Dynamics of Cough Frequency in Adults Undergoing Treatment for Pulmonary Tuberculosis.

Background: Cough is the major determinant of tuberculosis transmission. Despite this, there is a paucity of information regarding characteristics of cough frequency throughout the day and in response to tuberculosis therapy. Here we evaluate the circadian cycle of cough, cough frequency risk factors, and the impact of appropriate treatment on cough and bacillary load. Methods: We prospectively evaluated human immunodeficiency virus-negative adults (n = 64) with a new diagnosis of culture-proven, drug-susceptible pulmonary tuberculosis immediately prior to treatment and repeatedly until treatment day 62. At each time point, participant cough was recorded (n = 670) and analyzed using the Cayetano Cough Monitor. Consecutive coughs at least 2 seconds apart were counted as separate cough episodes. Sputum samples (n = 426) were tested with microscopic-observation drug susceptibility broth culture, and in culture-positive samples (n = 252), the time to culture positivity was used to estimate bacillary load. Results: The highest cough frequency occurred from 1 pm to 2 pm, and the lowest from 1 am to 2 am (2.4 vs 1.1 cough episodes/hour, respectively). Cough frequency was higher among participants who had higher sputum bacillary load (P < .01). Pretreatment median cough episodes/hour was 2.3 (interquartile range [IQR], 1.2-4.1), which at 14 treatment days decreased to 0.48 (IQR, 0.0-1.4) and at the end of the study decreased to 0.18 (IQR, 0.0-0.59) (both reductions P < .001). By 14 treatment days, the probability of culture conversion was 29% (95% confidence interval, 19%-41%). Conclusions: Coughs were most frequent during daytime. Two weeks of appropriate treatment significantly reduced cough frequency and resulted in one-third of participants achieving culture conversion. Thus, treatment by 2 weeks considerably diminishes, but does not eliminate, the potential for airborne tuberculosis transmission.

Rationing tests for drug-resistant tuberculosis - who are we prepared to miss?

BACKGROUND: Early identification of patients with drug-resistant tuberculosis (DR-TB) increases the likelihood of treatment success and interrupts transmission. Resource-constrained settings use risk profiling to ration the use of drug susceptibility testing (DST). Nevertheless, no studies have yet quantified how many patients with DR-TB this strategy will miss. METHODS: A total of 1,545 subjects, who presented to Lima health centres with possible TB symptoms, completed a clinic-epidemiological questionnaire and provided sputum samples for TB culture and DST. The proportion of drug resistance in this population was calculated and the data was analysed to demonstrate the effect of rationing tests to patients with multidrug-resistant TB (MDR-TB) risk factors on the number of tests needed and corresponding proportion of missed patients with DR-TB. RESULTS: Overall, 147/1,545 (9.5%) subjects had culture-positive TB, of which 32 (21.8%) had DR-TB (MDR, 13.6%; isoniazid mono-resistant, 7.5%; rifampicin mono-resistant, 0.7%). A total of 553 subjects (35.8%) reported one or more MDR-TB risk factors; of these, 506 (91.5%; 95% CI, 88.9-93.7%) did not have TB, 32/553 (5.8%; 95% CI, 3.4-8.1%) had drug-susceptible TB, and only 15/553 (2.7%; 95% CI, 1.5-4.4%) had DR-TB. Rationing DST to those with an MDR-TB risk factor would have missed more than half of the DR-TB population (17/32, 53.2%; 95% CI, 34.7-70.9). CONCLUSIONS: Rationing DST based on known MDR-TB risk factors misses an unacceptable proportion of patients with drug-resistance in settings with ongoing DR-TB transmission. Investment in diagnostic services to allow universal DST for people with presumptive TB should be a high priority.

Microscopic Observation Drug Susceptibility Assay for Rapid Diagnosis of Lymph Node Tuberculosis and Detection of Drug Resistance.

In this study, 132 patients with lymphadenopathy were investigated. Fifty-two (39.4%) were diagnosed with tuberculosis (TB). The microscopic observation drug susceptibility (MODS) assay provided rapid (13 days), accurate diagnosis (sensitivity, 65.4%) and reliable drug susceptibility testing (DST). Despite its lower sensitivity than that of other methods, its faster results and simultaneous DST are advantageous in resource-poor settings, supporting the incorporation of MODS into diagnostic algorithms for extrapulmonary TB.

Evaluation of bleach-sedimentation for sterilising and concentrating Mycobacterium tuberculosis in sputum specimens.

BACKGROUND: Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy. METHODS: This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method. RESULTS: M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative. CONCLUSIONS: Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies.

STAT3, p38 MAPK, and NF-kappaB drive unopposed monocyte-dependent fibroblast MMP-1 secretion in tuberculosis.

Tissue destruction characterizes infection with Mycobacterium tuberculosis (Mtb). Type I collagen provides the lung's tensile strength, is extremely resistant to degradation, but is cleaved by matrix metalloproteinase (MMP)-1. Fibroblasts potentially secrete quantitatively more MMP-1 than other lung cells. We investigated mechanisms regulating Mtb-induced collagenolytic activity in fibroblasts in vitro and in patients. Lung fibroblasts were stimulated with conditioned media from Mtb-infected monocytes (CoMTb). CoMTb induced sustained increased MMP-1 (74 versus 16 ng/ml) and decreased tissue inhibitor of metalloproteinase (TIMP)-1 (8.6 versus 22.3 ng/ml) protein secretion. CoMTb induced a 2.7-fold increase in MMP-1 promoter activation and a 2.5-fold reduction in TIMP-1 promoter activation at 24 hours (P = 0.01). Consistent with this, TIMP-1 did not co-localize with fibroblasts in patient granulomas. MMP-1 up-regulation and TIMP-1 down-regulation were p38 (but not extracellular signal-regulated kinase or c-Jun N-terminal kinase) mitogen-activated protein kinase-dependent. STAT3 phosphorylation was detected in fibroblasts in vitro and in tuberculous granulomas. STAT3 inhibition reduced fibroblast MMP-1 secretion by 60% (P = 0.046). Deletion of the MMP-1 promoter NF-κB-binding site abrogated promoter induction in response to CoMTb. TNF-α, IL-1ß, or Oncostatin M inhibition in CoMTb decreased MMP-1 secretion by 65, 63, and 25%, respectively. This cytokine cocktail activated the same signaling pathways in fibroblasts and induced MMP-1 secretion similar to that induced by CoMTb. This study demonstrates in a cellular model and in patients with tuberculosis that in addition to p38 and NF-κB, STAT3 has a key role in driving fibroblast-dependent unopposed MMP-1 production that may be key in tissue destruction in patients.

Prolonged infectiousness of tuberculosis patients in a directly observed therapy short-course program with standardized therapy.

BACKGROUND: Effective tuberculosis control is compromised by a lack of clarity about the timeframe of viable Mycobacterium tuberculosis shedding after treatment initiation under programmatic conditions. This study quantifies time to conversion from smear and culture positivity to negativity in unselected tuberculosis patients receiving standardized therapy in a directly observed therapy short-course (DOTS) program. METHODS: Longitudinal cohort study following up 93 adults initiating tuberculosis therapy in Lima, Peru. Baseline culture and drug susceptibility tests (DSTs) were performed using the MBBacT, proportion, and microscopic observation drug susceptibility (MODS) methods. Smear microscopy and MODS liquid culture were performed at baseline and weekly for 4 weeks then every other week for 26 weeks. RESULTS: Median conversion time from culture positivity to culture negativity of 38.5 days was unaffected by baseline smear status. Patients with fully susceptible tuberculosis had a median time to culture conversion of 37 days; 10% remained culture positive at day 60. Delayed culture conversion was associated with multidrug resistance, regardless of DST method used; non-multidrug resistance as defined by the proportion method and MODS (but not MBBacT) was also associated with delay. Persistent day 60 smear positivity yielded positive and negative predictive values of 67% and 92%, respectively, for detecting multidrug resistance. CONCLUSIONS: Smear and culture conversion in treated tuberculosis patients takes longer than is conventionally believed, even with fully susceptible disease, and must be accounted for in tuberculosis treatment and prevention programs. Persistent day 60 smear positivity is a poor predictor of multidrug resistance. The industrialized-world convention of universal baseline DST for tuberculosis patients should become the standard of care in multidrug resistance-affected resource-limited settings.

Evaluation of molecular tools for detection and drug susceptibility testing of Mycobacterium tuberculosis in stool specimens from patients with pulmonary tuberculosis.

Pulmonary tuberculosis diagnosis is difficult when patients cannot produce sputum. Most sputum is swallowed, and tuberculosis DNA can survive intestinal transit. We therefore evaluated molecular testing of stool specimens for detecting tuberculosis originating from the lungs. Paired stool and sputum samples (n=159) were collected from 89 patients with pulmonary tuberculosis. Control stool samples (n=47) were collected from patients without tuberculosis symptoms. Two techniques for DNA extraction from stool samples were compared, and the diagnostic accuracy of the PCR in stool was compared with the accuracy of sputum testing by PCR, microscopy, and culture. A heminested IS6110-PCR was used for tuberculosis detection, and IS6110-PCR-positive stool samples then underwent rifampin sensitivity testing by universal heteroduplex generator PCR (heteroduplex-PCR) assay. For newly diagnosed pulmonary tuberculosis patients, stool IS6110-PCR had 86% sensitivity and 100% specificity compared with results obtained by sputum culture, and stool PCR had similar sensitivities for HIV-positive and HIV-negative patients (P=0.3). DNA extraction with commercially available spin columns yielded greater stool PCR sensitivity than DNA extraction with the in-house Chelex technique (P=0.007). Stool heteroduplex-PCR had 98% agreement with the sputum culture determinations of rifampin resistance and multidrug resistance. Tuberculosis detection and drug susceptibility testing by stool PCR took 1 to 2 days compared with an average of 9 weeks to obain those results by traditional culture-based testing. Stool PCR was more sensitive than sputum microscopy and remained positive for most patients for more than 1 week of treatment. In conclusion, stool PCR is a sensitive, specific, and rapid technique for the diagnosis and drug susceptibility testing of pulmonary tuberculosis and should be considered when sputum samples are unavailable.

Microscopic-observation drug-susceptibility assay for the diagnosis of TB.

BACKGROUND: New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth. METHODS: In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on Löwenstein-Jensen medium with the proportion method. RESULTS: Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for Löwenstein-Jensen culture (P<0.001); the median time to culture positivity was 7 days, 13 days, and 26 days, respectively (P<0.001), and the median time to the results of susceptibility tests was 7 days, 22 days, and 68 days, respectively. The incremental benefit of a second MODS culture was minimal, particularly in patients at high risk for tuberculosis or multidrug-resistant tuberculosis. Agreement between MODS and the reference standard for susceptibility was 100% for rifampin, 97% for isoniazid, 99% for rifampin and isoniazid (combined results for multidrug resistance), 95% for ethambutol, and 92% for streptomycin (kappa values, 1.0, 0.89, 0.93, 0.71, and 0.72, respectively). CONCLUSIONS: A single MODS culture of a sputum sample offers more rapid and sensitive detection of tuberculosis and multidrug-resistant tuberculosis than the existing gold-standard methods used.

Sputum PCR-single-strand conformational polymorphism test for same-day detection of pyrazinamide resistance in tuberculosis patients.

Pyrazinamide is a first-line drug for treating tuberculosis, but pyrazinamide resistance testing is usually too slow to guide initial therapy, so some patients receive inappropriate therapy. We therefore aimed to optimize and evaluate a rapid molecular test for tuberculosis drug resistance to pyrazinamide. Tuberculosis PCR-single-strand conformational polymorphism (PCR-SSCP) was optimized to test for mutations causing pyrazinamide resistance directly from sputum samples and Mycobacterium tuberculosis isolates. The reliability of PCR-SSCP tests for sputum samples (n = 65) and Mycobacterium tuberculosis isolates (n = 185) from 147 patients was compared with four tests for pyrazinamide resistance: Bactec-460 automated culture, the Wayne biochemical test, DNA sequencing for pncA mutations, and traditional microbiological broth culture. PCR-SSCP provided interpretable results for 96% (46/48) of microscopy-positive sputum samples, 76% (13/17) of microscopy-negative sputum samples, and 100% of Mycobacterium tuberculosis isolates. There was 100% agreement between PCR-SSCP results from sputum samples and Mycobacterium tuberculosis isolates and 100% concordance between 50 blinded PCR-SSCP rereadings by three observers. PCR-SSCP agreement with the four other tests for pyrazinamide resistance varied from 89 to 97%. This was similar to how frequently the four other tests for pyrazinamide resistance agreed with each other: 90 to 94% for Bactec-460, 90 to 95% for Wayne, 92 to 95% for sequencing, and 91 to 95% for broth culture. PCR-SSCP took less than 24 hours and cost approximately $3 to $6, in contrast with the other assays, which took 3 to 14 weeks and cost $7 to $47. In conclusion, PCR-SSCP is a relatively reliable, rapid, and inexpensive test for pyrazinamide resistance that indicates which patients should receive pyrazinamide from the start of therapy, potentially preventing months of inappropriate treatment.

Improved diagnosis of pleural tuberculosis using the microscopic- observation drug-susceptibility technique.

Tests for pleural tuberculosis are insensitive and expensive. We compared nonproprietary microscopic-observation drug-susceptibility (MODS) culture with Löwenstein-Jensen culture for evaluation of pleural specimens. MODS culture was associated with greatly increased diagnostic sensitivity and shorter time to diagnosis, compared with Löwenstein-Jensen culture (sensitivity of culture of biopsy specimens, 81% vs.51%; time to diagnosis, 11 days vs. 24 days; P < .001). The MODS technique is inexpensive, allows drug-susceptibility testing, and is a considerably improved diagnostic method for pleural tuberculosis.

The infectiousness of tuberculosis patients coinfected with HIV.

BACKGROUND: The current understanding of airborne tuberculosis (TB) transmission is based on classic 1950s studies in which guinea pigs were exposed to air from a tuberculosis ward. Recently we recreated this model in Lima, Perú, and in this paper we report the use of molecular fingerprinting to investigate patient infectiousness in the current era of HIV infection and multidrug-resistant (MDR) TB. METHODS AND FINDINGS: All air from a mechanically ventilated negative-pressure HIV-TB ward was exhausted over guinea pigs housed in an airborne transmission study facility on the roof. Animals had monthly tuberculin skin tests, and positive reactors were removed for autopsy and organ culture for M. tuberculosis. Temporal exposure patterns, drug susceptibility testing, and DNA fingerprinting of patient and animal TB strains defined infectious TB patients. Relative patient infectiousness was calculated using the Wells-Riley model of airborne infection. Over 505 study days there were 118 ward admissions of 97 HIV-positive pulmonary TB patients. Of 292 exposed guinea pigs, 144 had evidence of TB disease; a further 30 were tuberculin skin test positive only. There was marked variability in patient infectiousness; only 8.5% of 118 ward admissions by TB patients were shown by DNA fingerprinting to have caused 98% of the 125 characterised cases of secondary animal TB. 90% of TB transmission occurred from inadequately treated MDR TB patients. Three highly infectious MDR TB patients produced 226, 52, and 40 airborne infectious units (quanta) per hour. CONCLUSIONS: A small number of inadequately treated MDR TB patients coinfected with HIV were responsible for almost all TB transmission, and some patients were highly infectious. This result highlights the importance of rapid TB drug-susceptibility testing to allow prompt initiation of effective treatment, and environmental control measures to reduce ongoing TB transmission in crowded health care settings. TB infection control must be prioritized in order to prevent health care facilities from disseminating the drug-resistant TB that they are attempting to treat.

Inter- and intra-assay reproducibility of microplate Alamar blue assay results for isoniazid, rifampicin, ethambutol, streptomycin, ciprofloxacin, and capreomycin drug susceptibility testing of Mycobacterium tuberculosis.

The intersample and intrasample variability of the results obtained with the microplate Alamar blue assay for the indirect drug susceptibility testing of Mycobacterium tuberculosis was investigated. Between 1.2 and 8.5% of paired MICs differed by more than one twofold dilution, resulting in discordant susceptible-resistant designations at frequencies between 0.6% (rifampin) and 18.9% (ethambutol).

Exhaled Nitric Oxide is Not a Biomarker for Pulmonary Tuberculosis.

To reduce transmission of tuberculosis (TB) in resource-limited countries where TB remains a major cause of mortality, novel diagnostic tools are urgently needed. We evaluated the fractional concentration of exhaled nitric oxide (FeNO) as an easily measured, noninvasive potential biomarker for diagnosis and monitoring of treatment response in participants with pulmonary TB including multidrug resistant-TB in Lima, Peru. In a longitudinal study however, we found no differences in baseline median FeNO levels between 38 TB participants and 93 age-matched controls (13 parts per billion [ppb] [interquartile range (IQR) = 8-26] versus 15 ppb [IQR = 12-24]), and there was no change over 60 days of treatment (15 ppb [IQR = 10-19] at day 60). Taking this and previous evidence together, we conclude FeNO is not of value in either the diagnosis of pulmonary TB or as a marker of treatment response.

The detection of airborne transmission of tuberculosis from HIV-infected patients, using an in vivo air sampling model.

BACKGROUND: Nosocomial transmission of tuberculosis remains an important public health problem. We created an in vivo air sampling model to study airborne transmission of tuberculosis from patients coinfected with human immunodeficiency virus (HIV) and to evaluate environmental control measures. METHODS: An animal facility was built above a mechanically ventilated HIV-tuberculosis ward in Lima, Peru. A mean of 92 guinea pigs were continuously exposed to all ward exhaust air for 16 months. Animals had tuberculin skin tests performed at monthly intervals, and those with positive reactions were removed for autopsy and culture for tuberculosis. RESULTS: Over 505 consecutive days, there were 118 ward admissions by 97 patients with pulmonary tuberculosis, with a median duration of hospitalization of 11 days. All patients were infected with HIV and constituted a heterogeneous group with both new and existing diagnoses of tuberculosis. There was a wide variation in monthly rates of guinea pigs developing positive tuberculin test results (0%-53%). Of 292 animals exposed to ward air, 159 developed positive tuberculin skin test results, of which 129 had laboratory confirmation of tuberculosis. The HIV-positive patients with pulmonary tuberculosis produced a mean of 8.2 infectious quanta per hour, compared with 1.25 for HIV-negative patients with tuberculosis in similar studies from the 1950s. The mean monthly patient infectiousness varied greatly, from production of 0-44 infectious quanta per hour, as did the theoretical risk for a health care worker to acquire tuberculosis by breathing ward air. CONCLUSIONS: HIV-positive patients with tuberculosis varied greatly in their infectiousness, and some were highly infectious. Use of environmental control strategies for nosocomial tuberculosis is therefore a priority, especially in areas with a high prevalence of both tuberculosis and HIV infection.

Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures.

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru.

Sputum induction, bronchoalveolar lavage, or gastric aspiration are often needed to produce adequate diagnostic respiratory samples from people with HIV in whom tuberculosis is suspected. Since these procedures are rarely appropriate in less-developed countries, we compared the performances of a simple string test and the gold-standard sputum induction. 160 HIV-positive adults under investigation for tuberculosis, and 52 asymptomatic HIV-positive control patients underwent the string test followed by sputum induction. The string test detected tuberculosis in 14 patients in whom this disease was suspected; sputum induction detected only eight of them (McNemar's test, p=0.03). These preliminary data suggest that the string test is safe and effective for retrieval of useful clinical specimens for diagnosis of pulmonary tuberculosis, and is at least as sensitive as sputum induction.

Tuberculosis mortality, drug resistance, and infectiousness in patients with and without HIV infection in Peru.

The effects of HIV co-infection and multi-drug resistant tuberculosis (MDRTB) on tuberculosis prognosis are poorly defined. Therefore, we studied infectiousness and mortality of 287 tuberculosis patients treated with standard, directly observed, short-course therapy in the Peruvian community. During 6-17 months of treatment, 49 (18%) of patients died, of whom 48 (98%) had AIDS and 28 (57%) had MDRTB; 17/31 (55%) of MDRTB-patients with AIDS died within 2 months of diagnosis, before traditional susceptibility testing would have identified their MDRTB. Most non-MDRTB became smear- and culture-negative within 6 weeks of therapy, whereas most MDRTB remained sputum-culture-positive until death or treatment completion. HIV-negative patients with non-MDRTB had good outcomes. However, MDRTB was associated with prolonged infectiousness and HIV co-infection with early mortality, indicating a need for greater access to anti-retroviral therapy. Furthermore, early and rapid tuberculosis drug-susceptibility testing and infection control are required so that MDRTB can be appropriately treated early enough to reduce mortality and transmission.

Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting.

One obstacle to wider use of rapid liquid culture-based tuberculosis diagnostics such as the microscopic observation drug susceptibility (MODS) assay is concern about cross-contamination. We investigated the rate of laboratory cross-contamination in MODS, automated MBBacT, and Lowenstein-Jensen (LJ) cultures performed in parallel, through triangulation of microbiologic (reculturing stored samples), molecular (spoligotype/RFLP), and clinical epidemiologic data. At least 1 culture was positive for Mycobacterium tuberculosis for 362 (11%) of 3416 samples; 53 were regarded as potential cross-contamination suspects. Cross-contamination accounted for 17 false-positive cultures from 14 samples representing 0.41% (14/3416) and 0.17% (17/10248) of samples and cultures, respectively. Positive predictive values for MODS, MBBacT (bioMérieux, Durham, NC), and LJ were 99.1%, 98.7%, and 99.7%, and specificity was 99.9% for all 3. Low rates of cross-contamination are achievable in mycobacterial laboratories in resource-poor settings even when a large proportion of samples are infectious and highly sensitive liquid culture-based diagnostics such as MODS are used.

A quantitative adaptation of the Wayne test for pyrazinamide resistance.

BACKGROUND: Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. METHODS: We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 µg/ml and 800 µg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. RESULTS: The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 µg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 µg/ml PZA were more accurate than 800 µg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. CONCLUSIONS: The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection.

Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)-positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58-92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures.