−−

−−

76M

ESp

Not

avai

labl

eE

ffec

tive

2063

/FII

3429

430

12.7

−−

−+

188

HC

−N

otav

aila

ble

Eff

ecti

ve21

53/M

III

7681

8216

.9−

−−

+25

92M

E,I

FMSp

1da

yE

ffec

tive

2251

/MII

I22

715

534

12.7

−−

−+

636

ME

,CV

D,

IFM

−1

day

Eff

ecti

ve

2366

/MII

I12

912

058

4.7

−−

−+

1600

ME

−4

day

Eff

ecti

ve24

2/F

II19

311

930

8.3

−−

−+

1444

ME

,HC

−N

otav

aila

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Eff

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Th

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ence

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regression analysis, significant correlation (R2 = 0.597,∗P < 0.0001) was demonstrated between M.Tb DNA copynumber and clinical stage of TBM (Figure 2(i)) [41]. Thesediachronic study results indicate that quantitative analysisby WR-QNRT-PCR assay is very useful for assessing theclinical course of TBM and ATT response [41]. To ourknowledge, there have been no previous studies that seriallyassessed the quantity of DNA or bacterial cell numbers ofM.Tb in CSF samples throughout the clinical course of CNStuberculosis [41]. In actual clinical application, the WR-QNRT-PCR assay demonstrated significant accuracy andreliability for quantitative detection of M.Tb DNA in CSFsamples owing to the development of NM-plasmid used asa new internal control. Therefore, this novel assay techniquecould be regarded as a useful and advanced method forrapidly and accurately diagnosing CNS tuberculosis [40, 41].

However, this novel assay technique is not widely usedin routine clinical examination. It may be that the twoconsecutive amplification steps of WR-QNRT-PCR assayare regarded as a complicated and laborious procedure.Therefore, the authors are developing a further novel and“simpler and low-cost” assay technique with quantificationand high-sensitivity almost equivalent to those with the WR-QNRT-PCR assay by one step of standard real-time PCRassay. This developing assay technique is a combinationof the whole genome amplification (WGA) method withthe real-time (TaqMan) PCR assay technique. The wholegenome amplification (WGA) method provides a possibilityof amplifying a small amount of high-quality DNA and thereare several such techniques using commercially available kitsat a reasonable price [50–52]. In particular, in these kits forthe WGA method, a kit based on the multiple displacementamplification (MDA) technique has been found in manystudies to provide the most balanced genome amplification[50–52]. The MDA technique is a superior method inwhich genomic DNA is continuously amplified by usingPhi29 DNA polymerase of bacteriophage origin and randomhexamer primers [50–52]. In this case, the authors used theGenomiPhi V2 kit (GE Healthcare Life Sciences, Uppsala,Sweden) based on the MDA technique. Because a smallamount of M.Tb DNA extracted and purified from a CSFspecimen for use as a template can be amplified directlyusing the GenomiPhi V2 kit, the first-step PCR in theWR-QNRT-PCR assay can be omitted. This assay techniquethat is currently being developed can markedly simplify theprocedure of WR-QNRT-PCR assay by innovation of theWGA method. Therefore, although it is yet to be reported, itswider use as a beneficial approach for clinical examination isexpected in actual clinical practice.

As described above, CNS tuberculosis including TBMis the severest form of M.Tb infection, causing death andserious sequelae [1–5]. In addition, owing to an increas-ing number of immunocompromised hosts caused by theprevalence of AIDS, increasing numbers of older people,and the wider use of immunosuppressive agents such ascorticosteroid, CNS tuberculosis remains a serious clinicaland social problem [1–5, 43–46]. In particular, in theunderdeveloped and developing countries in Asia and Africa,in which there are many exacerbating social problems such

as poverty, overcrowding, and malnutrition, and so forth,the epidemic of M.Tb infection including TBM is regardedas a serious social and demographic crisis [1, 2, 43–46]. Theauthors consider that the novel assay techniques that we havedeveloped are needed and should be used in these places.The wider use of our novel assay techniques will lead to anincrease of the number of definitively diagnosed cases withinthe early stage of TBM and the improvement of treatmentresults for TBM; therefore, they should make a significantmedical and social contribution in these countries. It ishoped that our efforts to develop novel and more rapid,accurate, highly sensitive, quantitative, simple, and low-costassay techniques for the diagnosis of CNS tuberculosis willhelp to improve the level of medical care globally, particularlyin underdeveloped or developing countries.

8. Conclusion

Recently, instead of the conventional “gold standard” basedon bacteriological techniques, various approaches have beenattempted for rapid and accurate diagnosis of CNS tuber-culosis with high sensitivity. In this paper, an overview ofrecent progress of the NAA methods, mainly highlightingthe PCR assay technique, was presented. In particular, theinnovation of nested PCR assay technique is worthy of notegiven its contribution to improve the diagnosis of CNStuberculosis. Although a novel assay technique, which isinternally controlled and combines the high sensitivity ofnested PCR with the accurate quantification of real-time(TaqMan) PCR, namely, “WR-QNRT-PCR assay,” is reportedas a rapid diagnostic method in TBM, it is not widely usedin routine clinical examination. This novel assay techniquewith high sensitivity in addition to accurate quantification isuseful for not only the rapid diagnosis of CNS tuberculosisbut also the prediction of prognosis and assessing the effectof ATT during the clinical course of TBM. Therefore, inactual clinical practice, its wider use for the diagnosis of CNStuberculosis is expected in the future.

Disclosure

All authors have not received any financial support fromanywhere, with regard to this paper. All authors have agreedand approved submission of this paper to this journal.

Conflict of Interests

There is no conflict of interest in connection with this paper.

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Clinical Study

Maintenance of Sensitivity of theT-SPOT.TB Assay after Overnight Storage ofBlood Samples, Dar es Salaam, Tanzania

Elizabeth A. Talbot,1, 2 Isaac Maro,3 Katherine Ferguson,4 Lisa V. Adams,1 Lillian Mtei,3

Mecky Matee,3 and C. Fordham von Reyn1

1 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, USA

Correspondence should be addressed to Elizabeth A. Talbot, [email protected]

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 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. Introduction

The 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 centralized

and 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 Methods

The ethical review boards of Dartmouth Medical School(Hanover NH), the National Institute for Medical Research


(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 × 105

cells 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.Sputum was 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. Results

A 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%) were HIV-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 known HIV 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. Discussion

T-SPOT.TB with delayed processing using T Cell Xtendamong patients with culture-confirmed tuberculosis showedhigh sensitivity, comparable to that reported for immediate


Screened143

Evaluable143 (100%)

Samples atambient

temperature57

Samples atcontrolled

temperature86

Pos40

(70.2%)

Neg11

(19.3%)

Invalid6

(10.5%)

Analyzed66

Failedto givesputum

1

RBCcontamination

19

Pos61

(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-infected male, 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 they

Table 1: Overall agreement between TB culture and T-SPOT.TB.

T-SPOT.TB

Positive Negative Invalid

CulturePositive 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. Conclusions

T-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 temperatures


<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’ Contributions

E. 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.

Acknowledgments

The 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, the manufacturer 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 OxfordImmunotec more 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 a modified 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.


Clinical Study

The Use of Interferon Gamma Release Assays in the Diagnosis ofActive Tuberculosis

Silvan M. Vesenbeckh,1 Nicolas Schonfeld,1 Harald Mauch,2 Thorsten Bergmann,2

Sonja Wagner,2 Torsten T. Bauer,1 and Holger Russmann2

1 Department of Pneumology, Lungenklinik Heckeshorn, HELIOS Klinikum Emil von Behring, 14165 Berlin, Germany2 Institute of Microbiology, Immunology and Laboratory Medicine, HELIOS Klinikum Emil von Behring, 14165 Berlin, Germany

Correspondence should be addressed to Silvan M. Vesenbeckh, [email protected]

Received 25 November 2011; Revised 5 January 2012; Accepted 6 January 2012


Copyright © 2012 Silvan M. Vesenbeckh 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.

Interferon gamma release assays (IGRAs) are in vitro immunologic diagnostic tests used to identify Mycobacterium tuberculosisinfection. They cannot differentiate between latent and active infections. The cutoff suggested by the manufacturer is 0.35 IU/mLfor latent tuberculosis. As IGRA tests were recently approved for the differential diagnosis of active tuberculosis, we assessedthe diagnostic accuracy of the latest generation IGRA for detection of active tuberculosis in a low-incidence area in Germany.Our consecutive case series includes 61 HIV negative, Mycobacterium tuberculosis culture positive patients, as well as 234 controlpatients. The retrospective analysis was performed over a period of two years. In 11/61 patients with active tuberculosis (18.0%) thetest result was <0.35 IU/mL, resulting in a sensitivity of 0.82. We recommend establishing a new cut-off value for the differentialdiagnosis of active tuberculosis assessed by prospective clinical studies and in various regions with high and low prevalence oftuberculosis.

1. Introduction

Tuberculosis (TB) remains a major public health problemaffecting one-third of the world’s population [1, 2]. Diagno-sis of TB is usually based on a combination of anamnesticsymptoms, clinical presentation, radiological and patho-logical changes, bacteriological findings of acid/alcohol-fastbacilli, and molecular tests [3]. Definitive TB diagnosisis based on the detection of Mycobacterium tuberculosis(MTB) in the culture, which usually takes four to six weeks.For decades, tuberculin skin test (TST) has been used asdiagnostic tool to support the physician’s decision process.With the introduction of interferon gamma release assays(IGRAs), a more specific method became available. Althoughprimarily developed for the diagnosis of latent TB, clinicianshave also been searching for improved diagnostic tools andexplored IGRAs for the immunodiagnosis of active TB.In 2010, the Centers for Disease Control and Prevention(CDC) updated their guidelines for testing for TB infection,concluding that IGRAs “may be used instead of a tuberculin

skin test in all situations in which the CDC recommends thetuberculin skin test as an aid in diagnosing M. tuberculosisinfection” [4, 5].

Nevertheless, with the cutoff for the diagnosis of latentTB as given by the producers, pooled sensitivity for thediagnosis of culture positive TB did not exceed 80% in themost recent meta-analyses [6, 7]. The present case series con-stitutes one of the largest reports of latest generation IGRAused in culturally proven HIV-negative TB cases in a low-prevalence country. Our study is meant to help evaluate thecutoff for the IGRA in the differential diagnosis of TB.

2. Study Population and Methods

This is a retrospective study performed on inpatients of aregional hospital specialized in lung diseases (LungenklinikHeckeshorn, Berlin). At least one IGRA is routinely per-formed on a blood sample of each TB-suspect patient, andevery suspicious sample (smear, lymph node biopsy, pleu-ral effusion, or biopsy) is routinely cultured for TB. All


Table 1: (a) Median and range for patient age and IGRA test result for all culture positive cases of active tuberculosis, by organ manifestation.(b) Median and range for age and IGRA test result for all controls, by diagnosis. “Others” includes other lung diseases such as asbestosis,aspergillosis, asthma, bronchiectasis, pleural effusion, haemoptysis, fibrosis, and sarcoidosis. (c) Study results for TB cases and controls. (n:sample size, CI: confidence interval).

(a)

MTB infection Male Female Total Median age, years (range) Median QFT, IU/mL (range)

Lung 28 25 53 45 (4–83) 2.40 (0,00–103,4)

Pleura 2 3 5 26 (17–76) 2,45 (0,96–300)

Lymph node 2 1 3 65 (25–69) 21,32 (3,78–86,8)

Total 32 29 61 46 (5–84) 3.46 (0,00–300)

(b)

Diagnosis Male Female Total Median age, years (range) Median QFT, IU/mL (range)

Malignancy 22 23 45 69 (39–90) 0,01 (0,00–23,9)

Bronchitis 27 13 40 64 (1–90) 0,00 (0,00–1,84)

Pneumonia 30 17 47 67 (1–99) 0,00 (0,00–75)

Others 53 49 102 62,5 (5–99) 0,02 (0,00–103,4)

Total 132 102 234 66 (1–99) 0,00 (0,00–103,4)

(c)

nQFT Age

Mean 95% CI P Mean 95% CI P

TB 61 14.13 3.44–24.82<0.05

47.8 41.88–53.79<0.05

Control 234 1.33 0.24–2.43 63 60.56–65.39

samples of patients included in this study were taken priorto initiation of antibiotic therapy. Over a two-year period(1/2008–1/2010), IGRA results of all MTB culture positivecases of active TB were analyzed. Patients with other lungdiseases than TB, including negative history for MTB in-fection and without radiological findings suggestive of MTBinfection in the past and with no signs of active disease,were chosen as control group. All HIV-positive patients wereexcluded from the database.

2.1. IGRA. We used the latest generation IGRA (Quanti-FERON-TB Gold in-Tube, Cellestis, Carnegie, Australia),later referred to as QFT-GIT, on all samples. Peripheral bloodsamples were obtained by trained personnel in specific bloodcollection tubes following the manufacturer’s instructions.All blood samples were processed within 4 h of phlebotomy.Otherwise, the test was performed as previously described[8].

2.2. TB Culture. Specimens were stained, processed, and cul-tured by standard procedures in mycobacteriology [9]. Theisolates were cultured for 4 weeks on Lowenstein-Jensen (L-J)medium at 37◦C and tested for growth rate, pigment pro-duction, and by biochemical testing using standard methods[10, 11].

2.3. Statistical Analysis. Data were analyzed using STATA12.0 (StataCorp, College Station, Texas, USA). The t-test forindependent samples (two-tailed) was carried out to assess

significance level of detected differences in both groups(IGRA test results, age).

3. Results

A total of 61 patients with active TB as ascertainedthrough positive MTB culture were examined using QFT-GIT between 1/2008 and 1/2010 (see Table 1(a)). 53 patientspresented with pulmonary TB, three patients with lymphnode TB, and five patients with tuberculous pleurisy. Themedian QFT-GIT value for all TB patients was 3.46 IU/mL,ranging from 0.00 to 300 IU/mL. In 11 patients, the test resultwas <0.35 IU/mL (see Figure 1). Assuming the cutoff forlatent TB suggested by the producer, these are false-negative(FN) test results (11/61 = 0.18; 18% FN). We calculated thesensitivity (the proportion of patients with active TB whoare correctly identified as such) as 1-FN (=82%). Only inone patient four months after therapy with adalimumab, theresult was 0.00 IU/mL, and in one other patient with lungcancer under concomitant chemotherapy, it was 0.02 IU/mL.All other patients showed values of 0.07 IU/mL or above.The median age of our patients was 46 years (range: 5–84years), in the group with a test result≥0.07 and <0.35 IU/mL,the median age was 80 years (range: 44–84 years), and, inthe group testing ≥0.35 IU/mL, it was 36 years (range: 5–84years).

Within the control group (see Table 1(b)), QFT-GIT val-ues ranged from 0.00 to 103.4 IU/mL in one patient withpneumonia. The median was 0.00 IU/mL. In 50% of all cases(116/234), no Interferon gamma (IFN-γ) release was mea-sured (0.00 IU/mL). In 51/234 patients (21%), test results


0

0.02

0.07

0.09

0.18

0.22

0.28

0.31

0

QFT

-GIT

res

ult

(IU

/mL)

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Figure 1: Range of QFT-GIT values below the cutoff of 0.35 IU/mLfor patients with active tuberculosis.

were above 0.35 IU/mL. The median age in the control groupwas 66 years with a range from 1 to 99 years.

Patients in the study group were significantly youngerthan in the control group and had significantly higher QFT-GIT test results (P < 0.05 for both, see Table 1(c)).

4. Discussion

IGRAs are in vitro immunologic diagnostic tests to identifyMTB infection. Latent and active infections are not dif-ferentiated. Several test systems are commercially available:QuantiFERON-TB Gold (QFT), QuantiFERON-TB Gold in-Tube (QFT-GIT), both Cellestis Limited, Chadstone, Aus-tralia, and T-SPOT.TB (Oxford Immunotec, Abingdon, UK).The QFT-GIT measures IFN-γ responses to the MTB specificantigens early secretory antigenic target 6 (ESAT-6), culturefiltrate protein 10 (CFP-10), and Rv2654 (TB 7.7). Their usein clinical practice is more and more widespread. Whereasthe test was initially conceived to support diagnosis of latentinfection, an increasing body of evidence is published onits use in detection of active TB infection [12–14]. Moreand more guidelines now include recommendations for oragainst the use of IGRAs in the differential diagnosis of activeTB [15].

Several systematic reviews and meta-analyses have re-cently been published specifically on the diagnostic accuracyof IGRAs in active TB [6, 16, 17]. Strong heterogeneitybetween study populations is a major limitation of thesemeta-analyses, and most studies had small sample sizes.Overall, few studies were done with the latest generationQFT-GIT in areas of low endemicity such as Germany (2studies in [17], 13 studies in [16]). Among those, even fewer

restricted sensitivity analysis to patients with culturally pro-ven MTB infection and confirmed HIV-negative status [12,18].

Specificity depends highly on the definition of the controlgroup. Pai et al. reported a pooled specificity of 99% amongnon-BCG vaccinated and 96% among BCG-vaccinated low-risk groups [17]. According to a recent meta-analysis thatdid not restrict studies on specificity to low-risk groups[6], a situation more compatible to our clinical setting, thespecificity of QFT-GIT was only 0.79 (95% CI 0.75–0.82). Inour study, 51 out of 234 control patients (21%) showed testresults above 0.35 IU/mL, indicative of latent TB accordingto the producer. However, this finding is also consistent withthe expected number of false positives assuming a specificityof around 0.8 according to Sester et al. [6]. The retrospectivedesign of our study does not allow any conclusion about thetest specificity in our study population.

Sensitivity in these studies was also found to be highlydependent on the study population, notably local TB preva-lence, and ranged from 0.58 in a high-prevalence country[19] to 1.00 in a low-prevalence country [20], when QFT-GIT was assessed. Diel et al. found a pooled sensitivity of0.84 (95% CI 0.81–0.87) when including only developedcountries [16], consistent with the results published by Sesteret al. (0.77, 95% CI 0.75–0.80) [6].

In our consecutive case series of 61 TB culture positive,HIV-negative patients in a low-prevalence setting in Ger-many, we found a sensitivity of 82.0% (95% CI: 0.696, 0.902)for the QFT-GIT, when the cutoff recommended for thediagnosis of latent TB was used (<0.35 IU/mL). In a low-prevalence country such as Germany with a TB prevalence of5.4/100,000 inhabitants [21], case finding is an outstandingpriority in the management of this disease. Therefore, highlysensitive test systems are needed for screening purposes. Ifthe use of IGRA in the differential diagnosis of active TBis recommended, at least under certain conditions, thenthe cut-off point for active TB should be lower comparedto latent TB, as previously discussed by Davidow [22].Prospective clinical studies in different defined regions, withhigh- and low-prevalence of active TB, should be performedto evaluate the use of IGRAs in the diagnostic workup of TBpatients. The cutoff for the detection of latent TB does notseem applicable for that purpose.

5. Conclusions

We suggest that the cutoff for the use of IGRA in the dif-ferential diagnosis of active TB in low incidence settings bereevaluated. Further prospective studies including clinicalcriteria for TB are needed to determine a new cutoff for activeTB.

Authors’ Contribution

H. Mauch, N. Schonfeld, and S. M. Vesenbeckh designed thestudy, T. Bergmann and S. Wagner managed data acquisitionand compilation, N. Schonfeld and S. M. Vesenbeckh anal-ysed the data, T. T. Bauer and S. M. Vesenbeckh did statistical


analysis, S. M. Vesenbeckh wrote the paper, and T. T. Bauer,H. Mauch, H. Russmann, and N. Schonfeld reviewed andedited the manuscript.

Funding

This work was funded by HELIOS Klinikum Emil von Beh-ring and received support by DZK (Deutsches Zentralkomi-tee zur Bekampfung der Tuberkulose) and OHH (StiftungOskar-Helene-Heim).

Conflict of Interests

The authors declare that there is no conflict of interests.

References

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Clinical Study

Comparison of Overnight Pooled and Standard SputumCollection Method for Patients with Suspected PulmonaryTuberculosis in Northern Tanzania

Stellah G. Mpagama,1, 2 Charles Mtabho,2 Solomon Mwaigwisya,2 Liberate J. Mleoh,1

I Marion Sumari-de Boer,2 Scott K. Heysell,3 Eric R. Houpt,3 and Gibson S. Kibiki2

1 Kibong’oto National Tuberculosis Referral Hospital, P.O. Box 12, Kilimanjaro, Tanzania2 Kilimanjaro Clinical Research Institute and Kilimanjaro Christian Medical College, Kilimanjaro Moshi, Tanzania3 Division of Infectious Diseases and International Health, University of Virginia, Charlotteville, VA 22908, USA

Correspondence should be addressed to Stellah G. Mpagama, [email protected]

Received 28 September 2011; Accepted 3 January 2012

Academic Editor: Catharina Boehme

Copyright © 2012 Stellah G. Mpagama 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.

In Tanzania sputum culture for tuberculosis (TB) is resource intensive and available only at zonal facilities. In this study overnightpooled sputum collection technique was compared with standard spot morning collection among pulmonary TB suspects atKibong’oto National TB Hospital in Tanzania. A spot sputum specimen performed at enrollment, an overnight pooled sputum, andsingle morning specimen were collected from 50 subjects and analyzed for quality, quantity, and time to detection in Bactec MGITsystem. Forty-six (92%) subjects’ overnight pooled specimens had a volume ≥5 mls compared to 37 (37%) for the combination ofspot and single morning specimens (P < 0.001). Median time to detection was 96 hours (IQR 87–131) for the overnight pooledspecimens compared to 110.5 hours (IQR is 137 right 137–180) for the combination of both spot and single morning specimens(P = 0.001). In our setting of limited TB culture capacity, we recommend a single pooled sputum to maximize yield and speedtime to diagnosis.

1. Background

Tuberculosis (TB) and HIV are among the global leaders ininfectious disease mortality [1]. Sub-Saharan Africa has oneof the highest burdens of TB and HIV coinfection [2].Prompt diagnosis of TB is critical to improve outcome, butdiagnosis of TB is challenging in HIV-infected patients andespecially in resource-limited settings [3]. HIV-infected pa-tients have a higher rate of extrapulmonary TB, atypicalchest radiographs and fewer pulmonary cavities [4–6]. As aconsequence, HIV patients may be more likely to have a neg-ative or paucibacillary sputum smear microscopy [7]. Sub-jects with paucibacillary specimens may be prone to beingdelayed in clinical diagnosis, either because acid-fast bacilliare not observed by microscopy or time to detection Myco-bacterium tuberculosis (MTB) culture is prolonged [8]. Bothpoor quality and low quantity of sputum have a significant

impact on TB detection rate [9, 10]. In settings reliant onsmear microscopy as the only means of TB diagnosis, thismay impact negatively the time to initiation of TB treatment.Ideal specimens should contain 5 mL or more of sputumwithout saliva. A previous study found that the quality andquantity of sputum were improved by pooling three versus asingle “spot” collection [11]. However, collection on multipledays may unnecessarily burden health facilities and maybe prone to contamination. In contrast, a single overnightpooled technique whereby a patient is given a sealable con-tainer in which to collect all expectorated sputum over thecourse of the night has been used in TB treatment trials andin assessment of early bactericidal activity of new TB medi-cations [12, 13]. However it has not been examined in rou-tine clinical practice.

At Kibong’oto National TB Hospital in Kilimanjaro, Tan-zania, HIV co infection among TB suspects is common and it


Enrolled: 50 subjectsAll subjects produced one of each specimen:

All specimens analyzedfor quality: comparison wasmade between overnightpooled specimen and thecombination of spot andfollow-up morning specimens

All specimens split for Ziehl-Neelsenmicroscopy, solid culture, and liquidculture (Bactec MGIT): comparison ofmicroscopy, culture yield, colony-formingunit count on solid culture, and time todetection in liquid culture was made between the overnight pooled specimen and both the spot specimens and follow-up morning specimens

(1) a spot specimen ( n = 50)(2) a overnight pooled specimen ( n = 50)(3) a follow-up morning specimen ( n = 50 )

Figure 1: Study diagram of enrollment and specimen analysis.

Table 1: Demographic and clinical characteristics.

Variables (N = 50) Measure

Sex

Male 36 (72%)

Age (years) median (IQR) 38.5 (30–46)

Weight (kg) median (IQR) 51 (47–58)

HIV status

Positive 8 (16%)

CD4 count (cells/µL) by HIV status inmedian (IQR)

HIV positive 71 (30.5–158)

HIV negative 540 (409–614)

Chest X-ray findings

Cavities 23 (46%)

has been observed that the minority of patients have cavitarychest radiographs. Furthermore it is a standard practice thateach TB suspect is given a sealable mug to collect all sputumas an infection control measure. Thus, among a population atrisk for lower bacillary yield, we sought to compare overnightpooled sputum in the sealable mugs to the standard spottechnique for sputum quality, quantity, and time to MTBdetection by culture in the Bactec MGIT system, a methodwhich has correlated well with conventional colony counts[14, 15].

2. Methods

Subjects suspected of pulmonary TB at Kibong’oto NationalTB Hospital were recruited for enrollment. Subjects wereeligible if they were 18 years and above and presenting withtwo weeks or more of cough. Each patient was asked to col-lect three sputum samples in calibrated wide mouthed con-tainer: (1) a spot specimen at the time of enrollment, (2)an overnight pooled specimen, and (3) a spot specimen

on the morning after enrollment (Figure 1). Subjects wereexcluded if initiated on any antituberculosis medication pri-or to sample collection. Sputum samples were sent to Kili-manjaro Clinical Research Institute (KCRI) laboratory forprocessing. The specimens were assessed for volume collect-ed, presence of blood, and color. Samples were analyzed forquality based on the following criteria: (1) volume equal to ormore than 5 mls, (2) sputum color was white, yellow, green,or red, (3) presence of blood, and (4) thick consistency. Eachsputum specimen was decontaminated with 4% NaOH andcentrifuged at 1500 g for 10 minutes. The sediment was splitequally for use in direct smear microscopy by Ziehl-Neelsenstaining, MTB culture on solid agar with Lowenstein-Jen-sen slant and Middlebrook 7H11 media, and in liquid me-dia using Bactec MGIT 960 machine (Becton Dickinson,USA). Positive culture growth was confirmed by secondaryZiehl-Neelsen stain. Colony-forming unit (CFU) count wasperformed on the Middlebrook 7H11 media.

Data were analyzed by Stata Version 11 statistical soft-ware (StataCorp, U.S.A). Values were presented as meanswith standard deviation (SD) or median with interquartilerange (IQR) for data that was not normally distributed. Chi-square was used for dichotomous variables and Spearman’scorrelation coefficient for continuous variables to investigatethe association with microscopy and culture yield. All testswere two sided with P values ≤ 0.05 regarded as statisticallysignificant.

All subjects provided written informed consent. Ethicalapproval for the study was given by the IRB of the Kiliman-jaro Christian Medical Center, Tumaini University and theNational Ethical Review Board.

3. Results

Fifty pulmonary TB suspects were enrolled for the study,each produced three specimens, hence 150 samples were usedfor analysis. The median age of patients was 38.5 years (IQR30–45) (Table 1). Thirty-six (72%) were male and 8 (16%)


Table 2: Comparison of overnight pooled and spot method of collection on quality and quantity of sputum.

MeasureCombination of spot and single morning

specimenOvernight pooled P value

Quantity (≥5 mls) 37/100 (37%) 46/50 (92%) P < 0.001

Color 92/100 (92%) 47/50 (94%) P = 0.79

Thick consistency 66/100 (66%) 41/50 (82%) P = 0.12

Presence of blood 11/100 (11%) 6/50 (12%) P > 0.99∗P values are similar if pooled is compared with spot or single morning specimen, thus we combined spot and single for this table.

were HIV-1-infected patients. Among the HIV-infected sub-jects, the median CD4 count was 71 cells/µL (IQR 30.5–158).The median CD4 count for HIV-seronegative patients was540 cell/µL (IQR 409–614). Twenty-three (46%) had cavitarydisease (Table 1).

Overnight pooled sputum yielded a specimen of ≥5 mLin 46 (92%) of subjects compared to 37 (37%) for the combi-nation of spot and single morning specimens (P < 0.001).Good quality of sputum specimen by color was observedin 47 (94%) of the patients for overnight pooled sputum—while combination of spot and single morning specimenswas 92 (92%) (P = 0.79). Thick consistency was observed in41 (82%) of patients for overnight pooled compared to com-bination of spot and single morning 66 (66%) (P = 0.12),and presence of blood was observed in 6 (12%) and 11(11%), respectively (P > 0.99) (Table 2).

Overall 100 (67%) specimens were positive by smear mi-croscopy. Thirty-four (68%) spot specimens were smear pos-itive, compared to 31 (62%) single morning specimens, and35 (70%) for overnight pooled (P = 0.28). Eighty-one (54%)culture-positive specimens were available for comparison oftime to detection in the MGIT system from 27 subjects; 49(33%) specimens were negative and bacterial contaminationprecluded analysis in 20 (13%). The MGIT contaminationrate did not differ from that of Lowenstein-Jensen, found in21 (14%) of all specimens. Furthermore, among MGIT spec-imens contamination did not vary by method of collection:spot (5.3%), single morning (3.3%) and overnight pooled(4.7%) (P > 0.99). However, the median time to detectionwas 96 hours (IQR 87–131) for the overnight pooledspecimens which was significantly faster compared to 118hours (IQR 98–167) for single morning specimen and 143hours (IQR 108–194) for the spot specimens (P < 0.005)(Figure 2).

Only a limited number of pooled specimens were TB-cul-ture positive on Middlebrook 7H11 to allow colony formingunit (CFU) count determination. The median CFU countamong subjects with cavities on chest radiograph was4.92 log/mL (IQR 4.3–6.28) compared to 3.95 log/mL (IQR3.61–4.84) in patients without cavities (P = 0.12). Amongthe 8 HIV-infected subjects, only 2 (25%) had overnightpooled specimens available for CFU count. The median CFUcount among HIV-infected subjects was 4.34 log/mL (IQR3.9–4.72) compared to 4.84 log/mL (IQR 4.30–5.86) for theHIV-uninfected subjects (P = 0.43). There was no cor-relation between CD4 counts with CFU (Spearman’s rho =−0.36) (P = 0.16).

50

100

150

200

250

Tim

e to

pos

itiv

ity

(hrs

)

Spot Morning Pooled

Figure 2: Median time-to-culture positivity among different collec-tion method. ∗P < 0.005 comparing pooled to morning or spot.

4. Discussion

The major finding of this study was that in routine prac-tice an overnight pooled sputum collection significantly im-proved the time to detection in the MGIT system among cul-ture-positive samples. This is an important finding amidstthe global scale-up of MTB culture.

Faster time to diagnosis in a TB suspect can speed drugsusceptibility testing, hasten proper treatment, and poten-tially decrease transmission to the community and withinthe hospital. In settings with increasing rates of multidrug-resistant (MDR) TB, faster drug susceptibility results havebeen postulated to be one of the most important means ofimproving MDR TB outcome [16].

Sputum collection is the first and most critical step in lab-oratory diagnosis of pulmonary TB. In resource-limited set-tings such as ours, multiple specimen collection has beenproposed to increase yield for rapid molecular probe diag-nostics [17, 18]; however it can be overwhelming to thelaboratory. Both microscopy and culture are time consumingand culture is costly. In Tanzania culture is recommended forsmear-negative cases, for treatment failures and for monitor-ing MDR-TB patients. Currently there are few laboratoriescapable of culture and specimens must be sent long distances.In this setting, maximizing yield by sending the optimal spec-imen will conserve resources. We would strongly advocatesending pooled specimens in our context.

Our findings of increased culture yield with volumes of aminimum of 5 mls of adequate quality are consistent withprevious studies that examined yield of microscopy [19, 20].


However, our microscopy yield did not approach theremarkable >95% rate of smear positivity reported by oth-ers when a 24-hour collection technique was used for diag-nosis of selected subjects with pulmonary TB [21]. This ispotentially contributed by the fact that we selected for pul-monary TB suspects and not confirmed cases. With an eyetowards molecular diagnostics in the future, given the dropin sensitivity of the rapid molecular diagnostics in smear-negative sputum samples [17], we would expect that over-night pooled sputum collection would be equal to or improveupon molecular diagnostic yield compared with a spot spec-imen.

Our study has several limitations. For the CFU analysison the overnight pooled specimens, fungal overgrowth lim-ited examination of nine different plates. The contaminationaffected subjects with and without cavities so it is unlikelyto have biased this finding; yet overall our total number ofpatients enrolled was small and may not have includedenough HIV-infected patients to appreciate differences inthis subgroup with regard to culture yield, CFU analysis, andtime to detection. Bacterial contamination also limited eval-uation of all samples in the MGIT, but the contaminationwas observed in all specimen collection techniques and hasbeen reported in other evaluations of liquid culture in MGIT[22]. Furthermore, the culture yield for all techniques mayhave been diminished by splitting specimens for both solidand liquid culture.

In conclusion, we found that overnight pooled sputumcollection improved the quantity of sputum without altera-tion in quality and led to faster time to detection. These find-ings can ultimately impact the decisions both of treatmentand infection control. Overnight pooled sputum should beconsidered as an alternative to standard spot testing in re-source-limited settings.

Acknowledgments

The authors would like to acknowledge the African PovertyRelated-Infection Oriented Research Initiative (APRIORI)which supported grant of training in master degree ofclinical research of which S. G. Mpagama is a recipient.S. G. Mpagama is also supported by the NIH Fogarty’sGlobal Infectious Disease Research Training Program (D43TW008270) which enabled final drafting of the paper.

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