Lora et al. BMC Infectious Diseases (2015) 15:222 DOI /s

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1 Lor et l. BMC Infectious Diseses (2015) 15:222 DOI /s RESEARCH ARTICLE Evlution of Microscopic Observtion Drug Susceptibility (MODS) nd the string test for rpid dignosis of pulmonry tuberculosis in HIV/AIDS ptients in Bolivi Meredith H. Lor 1*, Meliss J. Reimer-McAtee 2*, Robert H. Gilmn 3, Dniel Lozno 4, Ruth Srvi 5, Monic Pjuelo 6, Cryn Bern 7, Rosrio Cstro 4, Mgly Espinoz 8, My Vllejo 8, Mrco Solno 4, Roxn Chllp 5 nd Fustino Torrico 4 Open Access Abstrct Bckground: Tuberculosis (TB) is the most common opportunistic infection nd the leding cuse of deth in HIV-positive people worldwide. Dignosing TB is difficult, nd is more chllenging in resource-scrce settings where culture-bsed dignostic methods rely on poorly sensitive smer microscopy by Ziehl-Neelsen stin (ZN). Methods: We performed cross-sectionl study exmining the dignostic utility of Microscopic Observtion Drug Susceptibility liquid culture (MODS) versus trditionl Ziehl-Neelsen stining (ZN) nd Lowenstein Jensen culture (LJ) of pulmonry tuberculosis (TB) nd multidrug-resistnt tuberculosis (MDRTB) in HIV-infected ptients in Bolivi. For sputum scrce individuls we ssessed the vlue of the string test nd induced sputum for TB dignosis. The presence of Mycobcterium tuberculosis (Mtb) in the sputum of 107 HIV-positive ptients ws evluted by ZN, LJ, nd MODS. Gstric secretion smples obtined by the string test were evluted by MODS in 102 ptients. Results: The TB-HIV co-infection rte of HIV ptients with respirtory symptoms by sputum smple ws 45 % (48/107); 46/48 (96 %) were positive by MODS, 38/48 (79 %) by LJ, nd 30/48 (63 %) by ZN. The rte of MDRTB ws 9 % (4/48). Medin time to positive culture ws 10 dys by MODS versus 34 dys by LJ (p < ). In smer-negtive ptients, MODS detected TB in 17/18 ptients, compred to 11/18 by LJ (94.4 % vs 61.0 %, p = 0.03 %). In ptients unble to produce sputum smple without induction, the string test cultured by MODS yielded Mtb in of 9/11 (82 %) TB positive ptients compred to 11/11 (100 %) with induced sputum. Of the 10 ptients unble to produce sputum smple, 4 were TB-positive by string test. Conclusion: MODS ws fster nd hd higher Mtb detection yield compred to LJ, with greter difference in yield between the two in smer-negtive ptients. The string test is vluble dignostic technique for HIV sputum-scrce or sputum-bsent ptients, nd should be considered s n lterntive test to induced sputum to obtin smple for Mtb in resource-limited settings. Nine percent of our TB+ ptients hd MDRTB, which reinforces the need for rpid detection with direct drug susceptibility testing in HIV ptients in Bolivi. Keywords: Tuberculosis, HIV, AIDS, MODS, Bolivi, Resource-scrce, String test * Correspondence: meredith.holtz@gmil.com; mreimer2@tulne.edu Equl contributors 1 Deprtment of Medicine, University of Cliforni in Sn Frncisco, 505 Prnssus Ave, Rm 987, Sn Frncisco, CA , USA 2 Deprtment of Medicine, Tulne University School of Medicine, New Orlens, LA, USA Full list of uthor informtion is vilble t the end of the rticle 2015 Lor et l. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Public Domin Dediction wiver ( cretivecommons.org/publicdomin/zero/1.0/) pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 2 of 10 Bckground Tuberculosis (TB) is the most common opportunistic infection nd the leding cuse of deth in HIV-positive people worldwide [1]. Dignosing TB is difficult, nd is more chllenging in resource-scrce settings where those without timely culture-bsed dignostic methods rely on poorly sensitive smer microscopy using Ziehl-Neelsen stin (ZN) [1 3]. Co-infection with HIV nd TB ffects the dignosis of TB, s well s the morbidity, nd mortlity of ptients in multi-fceted mnner. TB dignosis in HIV is complicted by puci-bcillry infection nd typicl presenttions of disese [4]. TB hs been shown to increse HIV virl repliction nd my ccelerte HIV progression [5] nd increse mortlity [6 8]. In the HIV popultion, smer microscopy detects only % of culture-positive pulmonry TB infections [7, 9, 10], compred to % in HIV-negtive ptients [3]. Given the incresed incidence nd mortlity of smernegtive TB in HIV ptients, culture is recommended [2, 3], but not widely used. When performed, culture improves detection of ctive TB; however, commonly used solid culture methods, including Lowenstein Jensen culture (LJ), my tke 6 8 weeks, nd lck concurrent ntibiotic sensitivity dt [3, 11, 12]. Ultr rpid test GeneXpert is currently recommended by the WHO s first line TB test in the HIV popultion [13], but it is limited by lower sensitivity in smer negtive ptients nd cost. At $10/crtridge nd $17,000/mchine, cost remins brrier to widespred implementtion, therefore lterntive methods for rpid TB dignosis in HIV ptients re needed in resource limited res [14 16]. TB-HIV co-infection nd Multidrug-resistnt Tuberculosis (MDRTB) in Bolivi Bolivi hs the highest rte of ctive TB in South Americ, nd one of the highest in the world, with n estimted prevlence of 196 in 100,000 [1]. Although only 11,421 totl cses of HIV were reported in 2014, it ws estimted tht 15,000 persons were living with HIV in Bolivi, nd 1000 deths were ttributed to AIDS [17]. Despite the prevlence of TB infection in Bolivi, WHO reported only 170 totl ptients with HIV-TB co-infection in 2013 [1]. Worldwide prevlence of MDRTB is incresing, nd remins underdignosed [1]. It is estimted tht only 7 % of the 500,000 people with MDRTB re recognized s MDR, which my led to incresed tretment filure nd jeoprdize the success of TB control efforts [1]. The WHO reported 1.2 % of newly dignosed TB cses in Bolivi in 2013 were MDR [1]. In prts of the world where either MODS or GeneXpert re vilble, the dignosis of either MDRTB or Rifmpicin-resistnt TB is mde when the test returns positive for TB [13, 18, 19]. Currently, in Bolivi, however, dignosis of MDRTB relies on LJ followed by further ntimicrobil sensitivity testing, which cn dely drug susceptibility results for weeks fter initil dignosis [20]. Microscopic Observtion Drug Susceptibility Culture (MODS) The MODS ssy is low-cost, liquid culture used for the dignosis of TB nd direct drug susceptibility testing which uses commonly vilble lbortory mterils in order to permit its ppliction in resource-limited settings [18, 19]. MODS is bsed on the principles tht Mtb grows fster in liquid thn in solid medium nd chrcteristic cording formtion tht is specific for Mtb is microscopiclly observed erly in liquid medium [18]. The incorportion of Rifmpicin nd Isonizid during the initil stge of the culture permits concurrent nd direct drug-susceptibility testing, obviting the need for subculture sensitivity testing [18, 19]. Currently MODS costs $3 per test, nd the recent development of low-cost inverted microscope, utomted plte reders, nd stndrdized TB MODS KIT will stremline the cultivtion process, conserve lbor hours prepring necessry regents, nd my further decrese costs [21, 22]. In policy sttement in 2011, the WHO pproved MODS for drug susceptibility screening for MDRTB [23]. Scrce sputum production in HIV ptients Obtining n dequte sputum smple from ptients with scrce sputum production is chllenging in HIV-positive ptients. Lck of productive cough in AIDS ptients correltes poorly with the presence of pulmonry TB [24]. The most widely used option for smple obtinment is sputum induction. The string test, originlly demonstrted for the retrievl of enteropthogens from the upper gstrointestinl trct [25, 26], hs been shown to be sfe method of smple collection for the dignosis of pulmonry TB in HIV ptients [24]. Our study exmines the dignostic utility of Microscopic Observtion Drug Susceptibility liquid culture (MODS) versus trditionl Ziehl-Neelsen stining (ZN) nd Lowenstein Jensen culture (LJ) of pulmonry tuberculosis (TB) nd multidrug-resistnt tuberculosis (MDRTB) in HIV-infected ptients in Bolivi. For sputum scrce individuls we ssessed the vlue of the string test nd induced sputum for TB dignosis. Methods Study ptients nd setting This ws prospective nd consecutive cross-sectionl study conducted from Februry 2010 through August 2011 in Cochbmb, Bolivi, city identified s n endemic TB region with n incidence of smer-positive pulmonry TB of 41.4 per 100,000 in 2011 [2]. All study prticipnts were HIV-positive ptients, confirmed by Western blot, with respirtory symptoms such

3 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 3 of 10 s cough or dyspne. Constitutionl symptoms nd risk fctors for tuberculosis were noted on further evlution but not ssessed for study inclusion. Hospitlized subjects were evluted upon dmission to the infectious diseses wrd of the Universidd Sn Simon, the public hospitl in Cochbmb. Ambultory subjects were evluted upon presenttion to the clinic with respirtory symptoms. Subjects were excluded if they were under 18 yers of ge, unwilling or unble to provide informed consent or ctively receiving tuberculosis therpy for more thn seven dys. Study protocol nd consent forms were pproved by the institutionl review bords of Universidd Perun Cyetno Heredi, Asocición Benefic PRISMA, nd Johns Hopkins Bloomberg School of Public Helth. Smple collection Prticipnts were sked to submit three erly-morning sputum smples nd gstric secretion smple by the string test. The first smple ws divided into three liquots to be processed by ZN, LJ, nd MODS Two dditionl sputum smples were sent for ZN smer. Ptients unble to produce dequte sputum (defined s minimum volume of 1 ml/culture smple of wht mcroscopiclly ppered to be sputum) were induced by nebulizer with 3-5 % sline solution for up to three 5-min-intervls. Lter in the study, ll ptients were sked to submit both spontneous smple nd n induced sputum smple for comprison. Ptients ble to produce sputum spontneously were considered sputum productive. Ptients unble to produce spontneous sputum smple but with n dequte induced sputum smple were considered sputum-scrce. Ptients unble to produce sputum smple despite sputum induction were considered sputum-bsent. Those willing to tke prt in the string test swllowed geltin cpsule hnd-pcked with 90 cm string fter 8 hours of fsting. The triling end of the string ws ttched to the ptients cheek when swllowed. After one hour of intrgstric time [27] the string ws retrieved nd the intrgstric contents on the string were cultured by MODS s described below. If consented but unble to submit t lest one smple (either sputum or string), they were excluded from study. Lbortory methods String test smples were processed ccording to protocols described in previous publictions 25. Sputum nd gstric smples were stored t 4 C nd were cultured s soon s possible fter collection (Medin 3.5 dys, IQR 1 6). All specimens were decontminted by the stndrd NALC-NOH-N citrte method [28]. MODS ws performed s described in previous publictions [18, 19] nd cultures were exmined every other dy (excluding weekends) until dy 30. LJ cultures were performed ccording to the protocol set by the Ntionl TB Progrm of Bolivi, nd smples were inoculted t 37 C nd exmined twice weekly until dy 60. Rifmpin nd Isonizid susceptibilities in this study were ssessed through MODS lone. Sttisticl nlysis Dt were nlyzed using STATA 10, PRISM, nd Excel softwre. Comprisons of ctegoricl dt were performed with either the Chi-squred test or Fisher s exct test;comprisons of continuous dt were performed with either t- test or Wilcoxon rnk sum test. Comprisons of dignostic yield between the different methods were ssessed using McNemr s Chi-squre test or McNemr s exct test. As ll ptients hd respirtory symptoms, ptient with sputumor string test smple tht ws culture positive by Lowenstein Jensen or MODS ws considered TB positive ( definite TB ccording to 2012 NIH Consensus guidelines). As our study is evluting MODS, with known higher sensitivity thn the reference test, this composite culture positivity ws used s the reference stndrd to evlute ech test. As result, our results reflect dignostic yield of ech detection method; we re unble to comment on the true sensitivity or specificity of ech method. Stndrd nti-tuberculosis tretment ws dministered to ptients with positive result by ny of the bove dignostic methods by the ttending on service fter communiction of the positive results. Although this study ws not initilly designed to determine mortlity, we did note if ptients died by the time tht his or her culture results were vilble. We defined mortlity s deth within 60 dys of smple collection, the longest time period in which we would hve contcted ptient with culture results. Results Smples obtined nd ptient chrcteristics Of the 134 ptients enrolled in the study, 107 (79.8 %) subjects submitted sput tht were evluted by ZN, LJ, nd MODS. Additionlly, 24 of these 107 ptients (22.4 %) submitted both induced nd spontneous sputum smple for comprison. Of the 107 ptients who submitted sputum, 92 (85.9 %) lso submitted string smple. 10 ptients were unble to produce sputum but submitted string test. Of those who submitted string smple (n = 102), the ptients were grouped into sputum productive (68.6 %), sputum scrce (21.5 %), or sputum bsent (9.8 %), bsed on their degree of sputum production (See Fig.1). The medin ge of the ptients ws 32 yers (IQR 27 43). 101/107 ptients (94.4 %) reported cough; medin durtion of cough prior to presenttion ws 20.5 dys (IQR 7 60). 25/107 ptients reported hemoptysis (23.3 %). 65/107 ptients reported shortness of breth (60.7 %). 81/ 107 (75.7 %) of ptients reported fever, 68/107 (63.5 %) of ptients reported night swets. 56/107 (52.3 %) reported

4 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 4 of 10 Fig. 1 Recruitment of ptients, smples obtined, nd resulting groups of ptients for smple nlysis. 134 ptients were enrolled in the study. 7 ptients were unble to produce t lest one smple (either string or sputum) nd were excluded. 5 ptients were excluded s their cultures were contminted. 5 smples hd long delys in processing by lb (>12 dys); these ptients were excluded. 107 subjects submitted sputum smple sufficient for evlution by ZN, LJ, nd MODS tht tht were included in the nlysis. Additionlly, 24 of these ptients submitted both induced nd spontneous sputum smple for comprison. Of the 107 ptients who submitted n dequte sputum smple, 92 lso submitted string smple. 10 dditionl ptients submitted string smple but did not submit sputum smple. Of these102 ptients tht submitted string test smple, 70 ptients (68.6 %) were ble to produce sputum spontneously ( sputum productive ). 22 ptients (21.5 %) were only ble to produce sputum smple fter induction ( sputum-scrce ). Ten ptients (9.8 %) were unble to produce ny sputum ( sputum-bsent ). While we note the results of these 10 string smples, these ten ptients were not included in our lrger nlysis of TB dignostic methods s there ws no sputum for comprison both fevers nd night swets. The medin CD4 count for ll 107 ptients ws 98 cells/mm 3 ; for hospitlized ptients ws 76.5; for mbultory ptients ws 192 (Tble 1). Only 28 of the 107 ptients (26.1 %) were on ARVs t the time of smple collection. Of the TB+ ptients, 12/48 (25.0 %) were on ARVs. Rtes of TB nd Associted Mortlity of TB+ pts Of the 107 ptients, 44.9 % (48/107) were Mtb culture positive by MODS or LJ culture of the sputum or string test smple nd were considered TB+. The rtes of TB in hospitlized ptients (n = 35, 46.1 %) versus mbultory (n = 13, 41.9 %) were similr. The mortlity rte of ll TB+ ptients ws 45.8 % (22/48) (see Tble 1). Of those TB+ sensitive to both rifmpin nd isonizid, mortlity ws 35.0 % (14/40); of those TB+ with resistnce to one or more nti-tb drugs, mortlity ws 75 % (6/8);p=0.08. Of the 22 TB+ ptients tht died during the study, the medin CD4 count ws 59 (IQR ), compred to 174 (IQR ) in surviving TB+ ptients (n = 26). Between these two groups, their medin durtion of symptoms prior to presenttion ws 21 dys in decesed ptients (IQR ) versus 30 dys in surviving ptients (IQR ). The rtes of ntiretrovirl therpy t the time of enrollment in these two groups were similr (5/22 of decesed ptients, 6/26 of surviving ptients, p = 0.62). Mtb Detection nd Time to Positivity MODS detected Mtb in 46/48 TB+ ptients with dignostic yield of 95.8 % (95 % CI, ), nd detected Mtb in significntly more ptients thn LJ (38/48, 79.2 %, p = 0.022) nd ZN (30/48, 62.5 %, p < 0.001). In the mbultory ptients, MODS detected Mtb in 12/13 TB+ ptients (92.3 %) versus LJ in 7/13 (53.8 %), p = 0.06, nd the

5 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 5 of 10 Tble 1 Ptient Chrcteristics: Medin ges nd CD4 counts, rtes of TB positivity, nd ssocited mortlity of ll TB+ ptients tht submitted sputum for comprison, strtified by hospitlized nd mbultory groups n Medin Age (IQR) Medin CD4 count (IQR) TB+ (%) Mortlity of TB+ All ptients (27 43) 98 (33 249) 48/107 (44.9 %) 22/48 (45.8 %) Hospitlized ptients (27 46)* 76.5 ( )** 35/76 (46.1 %)*** 18/35 (51.4 %)**** Ambultory ptients (28 42)* 192 (64 339)** 13/31 (41.9 %)*** 4/13 (30.7 %)**** 107 ptients were included in nlysis. Medin ge of ll ptients s well s those hospitlized vs mbultory ws 32. Medin CD4 count for ll ptients ws 98. Medin CD4 count of hospitlized ptients ws 76 nd medin CD4 for mbultory ptients ws 192. Rtes of TB positivity (TB+), defined s ptient with sputum or gstric string smple tht ws culture positive by MODS or Lowenstein Jensen, ws 44.9 % in ll ptients, 46.1 % in hospitlized ptients, nd 41.9 % in mbultory ptients. Of those who were TB+, the mortlity rte overll ws 45.8 %. The mortlity rte for hospitlized TB+ ptients ws 51.4 % nd for mbultory TB+ ptients, it ws 30.7 % n number of ptients in ech group. Medins re displyed in yer. IQR Interqurtile rnge *The difference between the medin ge of hospitlized versus mbultory ptients ws not sttisticlly significnt (p = 0.87) **The difference between the medin CD4 count of hospitlized versus mbultory (76.5 vs 192) ws sttisticlly significnt (p = 0.01) **The difference between the rtes of TB of hospitlized vs mbultory ptients (46.1 % vs 41.9 %) ws not sttisticlly significnt (p = 0.70) ***The difference between the mortlity of the TB+ hospitlized ptients vs mortlity of TB+ mbultory ptients (51.4 % vs 30.7 %) ws not sttisticlly significnt (p = 0.08) detection yield of LJ ws similr to tht of ZN (53.8 % vs 61.5 %, p = 1.00). See Tble 2. Thirty-seven percent (18/48) of TB+ ptients were smer-negtive. In the smer-negtive group, MODS detected TB in 17/18 ptients, significntly more detected thn the 11/18 by LJ (94.4 % vs 61.0 %, p = 0.03 %). In the smer-positive group (30/48) the difference ws not significnt (MODS 96.6 % nd LJ 90.0 %, p = 0.63). See Tble 2. The medin time to sputum culture positivity ws significntly shorter for MODS thn for LJ (10 nd 34 dys, p < ). Within MODS, smer sttus significntly ffected time to positivity (medin 8 versus 13 dys in smer-positive nd negtive smples, p < ). This difference ws not seen in LJ by smer sttus. See Fig. 2. The string test: performnce in sputum-productive, sputum-scrce nd sputum-bsent ptients Sputum-scrce ptients hd lower medin CD4 count thn sputum productive ptients (55 versus 122; p = 0.021). 3 of 22 (13.6 %) sputum scrce ptients were on ARVs t the time of enrollment compred to 21 of 90 (23.3 %) of sputum productive ptients. The rte of TB+ in the sputum-scrce group ws 50 % (11/22), compred to 40 % (28/70) in the sputum-productive ptients (p = 0.41). Of those TB+, the sputum-scrce group hd higher mortlity rte (8/11, 72.7 %) thn the sputum-productive group (10/ %), p = See Tble 3. Cultures of string test smple nd sputum were compred by MODS. In the sputum-productive group (n = 70), of the 28 TB+ ptients, culture of the string test yielded Mtb in 18/28 (64.2 %) ptients compred to 27/ 28 (96.4 %) with sputum (p = 0.012). In the sputumscrce group (n = 22), of the 11 TB+ ptients, the string test yielded Mtb in 9/11 (81.8 %) ptients compred to 11/11 (100 %) with induced sputum (p = 0.50). Ten ptients with dry cough were sputum bsent, nd were only ble to submit string smple; culture of the string test yielded Mtb in 4/10 of these ptients. See Tble 3. In the sputum-productive group, time to positivity for sputum cultured by MODS ws significntly fster thn string test cultured by MODS (medin 9 vs 15 dys, p = Tble 2 Comprison of dignostic methods for the detection of Mtb in hospitlized versus mbultory nd in smer-positive versus smer-negtive ptients A. All ptients Hospitlized ptients Ambultory ptients TB+ (n = 48) Dignostic yield (95 % CI) TB+ (n = 35) Dignostic yield (95 % CI) TB+ (n = 13) Dignostic yield (95 % CI) MODS % ( ) % ( ) % ( ) LJ % ( ) % ( ) % ( ) ZN % ( ) % ( ) % ( ) B. All ptients Smer-positive Smer-negtive TB+ (n = 48) Dignostic yield (95 % CI) TB+ (n = 30) Dignostic yield (95 % CI) TB+ (n = 18) Dignostic yield (95 % CI) MODS % ( ) % ( ) % ( ) LJ % ( ) % ( ) % ( ) TB+ ws defined s ny ptient with sputum or gstric smple tht yielded positive culture result by LJ or MODS. As the reference stndrd is composite from ll tests tht re being evluted, we cn only comment on dignostic yield rther thn sensitivity nd specificity. Pnel A: Comprison of dignostic yield of MODS versus LJ nd ZN in ll ptients, hospitlized ptients, nd mbultory ptients who were TB positive. Pnel B: Subgroup comprison of the dignostic yield of MODS versus LJ in culture positive ptients, sub-grouped into ll ptients, smer positive ptients, nd smer negtive ptients n number of ptients in ech group; CI confidence intervl; TB + smples tested positive for Mtb by ech respective method (MODS, LJ, or ZN)

6 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 6 of 10 Fig. 2 Cumultive percentges of the time to culture positivity for culture-positive smples ccording to culture method (), ccording to smer sttus of sput (b nd c), nd ccording to smple type in sputum-productive nd sputum-scrce ptients (d nd e). A. Medin time to sputum culture positivity cultured by MODS vs LJ (10 dys vs 34 dys, p <0.0001). b. Within MODS culture of sput, effect of smer-positive vs smer-negtive on time to culture positivity (medin 8 vs 13 dys, p <0.001). c. Within LJ of sput, effect of smer-positive vs smer-negtive on time to culture positivity (medin 30 vs 40 dys, p = ) d. Within the sputum-productive cohort, time to culture positivity of MODS sputum vs MODS string test (medin 9 vs 13 dys, p = ). e Within the sputum-scrce cohort, time to culture positivity of MODS sputum vs MODS string test (medin 11 vs 14 dys, p = ). String smples were not neutrlized prior to storge nd this my hve ffected both dignostic yield nd time to positivity. Medin times to processing with interqurtile rnges for ech group of smples re below: All MODS Sputum Smples: Medin 3 dys (IQR 1 5). MODS Smer-positive Sputum Smples: Medin 4 dys (IQR 2 5). MODS Smer-negtive Sputum smples: Medin 2 dys (IQR ). LJ Sputum smples: Medin 5 dys (IQR 2 6). All MODS String Smples: Medin 3 dys (IQR 1 5). MODS Sputum-Productive Sputum Smples: Medin 3 dys (IQR 2 5). MODS Sputum-Productive String Smples (Medin 3 dys (IQR 2 5). MODS Sputum-Scrce Sputum Smples: Medin 4 dys (IQR 2 6). MODS Sputum-Scrce String Smples: Medin 4 dys (IQR 2 6) 0.006). In the sputum-scrce group, time to positivity of induced sputum ws similr to tht of string test (medin 11 vs 14 dys, p = 0.31). The string test smples were not neutrlized until just prior to culture, nd this long with the processing dely my hve negtively ffected both dignostic yield nd time to positivity. See Fig. 2. Detection of Mtb in induced sputum versus spontneous sputum using MODS culture 24 ptients submitted both spontneous nd induced sputum. In this group, 14 ptients were TB positive by t lest one of the smples obtined. Of these 14 ptients, MODS detected TB in 92.8 % (13/14) of the induced sputum smples vs 78.6 % (11/14) of the spontneous

7 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 7 of 10 Tble 3 String Test sub-nlysis: Ptient chrcteristics nd comprison of dignostic methods in sputum productive, sputum scrce nd sputum bsent ptient subgroups Ptient Chrcteristics (n = 102) Sputum-productive (n = 70) Sputum-scrce (n = 22) Sputum-bsent (n = 10) Medin ge (yers) CD4 count 122 (IQR ) 55 (IQR 23 86) 221 (IQR ) Rte of TB+ 28/70 (40 %) 11/22 (50 %) 4/10 (40 %) Mortlity of TB+ 10/28 (35.7 %) 8/11 (72.7 %) 0/4 (0 %) Test +Mtb (n = 28) Dignostic yield (95 % CI) +Mtb (n = 11) Dignostic yield (95 % CI) +Mtb (n = 4) Dignostic yield String test MODS % ( ) % ( ) 4 Sputum MODS % ( ) % ( ) Sputum LJ % ( ) % ( ) Sputum ZN % ( ) % ( ) TB positive ws defined s ny ptient with sputum or gstric smple with positive culture result from sputum or string test by LJ or MODS. This composite positivity ws used s the reference stndrd to determine dignostic yield. The ptients tht submitted string test (n = 102) were divided into three groups bsed on degree of sputum productivity. Sputum-productive ptients (n = 70, 68.6 %) were ble to produce spontneous sputum. Sputum-scrce ptients (n = 22, 21.5 %) required sputum induction to produce smple. Sputum-bsent ptients (n = 10, 9.8 %) were unble to produce sputum despite induction nd only submitted string smple. Of the sputum bsent ptients, 4 ptients were positive for Mtb by string test, but there re no smples for comprison since they were unble to produce sputum. The string test smples were not neutrlized prior to culture nd this my hve ffected dignostic yield n number of ptients in group; IQR interqurtile rnge; CI confidence intervl We re unble to provide these numbers s sputum bsent ptients did not submit sputum for comprison sputum smples (p = 0.63). The medin time to positivity for induced versus spontneous sputum ws not significntly different (11 vs 9 dys, p = 0.30). See Fig. 2. Rtes of MDRTB in TB-HIV co-infection The rte of both Rifmpin nd Isonizid resistnce (MDRTB) in our study ws 9.0 % (4/46). Rtes of resistnce to Rifmpin lone ws 4.0 % (2/46) nd to Isonizid lone ws 4.0 % (2/46), thus totl of 17.0 % (8/46) of ptients were resistnt to t lest one of these two medictions. Resistnce is determined concurrently with initil positive MODS culture, so time to resistnce prllels tht of time to positivity s described bove. Discussion The dt from our study stresses the need for rpid TB dignostic test in HIV ptients in Bolivi. The rte of TB in HIV ptients with respirtory symptoms ws very high (45 %), nd lmost hlf (46 %) of these ptients died within 60dys.Furthermore,therteofTB-HIVco-infectionin our study lone over 1.5 yer period (n = 48) does not correlte well with the rte of HIV-TB co-infection in ll of Bolivi reported in 2013 (n = 170) [1]; this dt rises concern tht this is problem of greter mgnitude thn is currently pprecited. Even more lrming is the prevlence of MDRTB in our popultion of HIV ptients (9 %), which is much higher thn the reported MDRTB rtes in Bolivi; nd tht 75 % of those with resistnce to one or more drugs died during the durtion of our study. A similr study by Kwi et l. in Peru showed tht MDRTB-HIV co-infection hdmortlityrtebove50%withintwomonthsnd ws ssocited with high rte of continued infectiousness [29]. Current methods of resistnce testing in Bolivi cn tke weeks beyond positive solid culture results, risking significnt delys of pproprite tretment. These dt strongly reinforce the need for the implementtion of rpid TB dignostic method with direct drug susceptibility testing in the HIV popultion in Bolivi. Additionlly, the discrepncy between the rtes of resistnce found in our study compred to previous reports, clls for dedicted study of ntibiotic resistnce in Bolivi in HIV ptients. Given the incresed rtes of MDRTB in HIV, the risk of nosocomil spred, nd the high mortlity ssocited with this co-infection, if these rtes of MDRTB re confirmed in future studies, MDRTB in Bolivi could quickly become greter thret to public helth. Our study demonstrtes tht MODS is vluble method for rpid dignosis of TB in the HIV popultion. In this study, the detection yield of MODS ws superior to LJ. Additionlly, consistent with prior studies bsed in centrlized MODS lbortories, Mtb ws observed in MODS in less thn one-third of the time of growth in LJ [18, 19, 23]. MODS incresed dignostic yield ws most profound in the smer-negtive HIV ptients, vulnerble group t highest risk for missed or delyed dignosis. Although smer-negtive sputum smples cultured by MODS grew lter thn MODS smer-positive smples, smer-negtive smples by MODS still grew significntly fster thn those by LJ. Our results emphsize the utility of MODS in the mbultory popultion. MODS detected lmost 30 % more disese thn LJ in this popultion, nd LJ did not identify ny more disese thn ZN lone, supporting use for MODS in ptients with milder presenttions. As this study highlights the need for widespred implementtion of rpid, sensitive TB dignostic method

8 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 8 of 10 in HIV ptients with direct drug susceptibility testing, it is importnt to mention the role of GeneXpert s compred to MODS. GeneXpert would lso be welcome lterntive to the current stndrd of cre in Bolivi for HIV ptients. However, t the current time, without committed long-term externl funding source, its cost ($10/crtridge nd $17,000/mchine) limits widespred implementtion in resource-limited settings like Bolivi [13]. MODS ($3/test) remins more cost-effective lterntive in these settings. Additionlly, given MODS performnce in smer negtive ptients s demonstrted in this study nd others, nd GeneXpert limittions in this domin [14, 15], we would lso recommend MODS s low cost lterntive for GeneXpert for smer negtive ptients. Our study provides dt bout sputum scrce HIV ptients with respirtory symptoms in TB endemic re. Despite miniml sputum production, the prevlence of TB in this popultion trended towrds higher thn the sputum-productive subjects nd the 60 dy mortlity rte of those TB+ ws greter in the sputum-scrce group thn the sputum productive ptients. Additionlly, we exmine the vlue of the string test s smple obtinment method for TB in sputum scrce or bsent ptients. In our sputum-scrce group, the string test detected over 80 % of TB cses, nd ws not significntly different thn the number of TB cses by induced sputum. Importntly, in the 10 ptients tht could not produce sputum smple despite induction, the string test detected 4 cses of TB tht would hve otherwise been missed; thus 40 % of ptients tht could not produce sputum smple were dignosed nd received tretment becuse of the string test. Our dignostic yield is lower thn in previous studies [24], this my be due to lck of neutrliztion before storge nd delyed processing of smples. Despite this, the string test provided n inexpensive, minimlly invsive lterntive for dignosis of TB in sputum-scrce nd sputum-bsent ptients without n ssocited biohzrd risk. Given the bove dt, we suggest tht the string test be considered s n lternte smple obtinment method used in conjunction with MODS in HIV ptients with little to no sputum production. Of the ptients with both induced nd spontneous sput (n = 24), lthough the difference of the results between the two groups were not sttisticlly significnt, two ptients tht were negtive for TB by spontneous sputum were TB+ by induced sputum. Although induction of sputum in the dignosis of TB is historiclly more common in the peditric popultion [30, 31], more recent studies evluting the utility in dults in HIV-endemic regions hve come to differing conclusions [32 35]. Smll studies hve promoted the use of induction of sputum by demonstrting n incresed dignostic yield when compred to spontneous sputum [32 34]; however, rndomized controlled tril of 418 ptients in South Afric did not show significnt difference between the results of helthcre workerinstructed versus induced sputum [35]. Furthermore, induction of sputum requires dditionl equipment, poses biohzrd risk, nd is n incresed cost to hospitls (US$2.14 vs $7.88) [35]. Additionlly, our study is the first in Bolivi to demonstrte tht MODS is superior to the current locl stndrds of tuberculosis dignosis. We showed tht lbortory with miniml prior experience with MODS is ble to produce results similr to tht of centrlized MODS lbortories, s in Peru, where MODS is now the stndrd of cre [23].. Furthermore, the implementtion of MODS cn be more simplified nd ffordble in the future, with the recent development of MODS kits nd vilbility of inexpensive inverted microscopes [21 23]. Limittions of our study include our incresed time to positivity compred to prior studies in Peru [18, 19]. This increse my hve been influenced by dely in processing times of the smples, nd the fct tht smples were checked less often (due to personnel vilbility), rther thn every dy. Although most smples were cultured within 4 dys of collection (Medin 3.5, IQR 1 6), some were stored for up to 11 dys, which my hve ffected growth of culture. The dignostic yield of the string test in this study is lower thn previous studies [24]; it is importnt to note our lck of neutrliztion of the smple before storge s well s delys in processing smples my hve decresed string test yield nd is limittion of our study. Our imperfect reference stndrd ws n dditionl limittion to optimlly evlute the dignostic performnce of MODS. The performnce of MODS would hve been more clerly defined if our stndrd included other sensitive dignostic methods such s MGIT or GeneXpert rther thn composite of ll of the tests being evluted. This ws due to logisticl limittions nd finncil constrints. Although the comprison of MODS to the stndrd solid culture medi for dignosis of TB in Bolivi provides importnt dt for the community tested, the incorportion of GeneXpert in our study would hve been vluble comprison, hd it been possibility. Additionlly, there is gp in our dt regrding the mortlity of the HIV ptients tht were negtive for TB. Regrettbly we did not record the mortlity dt for ll ptients without TB during the time of the study, thus we re not ble to mke comprison in mortlity between the TB-positive nd TB-negtive ptients. In mny prts of the world, HIV is no longer deth sentence, but hs become mngeble chronic disese due to incresed detection nd wider vilbility of effective tretments. The currently undertreted nd highly lethl HIV- TB co-infection is brrier to this becoming relity in

9 Lor et l. BMC Infectious Diseses (2015) 15:222 Pge 9 of 10 mny TB-endemic res such s Bolivi. Implementtion of MODS, or if economiclly fesible, nother rpid dignostic test such s GeneXpert, should become the stndrd for dignosis of TB in HIV popultions in resource-scrce settings. This would empower clinicins to tret ptients with effective ntimicrobils t erlier time points, which would led to significnt effects on both ptient mortlity nd public helth through decresed trnsmission of this dedly disese. Conclusions ThehighprevlencendmortlityofTB-HIVco-infection found in our study nd others highlights the need for more rpid dignostic test for TB tht includes direct drug susceptibility testing in HIV ptients in Bolivi nd in similr resource-limited settings. MODS culture performs well in this setting, nd should be considered s low cost lterntive in HIV ptients. In ddition, more effective pproch to symptomtic HIV ptients with little or no sputum production is needed; the string test could be vluble dignostic tool for this subset of ptients. Abbrevitions TB: Tuberculosis; ZN: Ziehl-Neelsen stin; MDRTB: Multidrug-resistnt tuberculosis; MODS: Microscopic Observtion Drug Susceptibility liquid culture; LJ: Lowenstein Jensen culture; Mtb: Mycobcterium tuberculosis; HIV:Humn Immunodeficiency Virus; AIDS: Acquired Immunodeficiency Syndrome. Competing interests The uthors declre tht they hve no competing interests. Authors contributions Conception of the study- MHL, RHG, DL, FT; Implementtion of the study- MHL, MRM, RC, DL, FT; Construction of the mnuscript- MRM nd MHL; Processing of smples- MHL, MRM, RS, RCh, ME, MV, MS; Dt nlysis- MP, CB; Editing of mnuscript- MHL, MRM, CB, RHG. All uthors red nd pproved the finl mnuscript. Acknowledgements We would like to thnk the lte Luz Cviedes for trining the lbortory technicins in Cochbmb, Bolivi, nd for being such n vilble nd grcious resource to everyone involved in the project. We would like to thnk Dr. Amilcr Apz, Dr. Ann Volz Glvez, nd Dr Mirey Clros t the TB progrm in Cochbmb for llowing the study to tke plce; Cst Phusi t L Escuel Técnic de Slud for her help processing the smples in the lb; nd J.B. Phu nd D. Sr for their technicl support. Finncil support This study ws supported by the following grnts through the Ntionl Institutes of Helth: T35-AI nd T35-AI Funders took no prt in the design or interprettion of the study. The content of this study is solely the responsibility of the uthors nd does not necessrily represent the views of the funding sources. Author detils 1 Deprtment of Medicine, University of Cliforni in Sn Frncisco, 505 Prnssus Ave, Rm 987, Sn Frncisco, CA , USA. 2 Deprtment of Medicine, Tulne University School of Medicine, New Orlens, LA, USA. 3 Johns Hopkins Bloomberg School of Public Helth, Bltimore, Mrylnd, USA. 4 Fcultd de Medicin, Universidd Myor de Sn Simón, Cochbmb, Bolivi. 5 Colectivo de Estudios Aplicdos y Desrrollo Slud y Medio Ambiente, Cochbmb, Bolivi. 6 Universidd Perun Cyetno Heredi, Lim, Peru. 7 University of Cliforni in Sn Frncisco, Sn Frncisco, USA. 8 L Escuel Técnic de Slud, Cochbmb, Bolivi. Received: 3 Februry 2015 Accepted: 28 My 2015 References 1. World Helth Orgniztion: Globl tuberculosis report. Genev; Accessed 31 My Ministerio de Slud y Deportes: Progrm Ncionl de Control de Tuberculosis. Boletín Informtivo, Bolivi Docs/Boletin/Boletin%20TB%202012%20I.pdf. Accessed 31 My Americn Thorcic Society. Dignostic stndrds nd clssifiction of tuberculosis in dults nd children. Consensus from n expert pnel. 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