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Decades of social and political unrest have contributed to the urbanization of the population of Peru with large-scale migration from rural Andean and Amazonian communities to overcrowded shantytowns around Lima. We administered a face-to-face survey questionnaire to 116 patients with suspected and proven tuberculosis (TB) in northern Lima to determine the extent to which the use of traditional therapies from indigenous regions persists and the impact of any such use on TB control. Sixty-three percent of participants reported some form of self-treatment prior to presentation to the National Tuberculosis Program; 52% of them used traditional remedies. Symptom duration was longer among self-remedy users than non-users (median = 25 versus 15 days; P = 0.07) and among those exclusively using western remedies rather than traditional remedies (median = 30 versus 15 days; P = 0.01). We thus found no evidence that use of traditional remedies has an appreciable effect on diagnostic delay in Lima.

The larval stage of the pork tapeworm (Taenia solium) infects the human nervous system, causing neurocysticercosis. This disease is one of the main causes of epileptic seizures in many less developed countries and is also increasingly seen in more developed countries because of immigration from endemic areas. Little information is available on the natural evolution of taeniasis or cysticercosis. Available therapeutic measures include steroids, treatments for symptoms, surgery, and, more controversially, antiparasitic drugs to kill brain parasites. Efforts to control and eliminate this disease are underway through antiparasitic treatment of endemic populations, development of pig vaccines, and other measures.

Summary Objectives Tuberculosis is frequent among poor and marginalized people whose limited tuberculosis-related knowledge may impair healthcare access. We characterised tuberculosis-related knowledge and associations with delayed treatment and treatment outcome. Methods Tuberculosis patients (n = 943), people being tested for suspected tuberculosis (n = 2020), and randomly selected healthy controls (n = 476) in 16 periurban shantytowns were interviewed characterizing: socio-demographic factors; tuberculosis risk-factors; and patients' treatment delay. Principle component analysis was used to generate a tuberculosis-related knowledge score. Patients were followed-up for median 7.7 years. Factors associated with tuberculosis treatment delay, treatment outcome and tuberculosis recurrence were assessed using linear, logistic and Cox regression. Results Tuberculosis-related knowledge was poor, especially in older people who had not completed schooling and had never been diagnosed with tuberculosis. Tuberculosis treatment delay was median 60 days and was more delayed for patients who were poorer, older, had more severe tuberculosis and in only unadjusted analysis with incomplete schooling and low tuberculosis-related knowledge (all p ≤ 0.03). Lower than median tuberculosis-related knowledge was associated with tuberculosis recurrence (unadjusted hazard ratio = 2.1, p = 0.008), and this association was independent of co-morbidities, disease severity and demographic factors (multiple regression adjusted hazard ratio = 2.6, p = 0.008). Conclusions Low tuberculosis-related knowledge independently predicted tuberculosis recurrence. Thus health education may improve tuberculosis prognosis.

BackgroundDiagnosing tuberculosis in children is challenging because specimens are difficult to obtain and contain low tuberculosis concentrations, especially with HIV-coinfection. Few studies included well-controls so test specificities are poorly defined. We studied tuberculosis diagnosis in 525 children with and without HIV-infection.Methods and findings'Cases' were children with suspected pulmonary tuberculosis (n = 209 HIV-negative; n = 81 HIV-positive) and asymptomatic 'well-control' children (n = 200 HIV-negative; n = 35 HIV-positive). Specimens (n = 2422) were gastric aspirates, nasopharyngeal aspirates and stools analyzed by a total of 9688 tests. All specimens were tested with an in-house hemi-nested IS6110 PCR that took 0.2) for HIV-positive versus HIV-negative cases. All specimens were also tested with auramine acid-fast microscopy, microscopic-observation drug-susceptibility (MODS) liquid culture, and Lowenstein-Jensen solid culture that took ≤6 weeks and had 100% specificity (all 2112 tests on 704 specimens from 235 well-controls were negative). Microscopy-positivity was rare (0.21%, 5/2422 specimens) and all microscopy-positive specimens were culture-positive. Culture-positivity was less frequent (P≤0.01) in HIV-infection: 1.2% (1/81) HIV-positive cases versus 11% (22/209) HIV-negative cases; caused by 0.42% (2/481) versus 4.7% (58/1235) of their specimens, respectively.ConclusionsIn HIV-positive children with suspected tuberculosis, diagnostic yield was so low that 1458 microscopy and culture tests were done per case confirmed and even in children with culture-proven tuberculosis most tests and specimens were false-negative; whereas PCR was so prone to false-positives that PCR-positivity was as likely in specimens from well-controls as suspected-tuberculosis cases. This demonstrates the importance of control participants in diagnostic test evaluation and that even extensive laboratory testing only rarely contributed to the care of children with suspected TB.Trial registrationThis study did not meet Peruvian and some other international criteria for a clinical trial but was registered with the ClinicalTrials.gov registry: ClinicalTrials.gov NCT00054769.

Toxoplasma gondii is a common life-threatening opportunistic infection. We used experimental murine T. gondii infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondii tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.

Tuberculosis (TB) remains a global health pandemic and greater understanding of underlying pathogenesis is required to develop novel therapeutic and diagnostic approaches. Matrix metalloproteinases (MMPs) are emerging as key effectors of tissue destruction in TB but have not been comprehensively studied in plasma, nor have gender differences been investigated. We measured the plasma concentrations of MMPs in a carefully characterised, prospectively recruited clinical cohort of 380 individuals. The collagenases, MMP-1 and MMP-8, were elevated in plasma of patients with pulmonary TB relative to healthy controls, and MMP-7 (matrilysin) and MMP-9 (gelatinase B) were also increased. MMP-8 was TB-specific (p

Tuberculosis viability microscopy predicted, within 1 hour, quantitative culture results that became available weeks later. Viability microscopy provides promising results for informing decisions concerning drug susceptibility testing, treatment changes, and infection control measures in resource-constrained settings where most tuberculosis occurs., Background. It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis. Methods. To assess treatment response, sputum samples (n = 124) from unselected patients (n = 35) with sputum microscopy–positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric first-line therapy. Tuberculosis quantitative viability microscopy with fluorescein diacetate, quantitative culture, and acid-fast auramine microscopy were all performed in triplicate. Results. Tuberculosis quantitative viability microscopy predicted quantitative culture results such that 76% of results agreed within ±1 logarithm (rS = 0.85; P < .0001). In 31 patients with non-multidrug-resistant (MDR) tuberculosis, viability and quantitative culture results approximately halved (both 0.27 log reduction, P < .001) daily. For patients with non-MDR tuberculosis and available data, by treatment day 9 there was a >10-fold reduction in viability in 100% (24/24) of cases and quantitative culture in 95% (19/20) of cases. Four other patients subsequently found to have MDR tuberculosis had no significant changes in viability (P = .4) or quantitative culture (P = .6) results during early treatment. The change in viability and quantitative culture results during early treatment differed significantly between patients with non-MDR tuberculosis and those with MDR tuberculosis (both P < .001). Acid-fast microscopy results changed little during early treatment, and this change was similar for non-MDR tuberculosis vs MDR tuberculosis (P = .6). Conclusions. Tuberculosis quantitative viability microscopy is a simple test that within 1 hour predicted quantitative culture results that became available weeks later, rapidly indicating whether patients were responding to tuberculosis therapy.

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Lowenstein-Jensen medium and tested by qPCR for the small mobile genetic element 1S6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost similar to 5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

Slc11a1 (formerly Nramp1) has many pleiotropic effects on macrophage (m) activation, including regulation of the CXC chemokine KC, interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), major histocompatibility complex (MHC) class II molecules, tumour necrosis factor α (TNFα), nitric oxide (NO) release, l-arginine flux, oxidative burst and tumoricidal as well as antimicrobial activity (reviewed by Blackwell and Searle, 1999; Blackwell et al., 2000). A naturally occurring Gly→Asp mutation at amino acid 169 of Slc11a1 makes mice as susceptible to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis as gene-disrupted mice (Vidal et al., 1995). Hence, the mutation is a functional null. This mutation also confers susceptibility to a range of other pathogens in mice, including Mycobacterium lepraemurium (Brown et al., 1982; Skamene et al., 1984), Mycobacterium intracellulare (Goto et al., 1989), Toxoplasma gondii (Blackwell et al., 1994), Candida albicans (Puliti et al., 1995) and Leishmania infantum (Leclercq et al., 1996). In man, SLC11A1 is linked or associated with multiple infectious (Shaw et al., 1997; Abel et al., 1998; Bellamy et al., 1998; Marquet et al., 1999; Cervino et al., 2000; Gao et al., 2000; Greenwood et al., 2000; Ryu et al., 2000; Mohamed et al., 2001) and autoimmune (Shaw et al., 1996; Hofmeister et al., 1997; Esposito et al., 1998; Maliarik et al., 2000; Sanjeevi et al., 2000; Singal et al., 2000; Yang et al., 2000) diseases. The infectious diseases include viral (HIV), bacterial (tuberculosis, leprosy, meningococcal meningitis) and protozoan (visceral leishmaniasis) pathogens. The autoimmune diseases include rheumatoid arthritis, juvenile rheumatoid arthritis, diabetes, sarcoidosis and Crohn's disease. Mutation in the closely related Slc11a2 (Nramp2) gene causes microcytic anaemia in mice (Fleming et al., 1997), but disease association in man has not been reported. Slc11a1 and Slc11a2 are polytopic integral membrane proteins with 10–12 putative membrane-spanning domains (Vidal et al., 1993; Gunshin et al., 1997). In both, the natural functional null mutation occurs in transmembrane domain 4 (Vidal et al., 1993; Fleming et al., 1997). Both Slc11a1 and Slc11a2 have protein kinase C (PKC) binding sites (Vidal et al., 1993; Barton et al., 1994; Gruenheid et al., 1995), but only Slc11a1 has a Pro–Ser-rich N-terminus (Barton et al., 1994). Here, we review current knowledge on the evolution, function and roles of Slc11a1/SLC11A1 in disease.

Cysticercosis is a frequent infection in developing countries where pigs are raised [1]. Population-based studies show substantially higher epilepsy rates in developing countries than in industrialized countries, a difference attributed in part to neurocysticercosis [2, 3]. Nevertheless, no estimate of the disease burden due to cysticercosis exists. In this article we estimate, for the first time, the burden of morbidity attributable to cysticercosis in a developing country. Cysticercosis is the larval tissue stage of the pork tapeworm Taenia solium. When humans consume undercooked cysticercotic pork, the larva attaches to the gut wall and grows into the intestinal tapeworm. The life cycle is maintained when pigs eat human feces containing tapeworm eggs and develop porcine cysticercosis, a process facilitated by husbandry practices in villages where pigs are free-roaming scavengers and human fecal contamination is widespread. These life-cycle stages are benign in comparison with those of human cysticercosis, defined by the presence of T. solium larvae in human tissues, which occurs when people ingest tapeworm eggs from food, drink, or soil contaminated by the feces of a tapeworm carrier. Seizures are the most common manifestation of symptomatic human cysticercosis [4–6]. The disease may also be associated with headache, hydrocephalus, chronic meningitis, or symptoms due to a space-occupying central nervous system (CNS) lesion [7]. Isolated nonneurological manifestations, such as ocular or dermal cysts, account for

Osteomyelitis, or bone infection, is a major worldwide cause of morbidity. Treatment is frequently unsatisfactory, yet little is known about pathogenesis of infection. Plasma tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 concentrations were measured before and after lipopolysaccharide stimulation of whole blood from patients with bacterial and tuberculous osteomyelitis and from controls. Patients with bacterial and tuberculous osteomyelitis mounted an acute-phase response and were anemic and febrile. However, plasma IL-6 concentrations were significantly elevated in only tuberculous osteomyelitis patients (vs. controls, P < .05). IL-6 concentrations correlated with erythrocyte sedimentation rate, C-reactive protein level, and plasma albumin concentration, all acute-phase markers. There were no other correlations between cytokine concentrations and clinical data. Following ex vivo stimulation, TNF, IL-6, and IL-8 were secreted equally by patients and controls. In summary, tuberculous osteomyelitis is characterized by elevated systemic IL-6 concentrations associated with an acute-phase response. For further insight into immunopathology of osteomyelitis, studies on infected bone are required.

Symptomatic neurocysticercosis, a major cause of epilepsy worldwide, results from inflammation around Taenia solium larvae, but the mechanisms are unknown. Eotaxin, not previously reported in cases of human infection, and interleukin-5 (IL-5) but not IL-8 concentrations were elevated in patient serum, and IL-5 levels were also elevated in cerebrospinal fluid (CSF). Eosinophil-selective mediators may be involved in the pathogenesis of cysticercosis. IL-6 concentrations were also elevated in patient CSF, possibly indicative of an acute-phase response.

Tom Wingfield and colleagues investigate the relationship between catastrophic costs and tuberculosis outcomes for patients receiving free tuberculosis care in Peru. Please see later in the article for the Editors' Summary, Background Even when tuberculosis (TB) treatment is free, hidden costs incurred by patients and their households (TB-affected households) may worsen poverty and health. Extreme TB-associated costs have been termed “catastrophic” but are poorly defined. We studied TB-affected households' hidden costs and their association with adverse TB outcome to create a clinically relevant definition of catastrophic costs. Methods and Findings From 26 October 2002 to 30 November 2009, TB patients (n = 876, 11% with multi-drug-resistant [MDR] TB) and healthy controls (n = 487) were recruited to a prospective cohort study in shantytowns in Lima, Peru. Patients were interviewed prior to and every 2–4 wk throughout treatment, recording direct (household expenses) and indirect (lost income) TB-related costs. Costs were expressed as a proportion of the household's annual income. In poorer households, costs were lower but constituted a higher proportion of the household's annual income: 27% (95% CI = 20%–43%) in the least-poor houses versus 48% (95% CI = 36%–50%) in the poorest. Adverse TB outcome was defined as death, treatment abandonment or treatment failure during therapy, or recurrence within 2 y. 23% (166/725) of patients with a defined treatment outcome had an adverse outcome. Total costs ≥20% of household annual income was defined as catastrophic because this threshold was most strongly associated with adverse TB outcome. Catastrophic costs were incurred by 345 households (39%). Having MDR TB was associated with a higher likelihood of incurring catastrophic costs (54% [95% CI = 43%–61%] versus 38% [95% CI = 34%–41%], p, Editors' Summary Background Caused by the infectious microbe Mycobacterium tuberculosis, tuberculosis (or TB) is a global health problem. In 2012, an estimated 8.6 million people fell ill with TB, and 1.3 million were estimated to have died because of the disease. Poverty is widely recognized as an important risk factor for TB, and developing nations shoulder a disproportionate burden of both poverty and TB disease. For example, in Lima (the capital of Peru), the incidence of TB follows the poverty map, sparing residents living in rich areas of the city while spreading among poorer residents that live in overcrowded households. The Peruvian government, non-profit organizations, and the World Health Organization (WHO) have extended healthcare programs to provide free diagnosis and treatment for TB and drug-resistant strains of TB in Peru, but rates of new TB cases remain high. For example, in Ventanilla (an area of 16 shantytowns located in northern Lima), the rate of infection was higher during the study period, at 162 new cases per 100,000 people per year, than the national average. About one-third of the 277,895 residents of Ventanilla live on under US$1 per day. Why Was This Study Done? Poverty increases the risks associated with contracting TB infection, but the disease also affects the most economically productive age group, and the income of TB-affected households often decreases post-diagnosis, exacerbating poverty. A recent WHO consultation report proposed a target of eradicating catastrophic costs for TB-affected families by 2035, but hidden TB-related costs remain understudied, and there is no international consensus defining catastrophic costs incurred by patients and households affected by TB. Lost income and the cost of transport are among hidden costs associated with free treatment programs; these costs and their potential impact on patients and their households are not well defined. Here the researchers sought to clarify and characterize TB-related costs and explore whether there is a relationship between the hidden costs associated with free TB treatment programs and the likelihood of completing treatment and becoming cured of TB. What Did the Researchers Do and Find? Over a seven-year period (2002–2009), the researchers recruited 876 study participants with TB diagnosed at health posts located in Ventanilla. To provide a comparative control group, a sample of 487 healthy individuals was also recruited to participate. Participants were interviewed prior to treatment, and households' TB-related direct expenses and indirect expenses (lost income attributed to TB) were recorded every 2–4 wk. Data were collected during scheduled household visits. TB patients were poorer than controls, and analysis of the data showed that accessing free TB care was expensive for TB patients, especially those with multi-drug-resistant (MDR) TB. Total expenses were similar pre-treatment compared to during treatment for TB patients, despite receiving free care (1.1 versus 1.2 times the same household's monthly income). Even though direct expenses (for example, costs of medical examinations and medicines other than anti-TB therapy) were lower in the poorest households, their total expenses (direct and indirect) made up a greater proportion of their household annual income: 48% for the poorest households compared to 27% in the least-poor households. The researchers defined costs that were equal to or above one-fifth (20%) of household annual income as catastrophic because this threshold marked the greatest association with adverse treatment outcomes such as death, abandoning treatment, failing to respond to treatment, or TB recurrence. By calculating the population attributable fraction—the proportional reduction in population adverse treatment outcomes that could occur if a risk factor was reduced to zero—the authors estimate that adverse TB outcomes explained by catastrophic costs and MDR TB were similar: 18% for catastrophic costs and 20% for MDR TB. What Do These Findings Mean? The findings of this study indicate a potential role for social protection as a means to improve TB disease control and health, as well as defining a novel, evidence-based threshold for catastrophic costs for TB-affected households of 20% or more of annual income. Addressing the economic impact of diagnosis and treatment in impoverished communities may increase the odds of curing TB. Study limitations included only six months of follow-up data being gathered on costs for each participant and not recording “dissavings,” such as selling of household items in response to financial shock. Because the study was observational, the authors aren't able to determine the direction of the association between catastrophic costs and TB outcome. Even so, the study indicates that TB is a socioeconomic as well as infectious problem, and that TB control interventions should address both the economic and clinical aspects of the disease. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001675. The World Health Organization provides information on all aspects of tuberculosis, including the Global Tuberculosis Report 2013 The US Centers for Disease Control and Prevention has information about tuberculosis Médecins Sans Frontières's TB&ME blog provides patients' stories of living with MDR TB TB Alert, a UK-based charity that promotes TB awareness worldwide, has information on TB in several European, African, and Asian languages More information is available about the Innovation For Health and Development (IFHAD) charity and its research team's work in Peru

In developing countries many enteric infections are caused by acid-sensitive pathogens. Failure of the gastric acid barrier to infection has been reported in cholera but gastric acid secretion has been little studied in other enteric infections. We therefore studied basal and stimulated gastric acid in 185 Bangladeshi men admitted to hospital for the treatment of enteric infection. Patients with dysentery (amoebiasis, n = 24 and shigellosis, n = 19) and culture-negative diarrhoea (n = 69) had similar mean gastric acid levels (basal, 3–5 mmol/h; stimulated, 11–17 mmol/h), which remained stable in those patients studied throughout 12 weeks of convalescence. In contrast, patients with secretory diarrhoea caused by cholera or enterotoxigenic Escherichia coli (ETEC) had low gastric acid levels (P < 0·05 compared with other groups) (cholera, n = 34: basal mean 1·8 mmol/h [SD = 2·2], stimulated mean 7·9 mmol/h [SD = 6·4]; ETEC, n = 39: basal mean 2·7 mmol/h [SD = 2·8], stimulated mean 9·4 mmol/h [SD = 7·5]). Cholera patients' gastric acid level rose during convalescence to similar levels to the dysentery patients'. Low gastric acid level was associated with severe disease in patients with cholera (P < 0·02) or ETEC (P < 0·05). Gastric acid level fell with increasing age (P < 0·007) but this did not account for the differences between groups. Gastric acid levels were not associated with Giardia duodenalis or Strongyloides stercoralis co-infection, fever, use of tobacco, or chewing betel nut. Cholera and secretory diarrhoea caused by ETEC may, therefore, partly result from a reduction in gastric acid level which does not occur during dysentery. Factors which impair gastric acid secretion may predispose to diarrhoeal disease in developing countries.

A cross-sectional, community-based study was performed in 2012 with 428 residents of periurban shantytowns in Lima, Peru to study risk factors for and changes in latent tuberculosis infection in age-stratified groups compared with our data from the same region in 1990 (N = 219) and 2005 (N = 103). Tuberculin skin test positivity in these communities was highly prevalent at 52% overall, increased with age (P < 0.01) and was similar to 2005 (53%) and 1990 (48%). From 1990 to 2012, the prevalence of tuberculin positivity decreased in 5-14 and 15-24 year old groups (to 17% and 34%, respectively, both P < 0.05). However, this may be explained by cessation of Bacille Calmette-Guerin revaccination during this period, because Bacille Calmette-Guerin revaccination doubled tuberculin positivity. Over the same 22-year period, tuberculin positivity in the ≥ 25 year old group remained high (71%, P = 0.3), suggesting that prevalent latent tuberculosis infection persists in the adult population despite improving medical care and socioeconomic development in this region.

Background Tuberculosis infection, disease and mortality are all less common at high than low altitude and ascent to high altitude was historically recommended for treatment. The immunological and mycobacterial mechanisms underlying the association between altitude and tuberculosis are unclear. We studied the effects of altitude on mycobacteria and antimycobacterial immunity. Methods Antimycobacterial immunity was assayed in 15 healthy adults residing at low altitude before and after they ascended to 3400 meters; and in 47 long-term high-altitude residents. Antimycobacterial immunity was assessed as the extent to which participants’ whole blood supported or restricted growth of genetically modified luminescent Bacille Calmette-Guérin (BCG) mycobacteria during 96 hours incubation. We developed a simplified whole blood assay that could be used by a technician in a low-technology setting. We used this to compare mycobacterial growth in participants’ whole blood versus positive-control culture broth and versus negative-control plasma. Results Measurements of mycobacterial luminescence predicted the number of mycobacterial colonies cultured six weeks later. At low altitude, mycobacteria grew in blood at similar rates to positive-control culture broth whereas ascent to high altitude was associated with restriction (p≤0.002) of mycobacterial growth to be 4-times less than in culture broth. At low altitude, mycobacteria grew in blood 25-times more than negative-control plasma whereas ascent to high altitude was associated with restriction (p≤0.01) of mycobacterial growth to be only 6-times more than in plasma. There was no evidence of differences in antimycobacterial immunity at high altitude between people who had recently ascended to high altitude versus long-term high-altitude residents. Conclusions An assay of luminescent mycobacterial growth in whole blood was adapted and found to be feasible in low-resource settings. This demonstrated that ascent to or residence at high altitude was associated with decreased mycobacterial growth in whole blood relative to controls, consistent with altitude-related augmentation of antimycobacterial cellular immunity.

Tartu, Estonia.To assess the performance and feasibility of the introduction of the thin-layer agar MDR/XDR-TB Colour Test (Colour Test) as a non-commercial method of drug susceptibility testing (DST).The Colour Test combines the thin-layer agar technique with a simple colour-coded quadrant format, selective medium to reduce contamination and colorimetric indication of bacterial growth to simplify interpretation. DST patterns for isoniazid (INH), rifampicin (RMP) and ciprofloxacin (CFX) were determined using the Colour Test for 201 archived Mycobacterium tuberculosis isolates. Susceptibilities were compared to blinded DST results obtained routinely using the BACTEC™ Mycobacteria Growth Indicator Tube™ (MGIT) 960 to assess performance characteristics.In all, 98% of the isolates produced interpretable results. The average time to positivity was 13 days, and all results were interpretable. The Colour Test detected drug resistance with 98% sensitivity for INH, RMP and CFX and 99% for multidrug-resistant tuberculosis. Specificities were respectively 100% (95%CI 82-100), 88% (95%CI 69-97) and 91% (95%CI 83-96) and 90% (95%CI 74-98). Agreement between the Colour Test and BACTEC MGIT 960 were respectively 98%, 96%, 94% and 97%.The Colour Test could be an economical, accurate and simple technique for testing tuberculosis strains for drug resistance. As it requires little specialist equipment, it may be particularly useful in resource-constrained settings with growing drug resistance rates.

The development, evaluation, and implementation of new and improved diagnostics have been identified as critical needs by human immunodeficiency virus (HIV) and tuberculosis researchers and clinicians alike. These needs exist in international and domestic settings and in adult and pediatric populations. Experts in tuberculosis and HIV care, researchers, healthcare providers, public health experts, and industry representatives, as well as representatives of pertinent US federal agencies (Centers for Disease Control and Prevention, Food and Drug Administration, National Institutes of Health, United States Agency for International Development) assembled at a workshop proposed by the Diagnostics Working Group of the Federal Tuberculosis Taskforce to review the state of tuberculosis diagnostics development in adult and pediatric populations.

BACKGROUND\ud \ud Because of the high global prevalence of latent TB infection (LTBI), a key challenge in endemic settings is distinguishing patients with active TB from patients with overlapping clinical symptoms without active TB but with co-existing LTBI. Current methods are insufficiently accurate. Plasma proteomic fingerprinting can resolve this difficulty by providing a molecular snapshot defining disease state that can be used to develop point-of-care diagnostics.\ud \ud METHODS\ud \ud Plasma and clinical data were obtained prospectively from patients attending community TB clinics in Peru and from household contacts. Plasma was subjected to high-throughput proteomic profiling by mass spectrometry. Statistical pattern recognition methods were used to define mass spectral patterns that distinguished patients with active TB from symptomatic controls with or without LTBI.\ud \ud RESULTS\ud \ud 156 patients with active TB and 110 symptomatic controls (patients with respiratory symptoms without active TB) were investigated. Active TB patients were distinguishable from undifferentiated symptomatic controls with accuracy of 87% (sensitivity 84%, specificity 90%), from symptomatic controls with LTBI (accuracy of 87%, sensitivity 89%, specificity 82%) and from symptomatic controls without LTBI (accuracy 90%, sensitivity 90%, specificity 92%).\ud \ud CONCLUSIONS\ud \ud We show that active TB can be distinguished accurately from LTBI in symptomatic clinic attenders using a plasma proteomic fingerprint. Translation of biomarkers derived from this study into a robust and affordable point-of-care format will have significant implications for recognition and control of active TB in high prevalence settings.

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.

Carlton Evans considers whether the new tuberculosis diagnostic test, GeneXpert, is the solution for TB control that it's said to be.

Carlton Evans considers whether the new tuberculosis diagnostic test, GeneXpert, is the solution for TB control that it's said to be.

The tuberculin skin test (TST) quantifies cell-mediated immunity to tuberculosis antigens. Helminths suppress cell-mediated immunity, so we studied the effect of helminth infection and deworming on the TST in a randomized, double-blind, placebo-controlled study in an indigenous Amazon community (N = 195). Stool microscopy diagnosed helminths in 98% and co-infection with multiple species in 24% of study subjects. The TST was positive (≥ 10 mm) for 49%, and responses increased with age (P < 0.001), Bacille Calmette Guerin (BCG) vaccination (P = 0.01), and tuberculosis contact (P = 0.05). TST results had no association with helminth-egg concentrations, species, or co-infections (all P > 0.1). One month after deworming with albendazole (three daily 400-mg doses), helminths were reduced, but 63% remained infected with helminths. Albendazole did not cause a change in TST size (P = 0.8) or positivity (P = 0.9) relative to placebo. Thus, TST reactions were unaffected by albendazole therapy that partially cured intestinal helminth infections, and TST interpretation was unaffected by high-burden helminth infections and co-infection with multiple helminth species.

BackgroundThere is evidence that female gender is associated with reduced likelihood of tuberculosis diagnosis and successful treatment. This study aimed to characterize gender-related barriers to tuberculosis control in Peruvian shantytowns.MethodsWe investigated attitudes and experiences relating gender to tuberculosis using the grounded theory approach to describe beliefs amongst key tuberculosis control stakeholders. These issues were explored in 22 semi-structured interviews and in four focus group discussions with 26 tuberculosis patients and 17 healthcare workers.ResultsWe found that the tuberculosis program was perceived not to be gender discriminatory and provided equal tuberculosis diagnostic and treatment care to men and women. This contrasted with stereotypical gender roles in the broader community context and a commonly expressed belief amongst patients and healthcare workers that female health inherently has a lower priority than male health. This belief was principally associated with men's predominant role in the household economy and limited employment for women in this setting. Women were also generally reported to experience the adverse psychosocial and economic consequences of tuberculosis diagnosis more than men.ConclusionsThere was a common perception that women's tuberculosis care was of secondary importance to that of men. This reflected societal gender values and occurred despite apparent gender equality in care provision. The greatest opportunities for improving women's access to tuberculosis care appear to be in improving social, political and economic structures, more than tuberculosis program modification.

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 IS 6110 -PCR was used for tuberculosis detection, and IS 6110 -PCR-positive stool samples then underwent rifampin sensitivity testing by universal heteroduplex generator PCR (heteroduplex-PCR) assay. For newly diagnosed pulmonary tuberculosis patients, stool IS 6110 -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.

This study aimed to determine the psychosocial factors associated with delayed test-seeking among tuberculosis patients. The duration of symptoms before seeking medical care was assessed by interview for 108 newly diagnosed pulmonary tuberculosis patients in the city of Iquitos in the Peruvian Amazon, which has high tuberculosis incidence. Beliefs associated with test-seeking behavior and delay was assessed in these patients. The median delay from symptom onset to seeking diagnostic testing was 61 days (inter-quartile range 30-91 days). The belief that tuberculosis is curable was associated with a 100% longer test-seeking delay; the perception that tuberculosis was common was associated with a 57% longer delay; male gender was associated with a 48% longer delay; and education less than complete secondary schooling was associated with a 44% longer delay. In conclusion, current health promotion activities that emphasize tuberculosis curability and high prevalence may paradoxically increase test-seeking delay and therefore require prospective evaluation.

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.

Background Institutional tuberculosis (TB) transmission is an important public health problem highlighted by the HIV/AIDS pandemic and the emergence of multidrug- and extensively drug-resistant TB. Effective TB infection control measures are urgently needed. We evaluated the efficacy of upper-room ultraviolet (UV) lights and negative air ionization for preventing airborne TB transmission using a guinea pig air-sampling model to measure the TB infectiousness of ward air. Methods and Findings For 535 consecutive days, exhaust air from an HIV-TB ward in Lima, Perú, was passed through three guinea pig air-sampling enclosures each housing approximately 150 guinea pigs, using a 2-d cycle. On UV-off days, ward air passed in parallel through a control animal enclosure and a similar enclosure containing negative ionizers. On UV-on days, UV lights and mixing fans were turned on in the ward, and a third animal enclosure alone received ward air. TB infection in guinea pigs was defined by monthly tuberculin skin tests. All guinea pigs underwent autopsy to test for TB disease, defined by characteristic autopsy changes or by the culture of Mycobacterium tuberculosis from organs. 35% (106/304) of guinea pigs in the control group developed TB infection, and this was reduced to 14% (43/303) by ionizers, and to 9.5% (29/307) by UV lights (both p < 0.0001 compared with the control group). TB disease was confirmed in 8.6% (26/304) of control group animals, and this was reduced to 4.3% (13/303) by ionizers, and to 3.6% (11/307) by UV lights (both p < 0.03 compared with the control group). Time-to-event analysis demonstrated that TB infection was prevented by ionizers (log-rank 27; p < 0.0001) and by UV lights (log-rank 46; p < 0.0001). Time-to-event analysis also demonstrated that TB disease was prevented by ionizers (log-rank 3.7; p = 0.055) and by UV lights (log-rank 5.4; p = 0.02). An alternative analysis using an airborne infection model demonstrated that ionizers prevented 60% of TB infection and 51% of TB disease, and that UV lights prevented 70% of TB infection and 54% of TB disease. In all analysis strategies, UV lights tended to be more protective than ionizers. Conclusions Upper-room UV lights and negative air ionization each prevented most airborne TB transmission detectable by guinea pig air sampling. Provided there is adequate mixing of room air, upper-room UV light is an effective, low-cost intervention for use in TB infection control in high-risk clinical settings., Using a guinea-pig detection model, Rod Escombe and colleagues find that upper-room UV lamps in hospital rooms can substantially reduce airborne transmission ofMycobacterium tuberculosis., Editors' Summary Background. Tuberculosis—a contagious infection, usually of the lungs—kills nearly 2 million people annually. It is caused by Mycobacterium tuberculosis, bacteria that are spread in airborne droplets when people with tuberculosis cough or sneeze. Most people infected with M. tuberculosis do not become ill—their immune system contains the infection. However, the bacteria remain dormant within the body and can cause disease years later if immunity declines because of, for example, infection with human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS). The symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Infection with M. tuberculosis is diagnosed using the tuberculin skin test. Tests for tuberculosis itself include chest X-rays and sputum cultures (in which bacteriologists try to grow M. tuberculosis from mucus brought up from the lungs by coughing). Tuberculosis can usually be cured by taking several powerful antibiotics daily for several months. Drug-resistant tuberculosis is much harder to cure, requiring multiple second-line antibiotics for up to two years or more. Tuberculosis transmission can be reduced by, for example, hospitalizing people with tuberculosis in isolation wards in which negative-pressure mechanical ventilation is used to reduce the concentration of infectious airborne droplets. Why Was This Study Done? After the development of antibiotics capable of killing M. tuberculosis in the mid 20th century, it seemed that tuberculosis would become a disease of the past. But in the mid 1980s, drug-resistant M. tuberculosis strains began to emerge, the HIV/AIDS epidemic took hold, and tuberculosis resurged to today's worrying levels. New ways of reducing tuberculosis transmission, particularly in health care settings and in resource-limited settings, are now urgently needed. The need for effective infection control measures is especially urgent in HIV care programs where highly susceptible individuals frequently mix with people with tuberculosis. In this study, the researchers use a guinea pig air-sampling model (which was first used in the 1950s to show that tuberculosis is an airborne infection) to investigate whether upper-room ultraviolet (UV) lights in patient rooms and negative air ionization can prevent airborne tuberculosis transmission. UV light kills M. tuberculosis; negative ionization gives airborne particles a charge that makes them stick to surfaces. What Did the Researchers Do and Find? The researchers exposed a group of control guinea pigs kept in a special air-sampling enclosure to untreated air from an HIV–TB ward in Lima (Perú). Another group of animals (the UV group) breathed air from the same ward, but only on the days that UV lights suspended near the ward's ceiling were turned on, together with mixing fans to mix up the room air. The “ionizer group” had a negative ionizer switched on in their enclosure when they were exposed to ward air (each group of animals was exposed to ward air every other day). The animals were tested monthly with the tuberculin skin test and all were examined for tuberculosis disease when they became infected with tuberculosis or at the end of the 535-day experiment. 35% of the control animals, 14% of the ionizer group animals, and 9.5% of the UV group animals developed M. tuberculosis infections. Tuberculosis disease was found in 8.6% of the control animals but in only 4.3% and 3.6% of the animals in the ionizer and UV groups, respectively. A “time-to-event analysis” also showed that UV lights and ionizers reduced tuberculosis infection and disease. Finally, an analysis of the data using an airborne infection model indicated that ionizers and UV lights prevented 60% and 70% of tuberculosis infections, respectively. What Do These Findings Mean? These findings indicate that upper-room UV lights, combined with adequate air mixing, or negative air ionization with special large-scale ionizers can prevent most airborne tuberculosis transmission to guinea pigs exposed to hospital room air. The effectiveness of these approaches in reducing tuberculosis transmission between people is likely to be similar, although remains to be tested. Nevertheless, this first study of the effect of upper-air UV light and of negative air ionization on airborne transmission in a clinical setting suggests that both approaches could be potentially important tuberculosis infection control measures. Furthermore, the UV light approach might provide a relatively low-cost intervention for possible use in waiting rooms and other overcrowded settings where patients with undiagnosed, untreated tuberculosis—individuals who tend to be highly infectious—are likely to come into contact with other susceptible patients, health care workers, and visitors. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000043. The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis, including multidrug-resistance tuberculosis, and on tuberculosis and HIV The US Centers for Disease Control and Prevention provide several fact sheets and other information resources about all aspects of tuberculosis, including Guidelines for preventing the Transmission of Mycobacterium tuberculosis in Health-Care Settings, 2005 (some information in Spanish is also available) The World Health Organization's 2008 report “Global Tuberculosis Control—Surveillance, Planning, Financing” provides a snapshot of the current state of the global tuberculosis epidemic and links to information about all aspects of tuberculosis and its control (in several languages) Tuberculosis Infection-Control in the Era of Expanding HIV Care and Treatment is another report from the World Health Organization HIVInsite provides detailed information about the combination of HIV infection and tuberculosis Avert, an international AIDS charity, also provides information about the interaction between HIV and tuberculosis GHD (Global Health Delivery) Online is an online resource dedicated to TB infection control, and is moderated by world experts

Pediatric pulmonary tuberculosis diagnosis is difficult because young children are unable to expectorate sputum samples. Testing stool for tuberculosis DNA from swallowed sputum may diagnose pulmonary tuberculosis. Hospitalized children with suspected tuberculosis had stool, nasopharyngeal, and gastric aspirates cultured that confirmed pulmonary tuberculosis in 16/236 patients. Twenty-eight stored stools from these 16 children were used to evaluate stool polymerase chain reaction (PCR) for tuberculosis diagnosis compared with 28 stool samples from 23 healthy control children. Two DNA extraction techniques were used: fast-DNA mechanical homogenization and Chelex-resin chemical extraction. DNA was tested for tuberculosis DNA with a hemi-nested IS6110 PCR. PCR after Fast-DNA processing was positive for 6/16 culture-proven tuberculosis patients versus 5/16 after Chelex extraction (sensitivity 38% and 31%, respectively). All controls were negative (specificity 100%). If sensitivity can be increased, stool PCR would be a rapid, non-invasive, and relatively bio-secure initial test for children with suspected pulmonary tuberculosis.

An 8-year-old girl from the rural eastern Amazon department of Loreto presented to her local health post with a 1-week history of fever, headache, and poorly localized abdominal discomfort. At the health post, the child had a temperature of 39°C. A thick smear was obtained, and the patient was started on chloroquine after parasites were observed on the thick smear. The smear was referred to a regional diagnostic center for further evaluation. There the smear was found to be negative for Plasmodium species but positive for

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., Using a guinea pig detection system above an HIV-tuberculosis ward, Rod Escombe and colleagues found that most transmitted tuberculosis originated from patients with inadequately treated multidrug-resistant tuberculosis., Editors' Summary Background. Every year, more than nine million people develop tuberculosis—a contagious infection usually of the lungs—and nearly two million people die from the disease. Tuberculosis is caused by Mycobacterium tuberculosis. These bacteria are spread in airborne droplets when people with the disease cough or sneeze. Most people infected with M. tuberculosis never become ill—their immune system contains the infection. However, the bacteria remain dormant within the body and can cause tuberculosis years later if host immunity declines. The symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Diagnostic tests for the disease include chest X-rays, the tuberculin skin test, and sputum cultures (in which bacteriologists try to grow M. tuberculosis from mucus brought up from the lungs by coughing). Tuberculosis can usually be cured by taking several powerful antibiotics daily for several months. Why Was This Study Done? Scientists performed definitive experiments on airborne tuberculosis transmission in the 1950s by exposing guinea pigs to the air from a tuberculosis ward. They found that a minority of patients actually transmit tuberculosis, that the infectiousness of transmitters varies greatly, and that effective antibiotic treatment decreases infectiousness. Since the 1950s, however, multidrug-resistant (MDR) and more recently extensively drug-resistant (XDR) strains of M. tuberculosis have become widespread. Treatment of drug-resistant tuberculosis is much more difficult than normal tuberculosis, requiring even more antibiotics, and for long periods, up to 2 years and beyond. In addition, HIV (the virus that causes AIDS) has emerged. HIV weakens the immune system so HIV-positive people are much more likely to develop active tuberculosis (and to die from the disease, which also speeds the development of HIV/AIDS) than people with a healthy immune system. Have these changes altered tuberculosis transmission between people? The answer to this question might help to optimize the control of tuberculosis infection, particularly in hospitals. In this study, the researchers investigate current patterns of tuberculosis infectiousness among HIV-positive patients by recreating the 1950s guinea pig model for tuberculosis transmission in a hospital in Lima, Perú. What Did the Researchers Do and Find? The researchers passed all the air from an HIV–tuberculosis ward over guinea pigs housed in an animal facility on the hospital's roof. The guinea pigs were tested monthly with tuberculin skin tests, and tissues from positive animals were examined for infection with M. tuberculosis. Sputum was also collected daily from the patients on the ward. The researchers then used the timing of patient admissions and guinea pig infections, together with the drug susceptibility patterns and DNA fingerprints of the M. tuberculosis strains isolated from the animals and the patients, to identify which patients had infected which guinea pigs. Finally, they used a mathematical equation to calculate the relative infectiousness of each patient in airborne infectious units (“quanta”) per hour. During the 505 study days, although 97 HIV-positive patients with tuberculosis were admitted to the ward, just ten patients were responsible for virtually all the characterized cases of tuberculosis among the guinea pigs. Six of these patients had MDR tuberculosis that had been suboptimally treated. The average patient infectiousness over the entire study period was 8.2 quanta per hour—six times greater than the average infectiousness recorded in the 1950s. Finally, the three most infectious patients (all of whom had suboptimally treated MDR tuberculosis) produced 226, 52, and 40 quanta per hour. What Do These Findings Mean? These findings show that a few inadequately treated HIV-positive patients with MDR tuberculosis caused nearly all the tuberculosis transmission to guinea pigs during this study. They also show for the first time that tuberculosis infectiousness among HIV-positive patients is very variable. The increase in the average patient infectiousness in this study compared to that seen in the 1950s hints at the possibility that HIV infection might increase tuberculosis infectiousness. However, studies that directly compare the tuberculosis infectiousness of HIV-positive and HIV-negative patients are needed to test this possibility. More importantly, this study demonstrates the potentially high infectiousness of inadequately treated MDR TB patients and their importance in ongoing TB transmission. These findings suggest that rapid, routine testing of antibiotic susceptibility should improve tuberculosis control by ensuring that patients with MDR TB are identified and treated effectively and quickly. Finally, they re-emphasize the importance of implementing environmental control measures (for example, adequate natural or mechanical ventilation of tuberculosis wards, or crowded waiting rooms or emergency departments where tuberculosis patients may be found) to prevent airborne tuberculosis transmission in health-care facilities, particularly in areas where many patients are HIV positive and/or where MDR tuberculosis is common. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050188. The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis, including multidrug-resistance tuberculosis, and on tuberculosis and HIV The US Centers for Disease Control and Prevention provide several fact sheets and other information resources about all aspects of tuberculosis (in English and Spanish) The World Health Organization's 2008 report on global tuberculosis control—surveillance, planning, financing provides a snapshot of the current state of the global tuberculosis epidemic and links to information about all aspects of tuberculosis and its control (in several languages) HIVInsite provides detailed information about coinfection with HIV and tuberculosis • Avert, an international AIDS charity, also provides information about the interaction between HIV and tuberculosis Tuberculosis Infection-Control in the Era of Expanding HIV Care and Treatment is a report from the World Health Organization

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.

Pediatric pulmonary tuberculosis diagnosis is difficult because young children are unable to expectorate sputum samples. Testing stool for tuberculosis DNA from swallowed sputum may diagnose pulmonary tuberculosis. Hospitalized children with suspected tuberculosis had stool, nasopharyngeal, and gastric aspirates cultured that confirmed pulmonary tuberculosis in 16/236 patients. Twenty-eight stored stools from these 16 children were used to evaluate stool polymerase chain reaction (PCR) for tuberculosis diagnosis compared with 28 stool samples from 23 healthy control children. Two DNA extraction techniques were used: fast-DNA mechanical homogenization and Chelex-resin chemical extraction. DNA was tested for tuberculosis DNA with a hemi-nested IS6110 PCR. PCR after Fast-DNA processing was positive for 6/16 culture-proven tuberculosis patients versus 5/16 after Chelex extraction (sensitivity 38% and 31%, respectively). All controls were negative (specificity 100%). If sensitivity can be increased, stool PCR would be a rapid, non-invasive, and relatively bio-secure initial test for children with suspected pulmonary tuberculosis.

We would like to thank the correspondents for their thoughtful contributions to this important public health topic [1]. As our abstract and article state, we measured natural and mechanical ventilation and then calculated the effects of these ventilation rates on estimated tuberculosis (TB) infection rates using a mathematical model of airborne infection. This appears to be the first published assessment of natural ventilation rates in health-care settings, and the novel conclusions of our article are that extremely high rates of dilutional ventilation can be achieved through natural ventilation at very little cost by simply opening windows and doors. Indeed, this natural ventilation was far in excess of even the best maintained mechanical ventilation systems used in health-care settings. Importantly, this natural ventilation greatly reduced the calculated risk of airborne infection. Measuring TB transmission itself is difficult, as rates in staff are confounded by exposures outside the workplace, and mechanical air sampling techniques have had limited success. We have established a guinea pig air sampling facility to directly measure TB transmission in a hospital ward in Lima, Peru [2] and have used this model to evaluate the effects of upper room ultraviolet light and negative air ionization on TB transmission. We plan to use this facility to further study natural ventilation, and its effect on actual TB transmission. The results of the current study cannot be generalized to regions too cold to tolerate enhanced natural ventilation and not every room may be as amenable to natural ventilation as the Peruvian rooms that we studied. However, the key conclusions are clear: high rates of natural ventilation were achieved even on days with little wind and even rooms without high ceilings and large windows were well ventilated, such that natural ventilation significantly exceeded mechanical ventilation. It is therefore clear that natural ventilation has an important role to play in the fight against institutional TB transmission in resource-limited settings. Mechanical ventilation is expensive to install, requires costly ongoing maintenance, may be dangerous if poorly maintained (for example, delivering positive instead of negative pressure), and is clearly inappropriate for the great majority of resource-limited settings where the burden of TB is highest. TB infection control is an urgent priority, underscored by the emergence of extreme drug-resistant TB strains and the increasing congregation in potentially high-risk overcrowded settings of persons living with HIV through the roll-out of enhanced HIV care. When infectious TB patients share rooms with others, opening windows and doors to enhance natural ventilation is a simple, inexpensive, and effective strategy in the fight against nosocomial TB transmission.

BACKGROUND: Institutional transmission of airborne infections such as tuberculosis (TB) is an important public health problem, especially in resource-limited settings where protective measures such as negative-pressure isolation rooms are difficult to implement. Natural ventilation may offer a low-cost alternative. Our objective was to investigate the rates, determinants, and effects of natural ventilation in health care settings. METHODS AND FINDINGS: The study was carried out in eight hospitals in Lima, Peru; five were hospitals of "old-fashioned" design built pre-1950, and three of "modern" design, built 1970-1990. In these hospitals 70 naturally ventilated clinical rooms where infectious patients are likely to be encountered were studied. These included respiratory isolation rooms, TB wards, respiratory wards, general medical wards, outpatient consulting rooms, waiting rooms, and emergency departments. These rooms were compared with 12 mechanically ventilated negative-pressure respiratory isolation rooms built post-2000. Ventilation was measured using a carbon dioxide tracer gas technique in 368 experiments. Architectural and environmental variables were measured. For each experiment, infection risk was estimated for TB exposure using the Wells-Riley model of airborne infection. We found that opening windows and doors provided median ventilation of 28 air changes/hour (ACH), more than double that of mechanically ventilated negative-pressure rooms ventilated at the 12 ACH recommended for high-risk areas, and 18 times that with windows and doors closed (p < 0.001). Facilities built more than 50 years ago, characterised by large windows and high ceilings, had greater ventilation than modern naturally ventilated rooms (40 versus 17 ACH; p < 0.001). Even within the lowest quartile of wind speeds, natural ventilation exceeded mechanical (p < 0.001). The Wells-Riley airborne infection model predicted that in mechanically ventilated rooms 39% of susceptible individuals would become infected following 24 h of exposure to untreated TB patients of infectiousness characterised in a well-documented outbreak. This infection rate compared with 33% in modern and 11% in pre-1950 naturally ventilated facilities with windows and doors open. CONCLUSIONS: Opening windows and doors maximises natural ventilation so that the risk of airborne contagion is much lower than with costly, maintenance-requiring mechanical ventilation systems. Old-fashioned clinical areas with high ceilings and large windows provide greatest protection. Natural ventilation costs little and is maintenance free, and is particularly suited to limited-resource settings and tropical climates, where the burden of TB and institutional TB transmission is highest. In settings where respiratory isolation is difficult and climate permits, windows and doors should be opened to reduce the risk of airborne contagion.

N-acetyltransferase 2 (NAT2), an important enzyme in clinical pharmacology, metabolizes antibiotics such as isoniazid and sulfamethoxazole, and catalyzes the transformation of aromatic and heterocyclic amines from the environment and diet into carcinogenic intermediates. Polymorphisms in NAT2 account for variability in the acetylator phenotype and the pharmacokinetics of metabolized drugs. Native Americans, settled in rural areas and large cities of Latin America, are under-represented in pharmacogenetics studies; therefore, we sequenced the coding region of NAT2 in 456 chromosomes from 13 populations from the Americas, and two from Siberia, detecting nine substitutions and 11 haplotypes. Variants *4 (37%), *5B (23%) and *7B (24%) showed high frequencies. Average frequencies of fast, intermediate and slow acetylators across Native Americans were 18, 56 and 25%, respectively. NAT2 intra-population genetic diversity for Native Americans is higher than East Asians and similar to the rest of the world, and NAT2 variants are homogeneously distributed across native populations of the continent.

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.

Here we put forward a roadmap that summarizes important questions that need to be answered to determine more effective and safer treatments. A key concept in management of neurocysticercosis is the understanding that infection and disease due to neurocysticercosis are variable and thus different clinical approaches and treatments are required. Despite recent advances, treatments remain either suboptimal or based on poorly controlled or anecdotal experience. A better understanding of basic pathophysiologic mechanisms including parasite survival and evolution, nature of the inflammatory response, and the genesis of seizures, epilepsy, and mechanisms of anthelmintic action should lead to improved therapies.

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

OBJECTIVE. The diagnosis of pulmonary tuberculosis presents challenges in children, because symptoms are nonspecific, sputa are not accessible, and Mycobacterium tuberculosis cultures and smears often are negative. The Microscopic Observation Drug Susceptibility technique is a simple, inexpensive method for Mycobacterium tuberculosis isolation with superior speed and sensitivity over Lowenstein-Jensen culture in studies of adults with pulmonary tuberculosis. The objective of this study was to determine whether Microscopic Observation Drug Susceptibility culture can improve the sensitivity and the speed of Mycobacterium tuberculosis recovery among Peruvian children with symptoms suggestive of pulmonary tuberculosis.METHODS. Two specimens of each type (gastric aspirate, nasopharyngeal aspirate, and stool specimens) were collected from each patient, examined by auramine stain, and cultured by Microscopic Observation Drug Susceptibility and Lowenstein-Jensen techniques. Patients (n = 165) were enrolled between April 2002 and February 2004 at the Instituto de Salud del Niño, the major pediatric hospital in Lima, Peru. Inclusion criteria were age ≤12 years, Stegen-Toledo clinical score ≥5 points, and absence of antituberculous therapy. The main outcome measurements were (1) proportion of specimens that were culture positive by Microscopic Observation Drug Susceptibility versus Lowenstein-Jensen and (2) days required for positive culture result, stratified by specimen type and auramine stain result.RESULTS. Fifteen (9%) patients had at least 1 positive Mycobacterium tuberculosis culture (from stool in 3 cases, nasopharyngeal aspirate in 8 cases, and gastric aspirate in 15 cases). Thirty-eight culture-positive specimens were obtained (22 gastric aspirate, 12 nasopharyngeal aspirates, and 4 stools). Microscopic Observation Drug Susceptibility provided significantly more positive cultures than Lowenstein-Jensen (33 of 38 specimens culture positive by Microscopic Observation Drug Susceptibility vs 21 of 38 by Lowenstein-Jensen). This was attributed to enhanced recovery of Mycobacterium tuberculosis from auramine-negative specimens (19 of 23 by Microscopic Observation Drug Susceptibility vs 9 of 23 by Lowenstein-Jensen), in contrast to similar detection rates for the 2 tests with auramine-positive samples. Similar results were found for analyses that were limited to gastric aspirates. Isolation was faster by Microscopic Observation Drug Susceptibility than Lowenstein-Jensen.CONCLUSIONS. Isolation of Mycobacterium tuberculosis from children with suspected pulmonary tuberculosis by Microscopic Observation Drug Susceptibility demonstrated greater yield and faster recovery than by Lowenstein-Jensen method, significantly improving local capabilities to detect pediatric tuberculosis in resource-poor settings.

Background Recent data demonstrate the utility of the string test for the diagnosis of sputum-scarce HIV-associated TB in adults. We hypothesized that, if well-tolerated by children, this simple tool might offer a breakthrough in paediatric TB diagnosis. Thus the objective of this study, undertaken in the paediatric service of the Hospital Nacional Dos de Mayo, Lima, Perú, was to determine the tolerability and acceptability of the string test to paediatric TB suspects, their parents and nursing staff. Methods 22 paediatric subjects aged 3–14 years (median 8) under investigation for TB were invited to undergo 2 string tests (four-hour downtime each). Subjective and objective pain and discomfort rating scales were used to assess the perception of the subject, parent and attending nurse. Results Patients as young as 4 years tolerated the procedure extremely well with 84% willing to undergo a second procedure. Peak discomfort at the time of swallowing and of string retrieval was mild (30% of maximum possible score) and brief as judged by visual analogue ratings and objective indicators. Good concordance of parent/child and objective/subjective ratings strengthened the validity of these findings. Conclusion The string test is well tolerated and achievable for most paediatric TB suspects as young as 4 years. A formal prospective paediatric efficacy study is now needed.

There is a paucity of HIV autopsy data from South America and none that document the postmortem findings in patients with HIV/AIDS in Peru. The purpose of this autopsy study was to determine the spectrum of opportunistic infections and the causes of mortality in HIV-positive patients at a public hospital in Lima. Clinico-epidemiological information regarding HIV infection in Peru is also reviewed. Sixteen HIV-related hospital postmortems, performed between 1999 and 2004, were included in this retrospective analysis. The primary cause of death was established in 12 patients: one died of neoplasia and 11 of infectious diseases, including 3 from pulmonary infection, 7 from disseminated infection, and 2 from central nervous system infection (one case had dual pathology). Opportunistic infections were identified in 14 cases, comprising cytomegalovirus, histoplasmosis, cryptococcosis, toxoplasmosis, Pneumocystis pneumonia, aspergillosis, tuberculosis, varicella zoster virus, and cryptosporidiosis. Fourteen patients had at least one AIDS-related disease that had been neither clinically suspected nor diagnosed premortem. Moreover, 82% of the diagnoses considered to be of important clinical significance had not been suspected antemortem. The spectrum and frequency of certain opportunistic infections differed from other South American autopsy studies, highlighting the importance of performing HIV/AIDS postmortems in resource-limited countries where locally specific disease patterns may be observed.

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.

Solute carrier family 11a member 1 (Slc11a1; formerly natural resistance-associated macrophage protein 1) encodes a late endosomal/lysosomal protein/divalent cation transporter, which regulates iron homeostasis in macrophages. During macrophage activation, Slc11a1 exerts pleiotropic effects on gene regulation and function, including generation of nitric oxide (NO) via inducible NO synthase (iNOS; encoded by Nos2A) and of reactive oxygen intermediates (ROI) via the phagocyte oxidase complex. As NO and ROI have potent antimicrobial activity in macrophages, it was assumed that their activities would contribute to Slc11a1-regulated innate resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani. By intercrossing mice with gene disruptions at Nos2A and Cybb (encoding gp91phox, the heavy chain subunit of cytochrome b-245 and an essential component of phagocyte NADPH oxidase) onto equivalent Slc11a1 wild-type and mutant genetic backgrounds, we demonstrate that neither iNOS nor gp91phox activity is required for Slc11a1-mediated innate resistance to either infection. Functional gp91phox and iNOS are required to control S. enterica serovar Typhimurium in non-Slc11a1-regulated phases of infection. For L. donovani, an organ-specific requirement for iNOS to clear parasites from the spleen was observed at 50 days post-infection, but neither iNOS nor gp91phox influenced late-phase infection in the liver. This contrasted with Leishmania major infection, which caused rapid lesion growth and death in iNOS knockout mice and some exacerbation of disease with gp91phox deficiency. This highlights the adaptive differences in tissue and cellular tropisms between L. donovani and L. major and the different genes and mechanisms that regulate visceral versus cutaneous forms of the disease.

There is an urgent need for new tools to improve our ability to diagnose tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in resource-poor settings. In a retrospective analysis undertaken in a region with a high incidence of TB, we evaluated the performance of the microscopic observation drug susceptibility assay (MODS), a novel assay developed in Perú which uses an inverted light microscope and culture in Middlebrook 7H9 broth to detect mycobacterial growth. MODS detected 94.0% of 1,908 positive sputum cultures, whereas Löwenstein-Jensen (LJ) culture detected only 86.9% ( P < 0.001). The median time to culture positivity was 8 days (compared to 16 days for the same 208 samples by LJ culture; P < 0.001, Wilcoxon signed rank test). The results obtained by direct susceptibility testing using MODS demonstrated excellent concordance for isoniazid and rifampin and the detection of multidrug resistance with those obtained by indirect colorimetric methods: the microplate Alamar Blue assay (MABA) and the tetrazolium microplate assay (TEMA) (agreement, 95, 98, and 94%; kappa values, 0.8, 0.7, and 0.7, respectively). The concordance of the susceptibility testing results for ethambutol and streptomycin was poor. MODS is a novel assay which can detect the organisms responsible for TB and MDR-TB directly from sputum inexpensively, rapidly, and effectively. A comprehensive prospective evaluation of MODS is under way in Perú, and independent validation in nonresearch laboratories should be undertaken at the earliest opportunity.