Patricia Sheen

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.

Upper-Room Ultraviolet Light and Negative Air Ionization to Prevent Tuberculosis Transmission

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.

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.

Improved detection of Mycobacterium tuberculosis in peruvian children by use of a heminested IS6110 polymerase chain reaction assay

A novel heminested IS6110 polymerase chain reaction (PCR) assay was evaluated as a tool for diagnosing tuberculosis in 222 children. In an analysis of 392 specimens (gastric aspirates, nasopharyngeal aspirates, and sputum samples), results of PCR were compared with those of 3 culture methods, acid-fast bacillus (AFB) staining, and clinical assessment by the Stegen-Toledo score. The sensitivity of PCR (67%) was comparable to that of the 3-culture method (71%) and was significantly higher than that of Löwenstein-Jensen culture (54%) or AFB stain (42%) for children with highly probable tuberculosis. PCR detection rates for culture-positive specimens were 100% for smear-positive samples and 76.7% for smear-negative samples. The specificity of PCR was 100% in control children. Compared with culture, PCR demonstrated a sensitivity of 90.4%, a positive predictive value of 89%, a specificity of 94%, and a negative predictive value of 95% (kappa=.85). With clinical assessment as the standard, PCR had a sensitivity of 71%, a positive predictive value of 92%, a specificity of 95%, and a negative predictive value of 79% (kappa=.67). PCR is a rapid and sensitive method for the early diagnosis of pediatric tuberculosis.

Detection of Myobacterium tuberculosis in nasopharyngeal aspirate samples in children

At the Institute de Salud del Nino in Lima, Peru, most cases of pulmonary tuberculosis are diagnosed and referred to peripheral health centres for treatment. Tuberculosis, however, caused 137 admissions in 1996, of whom 45% were children younger than 5 years. Paediatric tuberculosis is difficult to diagnose, and is generally made on the basis of clinical and epidemiological criteria, including signs and symptoms, chest radiography, percussion and postural drainage, and, most importantly, history of close contact with a patient with pulmonary tuberculosis. Bacteriological confirmation, however, is more difficult. Children are commonly infected with a small load of Mycobacterium tuberculosis and generally they cannot produce an adequate sputum sample. For this reason, 50–60% of children may be diagnosed with pulmonary tuberculosis in the absence of a positive culture or sensitivity testing. Because there is no efficient way to confirm the diagnosis, uninfected children may be treated unnecessarily, especially in areas with a low prevalence. Alternative ways of obtaining bronchial secretions are used to improve the yield of culture results;

Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTIn−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.

Effect of pyrazinamidase activity on pyrazinamide resistance in Mycobacterium tuberculosis

Resistance of Mycobacterium tuberculosis to pyrazinamide is associated with mutations in the pncA gene, which codes for pyrazinamidase. The association between the enzymatic activity of mutated pyrazinamidases and the level of pyrazinamide resistance remains poorly understood. Twelve M. tuberculosis clinical isolates resistant to pyrazinamide were selected based on Wayne activity and localization of pyrazinamidase mutation. Recombinant pyrazinamidases were expressed and tested for their kinetic parameters (activity, k(cat), K(m), and efficiency). Pyrazinamide resistance level was measured by Bactec-460TB and 7H9 culture. The linear correlation between the resistance level and the kinetic parameters of the corresponding mutated pyrazinamidase was calculated. The enzymatic activity and efficiency of the mutated pyrazinamidases varied with the site of mutation and ranged widely from low to high levels close to the corresponding of the wild type enzyme. The level of resistance was significantly associated with pyrazinamidase activity and efficiency, but only 27.3% of its statistical variability was explained. Although pyrazinamidase mutations are indeed associated with resistance, the loss of pyrazinamidase activity and efficiency as assessed in the recombinant mutated enzymes is not sufficient to explain a high variability of the level of pyrazinamide resistance, suggesting that complementary mechanisms for pyrazinamide resistance in M. tuberculosis with mutations in pncA are more important than currently thought.

Distinctiveness of Mycobacterium tuberculosis Genotypes from Human Immunodeficiency Virus Type 1-Seropositive and -Seronegative Patients in Lima, Peru

ABSTRACT Genotypic analysis of Mycobacterium tuberculosis isolates obtained from human immunodeficiency virus type 1 (HIV-1)-seropositive (n = 80) and -seronegative (n = 25) patients from Lima, Peru, revealed two distinct genotypes correlating with the host immune status. While the level of intrastrain diversity of DNA fingerprints of HIV-seropositive isolates was less pronounced, these isolates showed many clonal groupings.

Campylobacter Transmission in a Peruvian Shantytown: A Longitudinal Study Using Strain Typing of Campylobacter Isolates from Chickens and Humans in Household Clusters

Campylobacter jejuni is a major cause of pediatric diarrhea in developing countries-free-ranging chickens are presumed to be a common source. Campylobacter strains from monthly surveillance and diarrhea cases were compared by means of restriction-fragment length polymorphism (RFLP), rapid amplified polymorphic DNA, and Lior serotyping. RFLP analysis of 156 human and 682 avian strains demonstrated identical strains in chickens and humans in 29 (70.7%) of 41 families, and 35%-39% of human isolates from diarrhea and nondiarrhea cases were identical to a household chicken isolate. Isolation of the same RFLP type from a household chicken and a human within 1 month was highly protective against diarrhea (odds ratio, 0.07; P<.005). Campylobacter strains from symptomatic humans were unlikely to be identical to strains recently carried by household chickens, limiting the potential benefits from household-based control measures.

Evaluation of Molecular Tools for Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Stool Specimens from Patients with Pulmonary Tuberculosis

ABSTRACT 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.