Characteristics of Drug-sensitive and Drug-resistant Tuberculosis Cases among Adults at Tuberculosis Referral Hospitals in Indonesia.

As Indonesia's rifampin resistance testing rates are lower than global testing rates per the 2020 WHO global tuberculosis (TB) report, prevalence of multidrug-resistant TB may be underestimated. Our study aimed to evaluate prevalence and patterns of TB drug resistance (DR) within Indonesia. We conducted a cross-sectional analysis of baseline data collected from 2017-2018 as part of a cohort study of adults with presumed pulmonary TB at 7 DR-TB referral hospitals in Indonesia. Bacteriological examinations (acid-fast bacilli, GeneXpert, sputum culture) and drug-susceptibility testing were performed following the guidelines of the National TB Program. Of 447 participants with complete bacteriological examinations, 312 (69.8%) had positive sputum cultures for Mycobacterium tuberculosis. The proportion of MDR and pre-extensively drug-resistant was higher in previously treated compared with newly diagnosed participants (52.5% [73/139] versus 15% [26/173]). Compared with drug-sensitive case, drug-resistant TB was associated with cavities. Given the difference between rates of DR in TB referral hospitals from our study compared with the WHO survey in 2019 that showed 17.7% and 3.3% DR among previously treated and newly diagnosed participants globally, further characterization of Indonesia's TB epidemiology in the general population is needed. Strategies, including public policies to optimize case finding, strengthen capacity for resistance testing, and prevent loss to follow-up will be critical to reduce the burden of TB in Indonesia.

Correction: An observational prospective cohort study of the epidemiology of hospitalized patients with acute febrile illness in Indonesia.

[This corrects the article DOI: 10.1371/journal.pntd.0007927.].

Performance of Xpert MTB/RIF and sputum microscopy compared to sputum culture for diagnosis of tuberculosis in seven hospitals in Indonesia.

Introduction: Tuberculosis (TB) is a major public health concern in Indonesia, where the incidence was 301 cases per 100,000 inhabitants in 2020 and the prevalence of multi-drug resistant (MDR) TB is increasing. Diagnostic testing approaches vary across Indonesia due to resource limitations. Acid-fast bacilli (AFB) smear is widely used, though Xpert MTB/RIF has been the preferred assay for detecting TB and rifampicin resistance since 2012 due to higher sensitivity and ability to rapidly identify rifampicin resistance. However, <1,000 Xpert instruments were available in Indonesia as of 2020 and the Xpert supply chain has suffered interruptions. Methods: We compared the performance of Xpert MTB/RIF and AFB smear to facilitate optimization of TB case identification. We analyzed baseline data from a cohort study of adults with pulmonary TB conducted at seven hospitals across Indonesia. We evaluated sensitivity and specificity of AFB smear and Xpert MTB/RIF using Mycobacterium tuberculosis (Mtb) culture as the gold standard, factors associated with assay results, and consistency of Xpert MTB/RIF with drug susceptibility test (DST) in detecting rifampicin resistance. Results: Sensitivity of AFB smear was significantly lower than Xpert MTB/RIF (86.2 vs. 97.4%, p-value <0.001), but specificity was significantly better (86.7 vs. 73.3%, p-value <0.001). Performance varied by hospital. Positivity rate for AFB smear and Mtb culture was higher in subjects with pulmonary cavities and in morning sputum samples. Consistency of Xpert MTB/RIF with DST was lower in those with rifampicin- sensitive TB by DST. Discussion: Additional evaluation using sputa from primary and secondary Indonesian health centers will increase the generalizability of the assessment of AFB smear and Xpert MTB/RIF performance, and better inform health policy. Clinical trial registration: [https://clinicaltrials.gov/], identifier [NCT027 58236].

Prevalence of HIV Infection and Resistance Mutations in Patients Hospitalized for Febrile Illness in Indonesia.

HIV prevalence in Indonesia is increasing, and only 64% of infected individuals know their status. In a prospective cohort of 1,453 hospitalized patients with unexplained fever, 46 (3.2%) had HIV, including 15 (1.1%) patients without a prior HIV diagnosis. Among 31 subjects previously known to have HIV, 21 (68%) had been receiving combination antiretroviral therapy (cART) at the time of enrollment. Of 39 HIV cases with HIV RNA levels ≥ 100 copies/mL, sequencing for genotype analysis and resistance testing was successful in 30 (77%) subjects. The most common HIV subtypes were AE (90%) and B (10%). Five (16.7%) subjects had resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors, and all of them were on cART. No evidence of transmitted drug resistance was found in newly diagnosed individuals. Hospital-based screening may be an efficient method to expand HIV testing and identify a significant number of new cases. Access to care, close monitoring, expansion of anti-retroviral options, and ensuring availability of CD4 determinations, viral load testing, and genotyping are crucial to control of the epidemic in Indonesia.

Comparison of clinical characteristics of Zika and dengue symptomatic infections and other acute illnesses of unidentified origin in Mexico.

BACKGROUND: Our purpose was to provide a detailed clinical description, of symptoms and laboratory abnormalities, and temporality in patients with confirmed Zika and dengue infections, and other acute illnesses of unidentified origin (AIUO). METHODS/ PRINCIPAL FINDINGS: This was a two-year, multicenter, observational, prospective, cohort study. We collected data from patients meeting the Pan American Health Organization's modified case-definition criteria for probable Zika infection. We identified Zika, dengue chikungunya by RT-PCR in serum and urine. We compared characteristics between patients with confirmed Zika and dengue infections, Zika and AIUO, and Dengue and AIUO at baseline, Days 3,7,28 and 180 of follow-up. Most episodes (67%) consistent with the PAHO definition of probable Zika could not be confirmed as due to any flavivirus and classified as Acute Illnesses of Unidentified Origin (AIUO). Infections by Zika and dengue accounted for 8.4% and 16% of episodes. Dengue patients presented with fever, generalized non-macular rash, arthralgia, and petechiae more frequently than patients with Zika during the first 10 days of symptoms. Dengue patients presented with more laboratory abnormalities (lower neutrophils, lymphocytosis, thrombocytopenia and abnormal liver function tests), with thrombocytopenia lasting for 28 days. Zika patients had conjunctivitis, photophobia and localized macular rash more frequently than others. Few differences persisted longer than 10 days after symptoms initiation: conjunctivitis in Zika infections, and self-reported rash and petechia in dengue infections. CONCLUSIONS: Our study helps characterize the variety and duration of clinical features in patients with Zika, dengue and AIUO. The lack of diagnosis in most patients points to need for better diagnostics to assist clinicians in making specific etiologic diagnoses.

Etiologies of severe acute respiratory infection (SARI) and misdiagnosis of influenza in Indonesia, 2013-2016.

BACKGROUND: Severe acute respiratory infection (SARI) accounts for a large burden of illness in Indonesia. However, epidemiology of SARI in tertiary hospitals in Indonesia is unknown. This study sought to assess the burden, clinical characteristics, and etiologies of SARI and concordance of clinical diagnosis with confirmed etiology. METHODS: Data and samples were collected from subjects presenting with SARI as part of the acute febrile Illness requiring hospitalization study (AFIRE). In tertiary hospitals, clinical diagnosis was ascertained from chart review. Samples were analyzed to determine the "true" etiology of SARI at hospitals and Indonesia Research Partnership on Infectious Diseases (INA-RESPOND) laboratory. Distribution and characteristics of SARI by true etiology and accuracy of clinical diagnosis were assessed. RESULTS: Four hundred and twenty of 1464 AFIRE subjects presented with SARI; etiology was identified in 242 (57.6%), including 121 (28.8%) viruses and bacteria associated with systemic infections, 70 (16.7%) respiratory bacteria and viruses other than influenza virus, and 51 (12.1%) influenza virus cases. None of these influenza patients were accurately diagnosed as having influenza during hospitalization. CONCLUSIONS: Influenza was misdiagnosed among all patients presenting with SARI to Indonesian tertiary hospitals in the AFIRE study. Diagnostic approaches and empiric management should be guided by known epidemiology. Public health strategies to address the high burden of influenza should include broad implementation of SARI screening, vaccination programs, clinician education and awareness campaigns, improved diagnostic capacity, and support for effective point-of-care tests.

An observational prospective cohort study of the epidemiology of hospitalized patients with acute febrile illness in Indonesia.

BACKGROUND: The epidemiology of acute febrile illness, a common cause of hospitalization in Indonesia, has not been systematically studied. METHODOLOGY/PRINCIPAL FINDINGS: This prospective observational study enrolled febrile patients (temperature ≥38°C) aged ≥1 year from July 2013 until June 2016 at eight government referral teaching hospitals in seven provincial capitals in Indonesia. Patients were managed according to the hospital standard-of-care (SOC), and blood samples were drawn for molecular and serological assays. Clinical data, laboratory results, and specimens for additional tests were collected at enrollment, days 14-28, and at three months. Regular follow-up visits were then scheduled for every three months either until symptoms resolved or until one year. In total, this study included 1,486 adult and pediatric patients presenting with multi-organ (768, 51.7%), gastrointestinal (497, 33.0%), respiratory (114, 7.7%), constitutional (62, 4.2%), skin and soft-tissue (24, 1.6%), central nervous system (17, 1.1%), or genitourinary (4, 0.3%) manifestations. Microbiological diagnoses were found in 1,003/1,486 (67.5%) participants, of which 351/1,003 (35.0%) were not diagnosed during hospitalization using SOC diagnostic tests. Missed diagnoses included all cases caused by Rickettsia spp., chikungunya, influenza, and Seoul virus. The most common etiologic agents identified were dengue virus (467, 46.6%), Salmonella spp. (103, 10.3%), and Rickettsia spp. (103, 10.3%). The overall mortality was 89 (5.9%). CONCLUSIONS/SIGNIFICANCE: Febrile illness in Indonesia has various microbiologic etiologies and substantial overall mortality. Diagnostic limitations and lack of epidemiologic data resulted in potentially treatable, and at times fatal, diseases being missed.

Association of Mycobacterium africanum Infection with Slower Disease Progression Compared with Mycobacterium tuberculosis in Malian Patients with Tuberculosis.

Mycobacterium africanum (MAF) is known to endemically cause up to 40-50% of all pulmonary TB in West Africa. The aim of this study was to compare MAF with Mycobacterium tuberculosis (MTB) with regard to time from symptom onset to TB diagnosis, and clinical and radiological characteristics. A cross-sectional study was conducted in Bamako, Mali, between August 2014 and July 2016. Seventy-seven newly diagnosed pulmonary TB patients who were naive to treatment were enrolled at Mali's University Clinical Research Center. Sputum cultures were performed to confirm the diagnosis and spoligotyping to identify the mycobacterial strain. Univariate and multivariate analyses were used to identify factors associated with disease progression. Overall, the frequency of female patients was 25% in MAF infection and only 10.0% in MTB infection (OR = 2.9), and MAF was more represented in patients aged ≥ 30 years (57.1% versus 36.7% [OR = 2.3]). More MAF- than MTB-infected patients had a history of a prior TB contact (32.1% versus 14.3% [OR = 2.8]). The mean duration between cough onset and TB diagnosis was 111 days (∼3.7 months) for MAF and 72 days (∼2.4 months) for MTB (P = 0.007). In a multivariate regression, weight loss (body mass index [BMI] < 18.5 kg/m2) and cough duration (> 4 months) were strongly associated with MAF infection (OR = 5.20 [1.49-18.26], P = 0.010, and 4.74 [1.2-18.58], P = 0.02), respectively. Our data show that MAF infection was significantly associated with lower BMI and a longer time between symptom onset and TB diagnosis than MTB. This supports the concept that MAF infection may have slower disease progression and less severe cough symptoms than MTB.

Clinical risk factors associated with multidrug-resistant tuberculosis (MDR-TB) in Mali.

BACKGROUND: MDR-TB is a major threat to global TB control. In 2015, 580,000 were treated for MDR-TB worldwide. The worldwide roll-out of GeneXpert MTB/RIF® has improved diagnosis of MDR-TB; however, in many countries laboratories are unable to assess drug resistance and clinical predictors of MDR-TB could help target suspected patients. In this study, we aimed to determine the clinical factors associated with MDR-TB in Bamako, Mali. METHODS: We performed a cross-sectional study of 214 patients with presumed MDR-TB admitted to University of Bamako Teaching Hospital, Point-G between 2007 and 2016. We calculated crude and adjusted odds ratios for MDR-TB disease diagnosis using SPSS. RESULTS: We found that age ≤40years (OR=2.56. 95% CI: 1.44-4.55), two courses of prior TB treatment (OR=3.25, 95% CI: 1.44-7.30), TB treatment failure (OR=3.82, 95% CI 1.82-7.79), sputum microscopy with 3+ bacilli load (OR=1.98, 95% CI: 1.13-3.48) and a history of contact with a TB patient (OR=2.48, 95% CI: 1.11-5.50) were significantly associated with confirmation of MDR-TB disease. HIV was not a risk factor for MDR-TB (aOR=0.88, 95% CI: 0.34-1.94). CONCLUSION: We identified several risk factors that could be used to identify MDR-TB suspects and prioritize them for laboratory confirmation. Prospective studies are needed to understand factors associated with TB incidence and clinical outcomes of TB treatment and disease.

Diagnostic Performances of Three Rapid Diagnostic Tests for Detecting HIV Infections in Mali.

Diagnosis of HIV infections in resource-limited countries like Mali is based on Rapid Diagnostic Tests (RDTs). The RDTs are diagnostic assays designed for use at the Point-Of-Care (POC), which is quick, cost-effective and easy to perform. However, in these countries, the tests are commonly used without any initial evaluation or monitoring of their performance despite high levels of HIV strain diversity and rapid evolution of the virus. In this study, the reliability and accuracy of HIV RDTs (Determine™, Multispot™, SD Bioline™) used in Mali, where HIV-1 and HIV-2 co-exist, were evaluated from August 2004 to November 2017. A total of 1303 samples from new HIV-suspect patients in Bamako were tested for HIV-1 and HIV-2 using the RDT Determine™, followed by ELISA and Western Blot (WB). The Determine™ test showed a robust diagnostic sensitivity of 98.7% [CI 95: 97.59-99.37] and a diagnostic specificity of 99.2% [CI 95: 98.22-99.67]. The Multispot™ assay showed a diagnostic sensitivity of 98.77% [CI 95: 97.59-99.37] and a diagnostic specificity of 99.2% [CI 95: 98.22-99.67]. The diagnostic sensitivity and specificity of SD Bioline™ HIV-1/2 were 100% [CI 95:72.25-100] and 88.89% [CI 95: 56.50- 98.71], respectively. These data indicate excellent performance for HIV RDTs in Mali and we recommend the use of Determine™ HIV-1/2 for HIV screening and Multispot™ for discriminating HIV-2 from HIV-1 infections.

Differential HLA allele frequency in Mycobacterium africanum vs Mycobacterium tuberculosis in Mali.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC), however, the distribution and frequency of MTBC lineages and sublineages vary in different parts of the globe. Mycobacterium africanum, a member of MTBC is responsible for a large percentage of TB cases in West Africa, however, it is rarely identified outside of this part of the World. Whether or not differential HLA polymorphism (an important host factor) is contributing to the geographic restriction of M. africanum to West Africa is unknown. Here, we conducted a cohort study in Mali of newly diagnosed individuals with active pulmonary TB and normal healthy controls. The MTBC isolates were spoligotyped to determine the TB study groups (M. tuberculosis sensu stricto LAM10 and M. africanum), and HLA typing was performed on peripheral blood. Unlike previous reports on other populations, we found that HLA class-I alleles were significantly associated with active TB disease in this population. HLA-B alleles (B*07:02, B*08:01, B*14:02, B*15:03, B*15:10, B*18:01, B*42:01, B*42:02, B*51:01 and B*81:01) were significantly associated with M. africanum (40%-45%) and M. tuberculosis (75%) compared with healthy controls. Many HLA-A alleles (A*02:05, A*34:02, A*66:01 and A*68:02) were also associated with both TB groups (65%-70%). However, many class II HLA-DR variants were found to be associated with M. tuberculosis but not M. africanum with the exception of the DRB1*03:01, which was associated with both groups. The differential HLA distribution observed in this study might be at least partially responsible for the geographical restriction of M. africanum infections to West Africa.

The most frequent Mycobacterium tuberculosis complex families in mali (2006-2016) based on spoligotyping.

BACKGROUND: To identify strains of Mycobacterium tuberculosis complex (MTBc) circulating in Bamako region during the past 10 years. METHODS: From 2006 to 2016, we conducted a cross-sectional study to identify with spoligotyping, clinical isolates from tuberculosis (TB)-infected patients at different stages of their treatments in Bamako, Mali. RESULTS: Among the 904 suspected TB patients included in the study and thereafter tested in our BSL-3 laboratory, 492 (54.4%) had MTBc and therefore underwent spoligotyping. Overall, three subspecies, i.e., MTB T1 (31.9%) and MTB LAM10 (15.3%) from lineage 4 and M. africanum 2 (16.8%) from lineage 6 were the leading causes of TB in Bamako region during the past 10 years. Other spoligotypes such as MTB T3, MTB Haarlem 2, MTB EAI3, and MTB family 33 were also commonly seen from 2010 to 2016. CONCLUSION: This study showed a high genetic diversity of strains isolated in Bamako region and highlights that M. tuberculosis T1 strain was the most prevalent. Furthermore, the data indicate an increasing proportion of primary drug resistance overtime in Bamako.

Extensively drug resistant tuberculosis in Mali: a case report.

BACKGROUND: Drug resistant tuberculosis presents a major public health challenge. CASE PRESENTATION: We present here the first two patients diagnosed with extensively drug resistant tuberculosis in Bamako, Mali. Genotypic findings suggest possible nosocomial transmission from the first patient to the second one, resulting in superinfection of the second patient. After being diagnosed with extensively drug resistant tuberculosis in August 2016, the patients only started receiving appropriate treatment 10 months later. CONCLUSION: The identification of these patients highlights the need for improved diagnostic and treatment algorithms for better surveillance and management of drug resistance in Mali. In the interest of these as well as future patients suffering from resistant tuberculosis, all steps recommended for programmatic management of drug resistant tuberculosis must be urgently prioritized in order to strengthen the multidrug resistant tuberculosis program.

Laboratory Response to 2014 Ebola Virus Outbreak in Mali.

Aware of the rapid spread of Ebola virus (EBOV) during the current West African epidemic, Mali took several proactive steps to rapidly identify cases within its borders. Under the Mali International Center for Excellence in Research program, a collaboration between the National Institute of Allergy and Infectious Diseases and the Malian Ministry of Higher Education and Scientific Research established a national EBOV diagnostic site at the University of Sciences, Techniques and Technologies of Bamako in the SEREFO Laboratory. Two separate introductions of EBOV occurred in Mali from neighboring Guinea, but both chains of transmission were quickly halted, and Mali was declared "Ebola free" on 18 January 2015 and has remained so since. The SEREFO Laboratory was instrumental in the success of Mali's Ebola response by providing timely and accurate diagnostics. As of today, the SEREFO Laboratory has tested 103 samples from 88 suspected cases, 10 of which were EBOV positive, since the Ebola diagnostics unit started in April 2014. The establishment of Ebola diagnostics in the SEREFO Laboratory, safety precautions, and diagnostics are described.

Stool microbiome reveals diverse bacterial ureases as confounders of oral urea breath testing for Helicobacter pylori and Mycobacterium tuberculosis in Bamako, Mali.

Detection of bacterial urease activity has been utilized successfully to diagnose Helicobacter pylori (H. pylori). While Mycobacterium tuberculosis (M. tuberculosis) also possesses an active urease, it is unknown whether detection of mycobacterial urease activity by oral urease breath test (UBT) can be exploited as a rapid point of care biomarker for tuberculosis (TB) in humans. We enrolled 34 individuals newly diagnosed with pulmonary TB and 46 healthy subjects in Bamako, Mali and performed oral UBT, mycobacterial sputum culture and H. pylori testing. Oral UBT had a sensitivity and specificity (95% CI) of 70% (46-88%) and 11% (3-26%), respectively, to diagnose culture-confirmed M. tuberculosis disease among patients without H. pylori, and 100% sensitivity (69-100%) and 11% specificity (3-26%) to diagnose H. pylori among patients without pulmonary TB. Stool microbiome analysis of controls without TB or H. pylori but with positive oral UBT detected high levels of non-H. pylori urease producing organisms, which likely explains the low specificity of oral UBT in this setting and in other reports of oral UBT studies in Africa.

Case report: Weil's disease with multiple organ failure in a child living in dengue endemic area.

BACKGROUND: There were few reports in the literature of Weil's disease with multiple organ failures, especially in children living in dengue endemic areas. CASE PRESENTATION: A 12-year-old child was admitted to Tangerang district hospital with a provisional diagnosis of dengue infection. On the third day of hospitalization, dengue diagnostic tests were negative. As fever still remained and was followed by jaundice, decreasing hemoglobin, increasing bilirubin with abnormal value of liver enzymes; other causes of disease were investigated. Leptospirosis was confirmed by rapid IgM test (SD(®)) for leptospira; and micro-agglutination test which indicated Leptospira serogroup bataviae infection. The patient developed Weil's disease during the course of illness. Renal function was back to normal on the 21st day of hospitalization, while hemoglobin and bilirubin returned to normal three weeks after discharged. CONCLUSIONS: Our report highlights the importance of considering leptospirosis as a differential diagnosis in children with acute febrile illness; even when the signs and symptoms for the more common diagnoses such as dengue or typhoid fever were pathognomonic. A normal leukocyte count with neutrophilia and negative dengue NS1, dengue IgM, and Salmonella typhi IgM on admission should raise suspicion of leptospirosis, and prompt diagnostic assays for leptospirosis should be conducted.

Molecular identification of Mycobacterium bovis from cattle and human host in Mali: expanded genetic diversity.

BACKGROUND: Bovine tuberculosis (BTB) is a contagious, debilitating human and animal disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. The study objective were to estimate the frequency of BTB, examine genetic diversity of the M. bovis population in cattle from five regions in Mali and to determine whether M. bovis is involved in active tuberculosis (TB) in humans. Samples from suspected lesions on cattle at the slaughterhouses were collected. Mycobacterial smear, culture confirmation, and spoligotyping were used for diagnosis and species identification. Mycobacterium DNA from TB patients was spoligotyped to identify M. bovis. RESULTS: In total, 675 cattle have been examined for lesions in the five regions of Mali. Out of 675 cattle, 79 specimens presented lesions and then examined for the presence of M. bovis. Thus, 19 (24.1 %) were identified as M. bovis; eight (10.1 %) were non-tuberculous Mycobacterium (NTM). Nineteen spoligotype patterns were identified among 79 samples with five novel patterns. One case of M. bovis (spoligotype pattern SB0300) was identified among 67 TB patients. CONCLUSION: This study estimates a relatively true proportion of BTB in the regions of Mali and reveals new spoligotype patterns.

Nontuberculous Mycobacteria Isolated from Tuberculosis Suspects in Ibadan, Nigeria.

In Nigeria, one of the highest tuberculosis (TB) burdened nations, sputum smear microscopy is routinely employed for TB diagnosis at Directly Observed Treatment Short-Course (DOTS) Centers. This diagnostic algorithm does not differentiate Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM). Between December 2008 and January 2009, consecutive patients diagnosed with TB were screened for inclusion at 10 DOTS centers in Ibadan, Nigeria. To verify Mycobacterium species in patients diagnosed, we cultured and identified mycobacterial isolates using PCR, line probe assay, and spoligotyping techniques. From 48 patients screened, 23 met the inclusion criteria for the study. All the 23 study patients had a positive culture. Overall, we identified 11/23 patients (48%) with MTC only, 9/23 (39%) with NTM only, and 3/23 (13%) with evidence of both MTC and NTM. Strains of MTC identified were Latin American Mediterranean (LAM) genotype (n = 12), M. africanum (n = 1), and the genotype family T (n = 1). Four M. avium-intracellulare-M. scrofulaceum complexes, one M. chelonae complex, one M. abscessus, and one M. intracellulare were identified. Our findings underscore the need to incorporate molecular techniques for more precise diagnosis of TB at DOTS centers to improve clinical outcomes and safe guard public health, particularly in TB endemic countries.

Serological diagnosis of pulmonary Mycobacterium tuberculosis infection by LIPS using a multiple antigen mixture.

BACKGROUND: There is an urgent need for a simple and accurate test for the diagnosis of human Mycobacterium tuberculosis, the infectious agent causing tuberculosis (TB). Here we describe a serological test based on light emitting recombinant proteins for the diagnosis of pulmonary Mycobacterium tuberculosis infection. METHODS: Luciferase Immunoprecipitation Systems (LIPS), a fluid-phase immunoassay, was used to examine antibody responses against a panel of 24 different M. tuberculosis proteins. Three different strategies were used for generating the constructs expressing the recombinant fusion M. tuberculosis proteins with luciferase: synthetic gene synthesis, Gateway recombination cloning, and custom PCR synthesis. A pilot cohort of African pulmonary TB patients was used for initial antibody screening and confirmatory studies with selected antigens were performed with a cohort from Thailand and healthy US blood donors. In addition to testing M. tuberculosis antigens separately, a mixture that tested seven antigens simultaneously was evaluated for diagnostic performance. RESULTS: LIPS testing of a pilot set of serum samples from African pulmonary TB patients identified a potential subset of diagnostically useful M. tuberculosis antigens. Evaluation of a second independent cohort from Thailand validated highly significant antibody responses against seven antigens (PstS1, Rv0831c, FbpA, EspB, bfrB, HspX and ssb), which often showed robust antibody levels up to 50- to 1000-fold higher than local community controls. Marked heterogeneity of antibody responses was observed in the patients and the combined results demonstrated 73.5% sensitivity and 100% specificity for detection of pulmonary TB. A LIPS test simultaneously employing the seven M. tuberculosis antigen as a mixture matched the combined diagnostic performance of the separate tests, but showed an even higher diagnostic sensitivity (90%) when a cut-off based on healthy US blood donors was used. CONCLUSION: A LIPS immunoassay employing multiple M. tuberculosis antigens shows promise for the rapid and quantitative serological detection of pulmonary TB.