MODS for Tuberculosis Screening Prior to Isoniazid PreventiveTherapy in HIV-Infected Persons

Krishna P. Reddy, MD1,2,*, Mark F. Brady, MD, MPH1,3, Robert H. Gilman, MD1,4,5, JorgeCoronel, BSc1, Marcos Ñavincopa, MD6, Eduardo Ticona, MD6, Gonzalo Chavez, MD6,Eduardo Sánchez, MD7, Christian Rojas, MD7, Lely Solari, MD7, Jorge Valencia, MD8, YvettPinedo, MD8, Carlos Benites, MD8, Jon S. Friedland, FRCP, FMedSci5, and David A.J. Moore,MD1,4,5,#1Laboratorio de Investigación de Enfermedades Infecciosas, Universidad Peruana CayetanoHeredia, Lima, Peru2Harvard Medical School, Boston, Massachusetts, USA3Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA4Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore,Maryland, USA5Department of Infectious Diseases and Immunity and the Wellcome Trust Centre for ClinicalTropical Medicine, Imperial College London, United Kingdom6Hospital Nacional Dos de Mayo, Lima, Peru7Hospital Nacional Hipólito Unanue, Lima, Peru8Hospital Nacional General Arzobispo Loayza, Lima, Peru

AbstractBackground—Active tuberculosis (TB) must be excluded before initiating isoniazid preventivetherapy (IPT) in HIV-infected persons, but currently used screening strategies suffer from poorsensitivity and specificity and high patient attrition rates. Liquid TB culture is now recommendedfor the detection of Mycobacterium tuberculosis in TB suspects. This study compared the efficacy,effectiveness and speed of the microscopic-observation drug-susceptibility (MODS) assay withcurrently used strategies for tuberculosis screening prior to IPT in HIV-infected persons.

Methods—471 HIV-infected IPT candidates at three hospitals in Lima, Peru, were enrolled into aprospective comparison of tuberculosis screening strategies, including laboratory, clinical andradiographic assessments.

Results—Of 435 patients who provided two sputum samples, M. tuberculosis was detected in 27(6.2%) by MODS, 22 (5.1%) by Lowenstein-Jensen culture and 7 (1.6%) by smear. Of patients with

*Corresponding author: Krishna P. Reddy, MD, Department of Medicine, Massachusetts General Hospital, GRB 740, 55 Fruit Street,Boston, MA 02114, USA, Telephone: +1 617-935-5182, FAX: +1 617-724-7441, kreddy@post.harvard.edu.#Alternate corresponding author: David A.J. Moore, MD, Department of Infectious Diseases and Immunity, Wellcome Trust Centre forClinical Tropical Medicine, Imperial College London, DuCane Road, London W12 0NN, United Kingdom, Telephone: 0208 383 1943,FAX: 0208 383 3394, davidajmoore@msn.comContributors: KPR and DAJM had full access to all the data in the study and had final responsibility for the decision to submit forpublication. KPR, RHG, JSF and DAJM were involved in study concept and design and supervising study implementation. KPR wasthe main author. DAJM, MFB, RHG and JSF were involved in manuscript preparation. MFB assisted with data analysis and interpretation.JC supervised laboratory testing. MÑ, ET, GC, ES, CR, LS, JV, YV and CB recruited and examined participants.Conflict of Interest Statement: We declare that we have no conflict of interest.

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Published in final edited form as:Clin Infect Dis. 2010 ; 50: 988–996. doi:10.1086/651081.

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any positive microbiological test, a MODS culture was positive in 96% by 14 days and 100% by 21days. MODS simultaneously detected multidrug-resistant tuberculosis in two patients. Screeningstrategies involving combinations of clinical assessment, chest radiograph and sputum smear wereless effective than two liquid TB cultures in accurately diagnosing and excluding tuberculosis(p<0.01). Screening strategies that included non-culture tests had poor sensitivity and specificity.

Conclusions—MODS identified, and reliably excluded, cases of pulmonary tuberculosis moreaccurately than other screening strategies, while providing results significantly faster thanLowenstein-Jensen culture. The streamlining of TB rule-out through the use of liquid culture-basedstrategies could help facilitate the massive upscaling of IPT required to reduce HIV and TB morbidityand mortality.

Keywordstuberculosis diagnosis; preventive therapy; screening; HIV

IntroductionTuberculosis (TB) is the most common serious opportunistic disease and a leading cause ofdeath in HIV-infected persons. An important source of new TB cases is the reactivation oflatent tuberculosis infection (LTBI). This contribution has mushroomed in regions of high HIVprevalence because HIV greatly increases the risk for progression of LTBI to active TB disease[1]. This risk can be reduced by isoniazid preventive therapy (IPT), with benefits for both theindividual [2,3], and, by averting incident TB, public health [4,5]. Effectiveness has beendemonstrated in many populations [2,6-9], though not all [10,11], and it is generally acceptedthat the lifetime risk of reactivation TB can be reduced to 4% or less [12,13].

Isoniazid alone is appropriate treatment for LTBI but inadvertent isoniazid monotherapy foractive TB is ineffective and leads to drug resistance. Therefore, the pathway to commencingIPT must reliably exclude active TB. Surprisingly, international consensus guidelines on howthis should be achieved are lacking. Challenges [3,14] include procedural factors (therequirement for multiple investigations involving several visits leads to patient attrition of 20%[15,16]) and the poor sensitivity of conventional tests (clinical history [17], sputum smear andchest radiography [16,18,19]) for detection of HIV-associated TB in which cough may beunproductive and chest radiograph normal. This bottleneck to successfully ruling out activeTB is a significant obstacle to scale-up of IPT.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, liquid culturetool for TB culture and direct drug susceptibility testing (DST) developed for resource-limitedsettings. Diverse studies of MODS [20-22] have demonstrated high specificity and sensitivityfor diagnosis of TB and multidrug-resistant (MDR) TB in symptomatic patients. Importantlyfor TB rule-out, time to definitively negative culture with MODS is only 21 days; 99% ofpositive cultures are positive by 14 days.

These characteristics suggest that MODS might be a useful tool for the pre-IPT TB screeningof HIV-infected patients. We hypothesized that the high sensitivity of MODS would negatethe need for other investigations, and the rapid turnaround time would mitigate patient loss tofollow-up and thus facilitate a greater flow of patients onto IPT.

The Peruvian national TB rule-out strategy prior to IPT involves clinical evaluation by aphysician, chest radiography and analysis of two sputum samples by Ziehl-Neelsen smear andsolid media TB culture. In this study we investigated how the culture of one or two sputumsamples by MODS alone would compare to the Peruvian and other TB screening strategies interms of sensitivity, specificity, delay to diagnosis and cost. The intended outcome was the

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delivery to policymakers of robust comparative data to enable rational decision-making aboutresource allocation for TB rule-out.

MethodsStudy Setting and Population

HIV-infected candidates for IPT were consecutively recruited at the start of the TB rule-outwork-up at three public hospitals in Lima, Peru, from February through December 2007.Exclusion criteria were age under 15 years, current treatment with anti-tuberculosis agents,any IPT in the previous 12 months and inability to give written informed consent. Patients werenot paid for participation but travel costs were fully reimbursed.

Field ProceduresAll patients underwent clinical evaluation by a physician to screen for pulmonary andextrapulmonary TB. Physicians referred to the study only those patients for whom the purposeof TB rule-out was the initiation of IPT. Though no enrolled patient was strongly suspected ofhaving active TB, physicians were asked to assess the risk (none, low, moderate or high) ofactive TB relative to other IPT candidates, based on clinical history, symptoms and CD4 count.For subsequent analyses, “none” or “low” were grouped as “lower risk”; “moderate” or “high”were grouped as “higher risk.” After providing informed consent and interview to recorddemographic, clinical and socioeconomic data, patients were asked to provide two sputumsamples (on different days) for the study in addition to the two samples required by the hospitallaboratory for routine work-up.

Chest radiographs were interpreted by a single investigator blinded to patient details, who wasrequired to respond “yes” or “no” to the question, “Are there any findings suggestive of activeTB for which further testing would be recommended prior to IPT?”

In Peru no confirmation of LTBI (by tuberculin skin test [TST] or other means) is sought priorto IPT in HIV-infected persons. However, a positive TST is used to identify IPT candidates inother settings such as southern Africa [23], and was therefore included in this study. TST (5IUs PPD) was performed according to standard guidelines - transverse induration of ≥5 mmafter 48-72 hours was considered positive. TST was not undertaken if the patient declined orreported having had a TST in the previous six months. Automated CD4 cell count wasundertaken unless performed within the six months prior to enrollment, in which case this valuewas used.

Dedicated study personnel who rotated through all three study sites were trained collectivelyin data and specimen acquisition. Weekly team meetings ensured standardization of practicesacross the sites.

Laboratory ProceduresSputum samples were maintained at 4°C until same or next-day transport to the studylaboratory, where they were digested and decontaminated by the standard N-acetyl-L-cysteine-NaOH-sodium citrate method [24]. An aliquot was used for auramine smear microscopy, theremainder for parallel Lowenstein-Jensen (LJ) and MODS cultures, performed by threeexperienced laboratory technologists who were blinded to patient details and the results ofother tests. After inoculation LJ slants were incubated at 37°C and examined twice weeklyfrom day 7 through day 60 [24]. Contaminated slants were discarded and repeatdecontamination and culture were undertaken using a stored portion of the original sample.

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MODS CultureFive samples were each cultured in one 4-well column of a 24-well plate; a middle columnserved as a negative control. Each well contained 900 μL of decontaminated sputum,Middlebrook 7H9 broth, OADC and PANTA (full SOP available at www.modsperu.org).Plates were incubated at 37°C and examined daily (weekdays only) from days 5 to 15, then onalternate days to day 25, under an inverted microscope at 40× and 100× magnification. Tominimize cross-contamination and occupational exposure, plates were sealed inside plasticziploc bags and subsequently examined within the bag. If ≥ 2 colony-forming units ofcharacteristic morphology [25-27] were detected in each of the two drug-free wells, the samplewas considered positive. Growth in a drug-containing well indicated resistance to that drug. Ifthere was no evidence of growth by day 25, the culture was considered negative [20]. Fungalor bacterial contamination was recognized by rapid overgrowth or clouding; if detected, thestored portion of the original sample was decontaminated and cultured.

DefinitionsA TB-positive patient was defined as one for whom M. tuberculosis was detected by at leastone microbiological test (auramine stain, Lowenstein-Jensen culture or MODS). Indeterminatecultures were those that were persistently contaminated after repeat decontamination andculture. Patients with 2 smear and culture-negative sputum samples were considered TB-negative.

Estimated Initial Costs of Screening PackagesCosts of the screening packages (combinations of screening tests) were estimated by includingwholesale prices of test reagents including shipping, pro-rata costs of local medical personneltime and patient costs (median of transportation cost, personal lost income and family lostincome) as derived from a questionnaire. Costs for repeat culture due to contamination werealso included. Downstream costs, overhead and capital costs were not included. All costs wereconverted to US$2007 and based on current market costs for Lima public hospitals.

Data AnalysisData were analyzed utilizing Stata 9. To compare the equivalence of screening packages,McNemar's test was used with a significance level of α<0.05. Because some patients did notcomplete all strategies, due to either lack of follow-up or indeterminate results, separate tablesof sensitivity and specificity were derived to reflect the results from an intent-to-screenperspective and from the sub-group for whom all test results were available. Characteristics ofTB-confirmed and TB-excluded patients were compared using two-sided t tests and chi-squaretests with a significance level of α<0.05.

Ethical ReviewStudy protocol and consent and assent forms were approved by the ethics committees ofUniversidad Peruana Cayetano Heredia, Hospital Nacional Dos de Mayo, Hospital NacionalHipólito Unanue and Hospital Nacional General Arzobispo Loayza.

ResultsPatients and Samples

The characteristics of the population studied are shown in Table 1, and the flow chart for sputumtesting in Figure 1. Of 454 patients who provided at least one sputum sample, 129 (28%)reported taking highly active anti-retroviral therapy at the time of enrollment. Nineteen patientsprovided only one sample for the study; their culture results were included only when thesample was used as the unit of analysis. Of 889 sputum samples, 44 (4.9%) were culture-

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positive for M. tuberculosis by at least one method, of which 13 (30% of culture-positivesamples) were auramine-positive. By participant, 27 (6.2%) had at least 1 positive culture; 7(26% of culture-positive patients) had a positive auramine smear microscopy result. No smear-positive sample was culture-negative.

Table 2 shows comparative performances of culture methods. Sensitivities (and 95% CI) ofMODS and LJ by sample were 95% (85-99%) and 73% (57-85%), respectively (p=0.02,McNemar's test); and by patient were 100% (87-100%) and 81% (62-94%), respectively(p=0.07, McNemar's test).

ContaminationSixty-five (7.3%) samples were initially contaminated in MODS; repeat decontaminationyielded 64 definitively negative and one positive culture. In LJ, 192 (22%) samples wereinitially contaminated (versus MODS, p<0.001); repeat decontamination yielded 166 negative,four positive and 22 persistently contaminated and thus indeterminate cultures (2.5% of allsamples) (Figure 1).

Added Value of Second CultureOf 27 patients with at least one positive MODS culture, nine (33%) had only a positive secondsample; three (11%) had only a positive first sample. Of 22 patients with at least one positiveLJ culture, seven (32%) had only a positive second sample; five (23%) had only a positive firstsample.

Time to Culture Result, Including Delays Due To Contamination and Re-cultureMedian time to culture-positivity by MODS and LJ was 8 days (interquartile range 7-10 days)and 26 days (21-33 days), respectively (p<0.001, Wilcoxon test). Median time to definitiveculture-negativity by MODS and LJ was 28 days (26-31 days) and 63 days (61-66 days),respectively (p<0.001, Wilcoxon test). All TB-positive patients had a positive MODS culturewithin 21 days; 96% within 14 days.

Direct Drug-Susceptibility TestingSimultaneous MODS DST revealed that six of the 27 patients with culture-positive TB hadstrains resistant to isoniazid only, and two had strains resistant to both isoniazid and rifampin(multidrug-resistant).

Clinical Examination, Chest Radiography and Tuberculin Skin TestingPhysician-judged clinical evaluation assigned 394 patients as at lower risk and 60 patients asat higher risk of active TB, of which 15 (3.8%) and 12 (20%) respectively had culture-positiveTB. The presence or absence of two or more constitutional symptoms did not usefullydifferentiate between those with and without active TB (Table 1). Although clinical outcomeswere not a primary objective of this study, follow-up vital status data was available for 228baseline TB-negative patients representing 109,026 patient-days (297.12 patient-years); theremainder were lost to follow-up. Fourteen incident cases of TB were identified; mean time todiagnosis following negative screen was 298 days (median 178.5) and 79% (11/14) occurredmore than three months after screening.

The sensitivity of chest radiography for culture-positive TB in the 437 patients who completedx-ray was 56% (95% CI 35-75%). Culture-positive TB was demonstrated in 11 of 356 patients(3.1%) whose radiograph showed no evidence of active TB, and in 14 of 81 patients (17%)whose radiographic findings suggested further work-up was needed before initiating IPT.

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TST was performed in 445 patients, of whom 391 (88%) returned for reading in 2-3 days; TSTwas positive in 130 (33% of returners).

Comparison of MODS and Other Tests for TB Rule-OutPerformance characteristics of single tests and multi-test screening packages and theirassociated costs are presented in Table 3. Comparative times to correct assignment as TB-positive or TB-negative are plotted in Figure 2.

Costs of Initial Diagnostic PackagesThe costs per person intended to screen are presented in Table 3. Specific costs and assumptionsare in Supplemental Tables 1 and 2.

DiscussionThe enormous disconnect between international policy recommendations of IPT for HIV-infected persons and the implementation of this proven intervention (reaching <0.08% ofeligible people) owes much to the cumbersome nature of current TB rule-out strategies anduncertainty about optimal approaches. This study demonstrates that culture-based approachesare the only way to deliver optimum TB screening. MODS detected M. tuberculosis withgreater sensitivity and speed, and ruled out TB more quickly and with fewer indeterminateculture results, than LJ, consistent with previous data [20,28].

The strengths of this study derive from its prospective, real-world design, carried out at multiplesites. All tests were intended for all patients, and were interpreted by staff blinded to otherresults. Previous studies of TB screening strategies have used combinations of reportedsymptoms as a proxy for clinical assessment [29,30]. Here, referring clinicians directlyassessed the global likelihood of active TB in each patient, similar to an approach previouslydescribed for TB suspects [31]. This more accurately represents the real-world situationwherein a clinician decides based on overall assessment rather than a formalized score whethera patient should continue on the path to IPT.

IPT candidates have a lower prevalence of TB than the groups previously studied with MODS,who were largely active TB suspects [20-22]. Nevertheless, 6.2% of the patients in this studywere found to have culture-positive TB. If initiated on IPT, these patients are at risk fordeveloping drug-resistant TB [32]. Twenty of 27 culture-positive patients were smear-negativeand would have been administered IPT according to practices in which treatment decisions aremade before obtaining culture results.

Wider use of DST features in WHO STAG-TB recommendations [33]. MODS provides rapid,direct DST; results for the two patients with multidrug-resistant TB were available in six andnine days.

We previously demonstrated limited incremental value of a second MODS culture in TBsuspects and hospitalized, HIV-infected persons [20]. We predicted a similar finding in thepresent study, in a population with relatively low suspicion of TB. However, 50% more caseswere detected with a second culture. This likely reflects the pauci-bacillary nature of TB inHIV-infected persons with minimal symptoms, and the quality of sputum obtained. Theincreased yield of second sputum samples might be due to recruitment and follow-up practices:second samples were more likely than first samples to be provided in the early morning.Nevertheless, a single liquid culture still correctly assigned more people as TB-positive or TB-negative than any screening package that did not include MODS.

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Deciding upon the most important parameter when comparing screening packages is notstraightforward. Specificity indicates how well non-cases are identified but gives noinformation about cases misclassified as non-cases (having no screening at all and giving allcandidates IPT has a specificity of 100%). Negative predictive value describes how muchconfidence to put in a non-case result but is an unstable parameter modified by local prevalenceof cases. To capture the overall performance, we compared screening packages by determiningthe number of patients who would have been correctly assigned to IPT or active TB treatment.Interestingly, all currently recommended screening packages would have correctly assignedfewer patients than having no screening at all (i.e. assigning all patients referred to the studyas TB-negative); only the laboratory-based strategies correctly assigned more patients than notesting at all

As previously described [16,18], chest radiography appears to be a particularly costly andineffective tool; low sensitivity for detecting culture-positive cases indicates that a negativeradiograph is not sufficient grounds to start IPT. These data suggest, perhaps controversially,that chest radiography could be dropped from TB rule-out strategies in settings where onlypauci-symptomatic patients enter the algorithm.

Attrition rates – assessed by how many patients failed to undergo chest radiography, or providea second sputum sample – were low. Attrition is likely to be higher in non-study settings wherepatients assume transportation and test costs and follow-up supervision is less rigorous.

The 19 patients who did not provide a second sputum sample were excluded from analyses ofstrategies because all had a single negative culture, insufficient for definitive classification asTB-negative. However, these would be considered failures of the two MODS strategy.

We defined active TB as any positive microbiological result. As the primary purpose was TBrule-out, positive results from any test with proven high specificity, including those underevaluation for this new indication (such as MODS) cannot be ignored, particularly when thetest is known to be more sensitive than existing reference standards [20-22]. High specificityand infrequent cross-contamination of MODS have been previously demonstrated, whichsomewhat attenuates the threat of incorporation bias [20].

Of 14 baseline TB-negative patients subsequently diagnosed with TB, only three occurredwithin three months of negative screen. Though IPT initiation and monitoring were beyondthe scope of the study, it was determined that many patients had not initiated IPT, or had notbeen adherent. While the 11 later diagnoses almost certainly represent either newly acquireddisease or reactivation of pre-existing LTBI in patients truly disease-free at baseline, it ischallenging to determine whether this also applies to the three early cases or if their diseaserepresents progression of previously undetected active TB, and thus a false-negative screen.

The high rate of initially-contaminated Lowenstein-Jensen cultures is not easily explained;when the same sputum decontaminate is used for both LJ and MODS, as here, contaminationin LJ usually only slightly exceeds MODS. Nevertheless, after repeat decontamination only2.5% of all samples yielded an indeterminate LJ culture, so although turnaround times wereprolonged the detection performance of LJ was not significantly compromised.

An important consideration is the assumed validity of our definition of non-TB (negativemicrobiologic tests for two sputum samples). Obvious weaknesses are that poor sputum samplequality can compromise smear and culture performance and sputum testing cannot detectisolated extrapulmonary TB. False-positive MODS cultures are extremely unusual [28], sospecificity and positive predictive value are high.

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IPT has the potential to save millions of lives and contribute importantly to TB control in HIVhigh-burden regions. TB rule-out is a major bottleneck to implementation. Strategies tostreamline and expedite this process could get millions of eligible HIV+ patients onto the IPTthey require. Increasing the proportion of eligible patients that are screened from the current<0.1% to just 10% could avert over one million new cases of TB.

Based on the results of this study, we propose that liquid culture of two sputum samples alonecan be used as an effective screening strategy for pulmonary TB prior to IPT in HIV-infectedpersons. The worldwide movement towards greater use of liquid culture and TB laboratorycapacity building in resource-limited settings provides an opportunity for roll-out that couldtranslate this research into large-scale practice. Clinical history and examination can provideadditional information about the risk of extrapulmonary TB but they are not a useful filter forpulmonary TB screening. Future studies can address the utility of improved sputum collection,perhaps after instruction [34], as a means to improve the sensitivity of a single culture in thispopulation.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank all study participants and the following: Beatriz Castro, Fanny García, Virginia Huancaré, Miriam Huayta,Ruth Limascca, Sonia López, Jeny Rodríguez, Eleana Sánchez, Esther Soto, Rosa Yataco and Christian Solis for fieldassistance and data collection and verification; Paula Maguina for administrative support; Don Juanito; Sara Benitesfor database management; Pilar Navarro, Gianina Luna, William Roldán, Mirko Diaz and Jesús Rojas for assistancewith laboratory studies; Yuri García, MD, Jaime Soria, MD, Leonel Martínez, MD, Eduardo Matos, MD, Lisset García,MD, and Miriam Calderón, MD, for patient referral; and Aldo Vivar, MD, Jorge Arévalo, MD, Victor Chavez, MD,and Carlton Evans, MD, PhD, for protocol review and guidance.

This work was supported by Wellcome Trust Career Development Fellowship 078067/Z/05 (DAJM), NationalInstitutes of Health/Fogarty Global Research Training Grant 3D43 TW006581-04S1 (RHG) and National Institutesof Health/Fogarty International Clinical Research Scholar Awards (KPR and MFB). DAJM and JSF are grateful forsupport from the NIHR Biomedical Research Centre funding scheme.

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30. Mohammed A, Ehrlich R, Wood R, Cilliers F, Maartens G. Screening for tuberculosis in adults withadvanced HIV infection prior to preventive therapy. Int J Tuberc Lung Dis 2004;8:792–5. [PubMed:15182152]

31. Catanzaro A, Perry S, Clarridge JE, et al. The role of clinical suspicion in evaluating a new diagnostictest for active tuberculosis: results of a multicenter prospective trial. JAMA 2000;283:639–45.[PubMed: 10665704]

32. Balcells ME, Thomas SL, Godfrey-Faussett P, Grant AD. Isoniazid preventive therapy and risk forresistant tuberculosis. Emerg Infect Dis 2006;12:744–51. [PubMed: 16704830]

33. Strategic & Technical Advisory Group for Tuberculosis (STAG-TB) Seventh Meeting: Report onConclusions and Recommendations. Geneva: World Health Organization; 2007.

34. Khan MS, Dar O, Sismanidis C, Shah K, Godfrey-Faussett P. Improvement of tuberculosis casedetection and reduction of discrepancies between men and women by simple sputum-submissioninstructions: a pragmatic randomised controlled trial. Lancet 2007;369:1955–60. [PubMed:17560448]

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Figure 1.Patient flow chart for sputum testing by MODS and by Lowenstein-Jensen culture (LJ)a15 patients had two positive MODS cultures. Twelve patients had one positive and onenegative MODS culture; of these, three patients had no positive LJ culture or auramine stain.bThree of these LJ-negative patients had at least one positive MODS culture.c10 patients had two positive LJ cultures, 10 had one positive and one negative culture and twohad one positive and one contaminated culture. All 22 of these patients had at least one positiveMODS culture.dTwo of these patients had at least one positive MODS culture.eDemographic data was available for 131 of the 178 patients referred to the study but ultimatelynot part of the inclusion group: 37.4% were women (p=0.97 when compared with participants),and mean age ± standard deviation was 36.7 ± 10.8 (p=0.02). CD4 count was available for 101of these patients: mean ± standard deviation was 261 ± 170 (p=0.31).

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Figure 2.Time to correct assignment of patients by the various screening packages. This graph representsthe time until the 435 patients who provided two sputum samples would have been correctlyassigned as TB-positive or TB-negative by the various screening packages. Each package isrepresented by a distinct line, with the maximum height on the y-axis signifying the numberof patients correctly assigned by that package. A patient was considered “correctly assigned”if and when 1) any component of the screening package was positive and any microbiologictest result was positive or 2) all components of the screening package were negative and allmicrobiologic test results were negative. Patients who failed to complete all components of apackage were considered “not correctly assigned,” unless at least one component test waspositive in the context of a positive microbiologic test result. Packages delivering the bestperformance in correct patient assignment reach furthest up the y axis, whilst the speed withwhich results are available is reflected on the x axis. An ideal package would be representedby a straight vertical line which ended in the top left hand corner. Re-culturing times forcontaminated samples are included. Exam=clinical exam by referring physician. CXR=chestradiograph. LJ=Lowenstein-Jensen.

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Reddy et al. Page 13

Table 1

Participant characteristics

Patients with no demonstratedTBa (n=408)

Patients with active TBb(n=27) p-valuec

Age in years, mean ± SD 34.1 ± 10.7 34.7 ± 10.3 0.78

Women 158 (39%) 4 (15%) 0.01

Previous TBd 60 (15%) 2 (7%) 0.29

Previous isoniazid preventive therapye 58 (14%) 4 (15%) 0.94

Recent TB contactd 125 (31%) 8 (30%) 0.91

Positive tuberculin skin test (≥5 mm)f 116 (32%) 13 (59%) 0.008

CD4 cells/μL, mean ± SDg 246 ± 190 164 ± 96 0.06

Two or more symptoms (cough, fever, weight loss, night sweats)for ≥ 14 daysh,i

60 (15%) 8 (30%) 0.08

Data are presented as number (percentage), unless otherwise noted. Nineteen patients who provided less than two sputum samples are excluded. Fivepatients were hospitalized at the time of enrollment; the remainder were ambulatory.

aDefined as having only negative microbiologic tests.

bDefined as having at least one positive microbiologic test.

cChi-square for categorical variables, t-test for continuous variables.

dSelf-reported, one non-response.

eSelf-reported, two non-responses.

f368 TB-negative patients and 22 TB-positive patients returned for reading of a tuberculin skin test placed at the time of enrollment.

g375 TB-negative patients and 20 TB-positive patients had a documented CD4 count within six months of study enrollment.

hSelf-reported by 404 TB-negative patients and 27 TB-positive patients.

iThe positive predictive value (PPV) of having two or more of these symptoms for ≥14 days was 11.8%, the negative predictive value (NPV) was

94.8%, and the positive likelihood ratio (LR) was 1.7; for those with CD4<200, PPV was 10.0% (p=0.75), while for those with CD4≥200, PPV was4.5% (p=0.62). The PPV of having any of these symptoms for any period of time was 8.0%, the NPV was 97.7%, and the positive LR was 3.5. Threepatients with active TB were asymptomatic. Because of the low PPVs, and because symptoms do not figure in Peruvian screening guidelines, nofurther evaluations of symptoms as a screening tool were undertaken.

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Reddy et al. Page 14

Tabl

e 2

Com

paris

on o

f MO

DS

and

Low

enst

ein-

Jens

en c

ultu

re re

sults

Low

enst

ein-

Jens

en c

ultu

re r

esul

t

Per s

ampl

ePe

r pat

ient

c

TB

Not

TB

TB

Not

TB

MO

DS

TB

3012

225

resu

ltN

ot T

B2a

845b

040

8d

a For t

he 2

sam

ples

that

wer

e LJ

-pos

itive

but

MO

DS-

nega

tive,

the

othe

r sam

ple

from

eac

h pa

tient

was

MO

DS-

posi

tive;

poo

r sam

ple

qual

ity o

r div

isio

n m

ight

exp

lain

the

disc

repa

ncy.

b Incl

udes

24

sam

ples

that

wer

e co

ntam

inat

ed (i

ndet

erm

inat

e) in

Low

enst

ein-

Jens

en m

edia

.

c Excl

udes

19

patie

nts w

ho p

rovi

ded

only

one

sput

um sa

mpl

e.

d Incl

udes

20

patie

nts w

ho h

ad o

ne c

onta

min

ated

(ind

eter

min

ate)

and

one

neg

ativ

e cu

lture

in L

owen

stei

n-Je

nsen

med

ia.

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Reddy et al. Page 15

Tabl

e 3

Tube

rcul

osis

scre

enin

g pa

ckag

e pe

rfor

man

ce c

ompa

rison

(ord

ered

by

prop

ortio

n of

pat

ient

s cor

rect

ly a

ssig

ned)

Com

plet

ed p

acka

gea

(n v

arie

s)In

tent

-to-s

cree

nb (n

=435

)

Pack

age

Patie

nts c

orre

ctly

assi

gned

c (n

=435

)Se

nsiti

vity

of t

ests

%Sp

ecifi

city

of t

ests

%Po

sitiv

e pr

edic

tive

valu

e of

pac

kage

Neg

ativ

e pr

edic

tive

valu

eof

pac

kage

Faile

d to

com

plet

epa

ckag

ed (n

)T

B-p

ositi

veca

ses m

isse

d (n

)U

S$20

07 p

er p

erso

nin

tend

ed-to

-scr

eene

Mor

e pa

tient

sco

rrec

tly a

ssig

ned

than

no

scre

enin

g

2 M

OD

S43

5 (1

00%

)27

/27

(100

%)

408/

408

(100

%)

27/2

7 (1

00%

)40

8/40

8 (1

00%

)19

0$1

3.62

1 M

OD

S42

6 (9

7.9%

)18

/27

(67%

)40

8/40

8 (1

00%

)18

/18

(100

%)

408/

417

(97.

8%)

09

$7.3

1

2 Sm

ears

415

(95.

4%)

7/27

(26%

)40

8/40

8 (1

00%

)7/

7 (1

00%

)40

8/42

8 (9

5.3%

)19

20$5

.57

1 L

J41

4 (9

5.2%

)15

/25

(60%

)39

9/39

9 (1

00%

)15

/15

(100

%)

408/

420

(97.

1%)

1112

$6.3

9

2 L

J41

2 (9

4.7%

)22

/25

(88%

)39

0/39

0 (1

00%

)22

/22

(100

%)

408/

413

(98.

8%)

395

$11.

77

No

Scre

enin

g (a

ll w

ould

get I

PT)

408

(93.

8%)

0/27

(0%

)40

8/40

8 (1

00%

)n/

a40

8/43

5 (9

3.8%

)0

27$0

.00

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Reddy et al. Page 16

Com

plet

ed p

acka

gea

(n v

arie

s)In

tent

-to-s

cree

nb (n

=435

)

Pack

age

Patie

nts c

orre

ctly

assi

gned

c (n

=435

)Se

nsiti

vity

of t

ests

%Sp

ecifi

city

of t

ests

%Po

sitiv

e pr

edic

tive

valu

e of

pac

kage

Neg

ativ

e pr

edic

tive

valu

eof

pac

kage

Faile

d to

com

plet

epa

ckag

ed (n

)T

B-p

ositi

veca

ses m

isse

d (n

)U

S$20

07 p

er p

erso

nin

tend

ed-to

-scr

eene

Few

er p

atie

nts

corr

ectly

ass

igne

dth

an n

o sc

reen

ing

Clin

ical

Exa

m +

2 M

OD

S39

3 (9

0.3%

)27

/27

(100

%)

366/

408

(89.

7%)

27/6

9 (3

9.1%

)36

6/36

6 (1

00%

)19

0$1

4.14

Clin

ical

Exa

m +

2 S

mea

rs38

0 (8

7.4%

)14

/27

(52%

)36

6/40

8 (8

9.7%

)14

/56

(25%

)36

6/37

9 (9

6.6%

)19

13$6

.09

Clin

ical

Exa

m37

8 (8

6.9%

)12

/27

(44%

)36

6/40

8 (8

9.7%

)12

/54

(22%

)36

6/38

1 (9

6.1%

)0

15$1

.52

Clin

ical

Exa

m +

2 S

mea

rs+

2 L

J37

1 (8

5.3%

)23

/25

(92%

)34

8/39

0 (8

9.2%

)23

/65

(35%

)36

6/37

0 (9

8.9%

)39

4$1

4.86

CX

R35

1 (8

0.7%

)14

/25

(56%

)33

7/39

9 (8

4.5%

)14

/76

(18%

)34

6/35

9 (9

6.4%

)17

13$9

.17

Sout

hern

Afr

ica

Alg

orith

mf (

430

tota

lpa

tient

s)

350

(81.

4%)

11/2

1 (5

2%)

338/

358

(94.

4%)

12/3

3 (3

6%)

382/

397

(96.

2%)

8715

$17.

03

CX

R +

Clin

ical

Exa

m33

0 (7

5.9%

)18

/25

(72%

)31

0/39

9 (7

7.7%

)20

/111

(18%

)31

7/32

4 (9

7.8%

)17

7$9

.70

CX

R +

Clin

ical

Exa

m +

2Sm

ears

330

(75.

9%)

18/2

5 (7

2%)

310/

399

(77.

7%)

20/1

11 (1

8%)

317/

324

(97.

8%)

307

$13.

26

CX

R +

Clin

ical

Exa

m +

2Sm

ears

+ 2

LJ

320

(73.

6%)

22/2

3 (9

6%)

295/

381

(77.

4%)

25/1

16 (2

2%)

317/

319

(99.

4%)

502

$22.

03

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Reddy et al. Page 17TB

-pos

itive

cas

es w

ere

defin

ed a

s pat

ient

s hav

ing

any

posi

tive

mic

robi

olog

ic te

st. P

atie

nts w

ho p

rovi

ded

few

er th

an tw

o sp

utum

sam

ples

did

not

hav

e an

ade

quat

e cu

lture

refe

renc

e st

anda

rd a

nd th

us w

ere

excl

uded

from

the

tabl

e. P

ositi

ve c

linic

al e

xam

=hig

her r

isk

of a

ctiv

e TB

,

acco

rdin

g to

refe

rrin

g cl

inic

ian.

Pos

itive

che

st ra

diog

raph

(CX

R)=

findi

ngs c

onsi

sten

t with

act

ive

TB fo

r whi

ch fu

rther

test

ing

wou

ld b

e re

com

men

ded

prio

r to

ison

iazi

d pr

even

tive

ther

apy.

Sou

ther

n A

fric

a al

gorit

hm27

=pre

limin

ary

scre

enin

g by

tube

rcul

in sk

in te

st (T

ST);

then

, for

only

thos

e w

ith p

ositi

ve T

ST: c

linic

al e

xam

, che

st ra

diog

raph

, tw

o sm

ears

, and

two

LJ.

a Excl

udes

pat

ient

s who

did

not

und

ergo

one

or m

ore

com

pone

nts o

f the

pac

kage

(e.g

. CX

R),

or w

ho h

ad o

ne c

onta

min

ated

(ind

eter

min

ate)

and

one

neg

ativ

e LJ

; den

omin

ator

s var

y ac

cord

ing

to c

ompl

etio

n ra

te o

f the

pac

kage

. Thi

s is t

he “

as sc

reen

ed”

anal

ysis

whi

ch p

rovi

des

effic

acy

data

for c

ompl

eted

pac

kage

s.

b With

in a

pac

kage

, a p

ositi

ve re

sult

for a

ny c

ompo

nent

was

con

side

red

to b

e a

posi

tive

resu

lt fo

r the

pac

kage

. Thi

s “in

tent

ion-

to-s

cree

n” a

naly

sis d

escr

ibes

ove

rall

perf

orm

ance

in a

ll th

ose

who

ent

ered

into

scre

enin

g, a

nd in

corp

orat

es th

e ef

fect

of l

oss-

to-f

ollo

w-u

p.

c Indi

cate

s tho

se c

orre

ctly

ass

igne

d as

TB

-pos

itive

or T

B-n

egat

ive

by th

e pa

ckag

e, w

ith th

e re

fere

nce

stan

dard

bei

ng a

ny p

ositi

ve m

icro

biol

ogic

test

. Pat

ient

s who

faile

d to

com

plet

e al

l com

pone

nts o

f a p

acka

ge w

ere

cons

ider

ed n

ot c

orre

ctly

ass

igne

d, u

nles

s at l

east

one

com

pone

ntte

st w

as p

ositi

ve in

the

cont

ext o

f a p

ositi

ve c

ultu

re re

sult.

d This

is th

e on

ly c

olum

n th

at in

clud

es th

e 19

pat

ient

s who

pro

vide

d on

ly o

ne sp

utum

sam

ple;

thes

e pa

tient

s wer

e co

nsid

ered

to h

ave

faile

d to

com

plet

e al

l pac

kage

s inv

olvi

ng tw

o sp

utum

sam

ples

.

e This

val

ue w

as a

ttain

ed b

y su

mm

ing

the

cost

of d

iagn

osis

and

pat

ient

cos

ts fr

om S

uppl

emen

tal T

able

s 1 a

nd 2

, the

n di

vidi

ng th

e re

sult

by th

e 43

5 pa

tient

s pre

sent

ed in

Tab

le 3

. The

med

ian

of th

e pa

tient

cos

ts, i

nclu

ding

tran

spor

t, w

as u

sed.

f Five

pat

ient

s did

not

und

ergo

TST

and

thus

wer

e ex

clud

ed fr

om a

naly

sis o

f thi

s pac

kage

.

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