Introduction Seminal experiments demonstrating airborne tuberculosis (TB) transmission by droplet nuclei were performed by Riley and coworkers in the 1950s–1960s [1,2]. Guinea pigs acquired TB by breathing exhaust air from a TB ward. The studies demonstrated TB transmission from a minority of patients, marked variability in patient infectiousness, and reduced infectiousness following initiation of effective chemotherapy [1–3]. These classic studies were recently recreated in Lima, Perú, in the modern era of HIV infection and multidrug-resistant (MDR) TB, and again showed striking variability in patient infectiousness [4]. The strongest predictor of TB patient infectiousness is sputum smear status [5–7]. However, considerable variation exists in TB prevalence amongst contacts of smear-positive patients [5], and the importance of smear-negative transmission has also been demonstrated [8]. Other determinants of infectiousness include lung cavitation and cough frequency [9], and cough-inducing procedures have been associated with extensive transmission [10–12]. Additional factors are likely and may include sputum volume or consistency, and TB strain variables such as ability to survive in the airborne state. Recent studies detected culture-positive cough-generated aerosols in only 25% of new TB patients [13]. The relative infectiousness of MDR versus drug-susceptible TB remains controversial. The patients with drug-susceptible TB studied by Riley were four to eight times more likely to infect guinea pigs than were those with drug-resistant disease [1–3]. Epidemiological studies of contacts of both drug-resistant and drug-susceptible TB infected patients have shown no difference in relative transmissibility [14–16], whilst studies utilising molecular epidemiology have had conflicting results [17–20]. Similarly, the effect of HIV infection on TB infectiousness remains disputed [21]. The beneficial effect of anti-TB chemotherapy on reducing TB infectiousness is well known, but this presupposes that the treatment administered is effective. The inadequate treatment of MDR TB and the emergence of extensively drug-resistant (XDR) TB therefore have important consequences for hospital TB infection control policies. In this paper we report the results of molecular fingerprinting to determine which patients had infected which guinea pigs in our in vivo air sampling model above a TB ward. We investigate factors associated with TB transmission from this group of HIV-coinfected TB patients with a high prevalence of MDR TB. Methods Setting An airborne infection study facility was constructed on the roof of a TB-HIV ward at Hospital Nacional Dos de Mayo, Lima, Perú, as previously described [4]. All air from two 4-bedded negative-pressure patient rooms passed over guinea pigs in the study facility before being exhausted into the atmosphere. Airflow from the ward and into the animal facility was measured using a capture hood (Alnor, Shoreview, United States) at air injection and extraction vents. Patients The ward operated as the hospital TB-HIV service, and patient admission, management, and duration of stay were not influenced by the study. All patients were invited to join the study through written consent. An admission questionnaire recorded daily symptoms including cough, haemoptysis, and fever. Twenty-four-hour sputum collections were made daily for auramine microscopy [22] and TB culture using MODS [23]. For nonconsenting patients, anonymised unlinked information routinely available for ward infection control, including sputum microscopy and treatment, was recorded. Experimental Protocol Outbred Peruvian guinea pigs were maintained in quarantine for 1–3 mo. Animals were skin tested monthly as described [4] and induration measured 48 h later. Animals were transferred to the hospital following at least two negative skin tests to ensure freedom from TB. In May 2002, 144 animals began ward air exposure, and 6 mo later, 148 animals were added. Monthly skin tests continued throughout 505 d exposure to ward air, and positive reactors were removed for humane killing and autopsy. Evidence of TB infection was sought in lungs, mediastinal lymph nodes, spleen, and liver. Tissues were homogenised and cultured for TB as described [4]. Forty negative control guinea pigs were maintained separately, breathing fresh air. Determination of Patient Infectiousness TB drug-susceptibility testing of animal and patient strains for susceptibility to isoniazid, rifampicin, streptomycin, ethambutol, capreomycin, and ciprofloxacin was performed using the tetrazolium microplate assay [24] and DNA fingerprinting performed using spoligotyping [25]. Linkage of source patient with infected guinea pigs relied upon genotypic (identical spoligotype) and phenotypic (drug-susceptibility pattern) concordance and patient ward occupancy 3–9 wk prior to animal PPD conversion, reflecting TB incubation in these guinea pigs [26]. Individual patient infectiousness was determined using the Wells-Riley airborne infection equation (see Text S1) [27]. Determinants of patient infectiousness were assessed by univariate and multiple logistic regression in which nonsignificant factors were sequentially dropped from the model using SPSS and SAS statistical software [28]. Ethical Approval The study was approved by the Institutional Review Boards at Hospital Dos de Mayo, Asociación Benéfica PRISMA, Perú, and Imperial College London, Hammersmith Hospital Campus, UK. Animal ethical approval was obtained from the Veterinary Medicine Faculty, Universidad Nacional Mayor San Marcos, Lima, Perú, which supervised all animal work. Results Patient Characteristics There were 185 ward admissions by 161 HIV-positive patients, resulting in 2,667 patient days, comprising 118 admissions of 97 pulmonary TB patients (1,798 [67%] patient days), 33 admissions of 30 extrapulmonary TB patients (609 [23%] patient days), and 34 admissions of 34 TB suspects who subsequently had no laboratory evidence of TB (260 [10%] patient days). Of the 64 extrapulmonary disease patients or TB suspects, 59 able to produce sputum were acid-fast-bacillus smear and TB culture negative. Median length of stay was 11 d (interquartile range [IQR] 6–21 d). Monthly variations in pulmonary TB patient days according to sputum status are shown in Figure 1A. Of 66 sputum culture–positive pulmonary TB admissions, 35 (53%) were sputum smear positive and 31 (47%) were sputum smear negative. Figure 1 Pulmonary TB Patient Bed Days and TB Strain Spoligotype Pattern Compared with TB Infection in Guinea Pigs by Study Month (A) Number of bed days in each study month resulting from pulmonary TB patients, who were either smear positive (patterned bars, “+ve”), or smear negative (white bars, “−ve”) at the time of admission. (B) Number of bed days in each study month resulting from pulmonary TB patients for whom a TB strain spoligotype pattern was available. Each block of colour corresponds to one patient, and each colour to one of the eight spoligotype patterns observed in the guinea pigs. Pale yellow represents the remaining 19 patterns observed in patients whose TB was not seen in the guinea pigs. If a patient resided on the ward for a period spanning more than one study month, that patient is included in the month where they accounted for more smear positive patient bed days. The coloured blocks containing numbers correspond to the ten identified infectious patients, numbered 1 to 10 in Table 2. (C) Percentage of animal colony skin tested each study month that were PPD positive. Each colour represents one spoligotype pattern, except for pale blue, which represents ten guinea pigs culture positive for TB but for which spoligotype patterns were unavailable. White represents animals that were PPD positive but TB culture negative. Animals culture positive for TB that were PPD false negatives or that died between skin tests were included in the subsequent month's skin test. Pulmonary TB patients composed a heterogeneous group of new and existing TB diagnoses, admitted for diagnosis and treatment, adverse treatment effects, or other complications of TB or HIV infection. All patients were HIV positive, none were taking combination antiretroviral therapy, and CD4+ T cell counts were not available. Twelve pulmonary TB patients had isoniazid- or rifampicin-monoresistant strains (251 [14%] pulmonary TB patient days); 21 patients had confirmed MDR TB (434 [24%] pulmonary TB patient days); and 11 patients had presumed MDR TB (treated empirically for drug-resistant disease due to treatment failure; 143 [8.0%] pulmonary TB patient days). No patients had XDR TB. Thirty-four patients had confirmed drug-susceptible TB (687 [38%] pulmonary TB patient days); and 20 patients had presumed drug-susceptible TB treated empirically without drug-susceptibility results (275 [15%] patient days). Two patients with drug-susceptible TB acquired MDR TB with a new spoligotype pattern, which was not demonstrated to have been acquired on the ward. For MDR TB patients, significantly more patient bed days were accounted for by sputum culture-positive patients than by sputum culture-negative patients; in contrast, for non-MDR TB patients, significantly more patient bed days were accounted for by sputum culture-negative patients than by sputum culture-positive patients (both p 0.6), which may reflect low power due to the small sample. Table 3 Determinants of Patient Infectiousness: Analysis of Infectious Versus Noninfectious Patients Table 4 Determinants of Patient Infectiousness: Analysis of the Degree of Infectiousness Multiple regression analysis of data from all patients identified as having caused TB transmission demonstrated that the degree of patient infectiousness was independently significantly associated with MDR TB; patient infectiousness appeared possibly to increase with sputum-smear positivity and days on the ward without treatment, but these associations were not found to be statistically significant (Table 4). This statistical model allowed patient characteristics to be used to predict the number of infectious quanta produced per hour (q). The regression model (Table 4) indicated that: non-MDR TB, smear-negative patients with undelayed treatment had a predicted infectiousness q = 0.3; patients with MDR TB or sputum-smear positivity without treatment delay had a predicted q = 3.9; more than 2 d of treatment delay resulted in an increase in q of 1.7; sputum-smear positive MDR TB had a predicted q = 24; MDR TB and (more than 2 d of delayed treatment or sputum-smear positivity) had predicted q = 14; and sputum-smear positive MDR TB with more than 2 d of delayed treatment had a predicted q = 54. Discussion This study provides novel characterization of the heterogeneity and determinants of infectiousness of HIV-positive TB patients by applying molecular strain characterization to track airborne TB transmission to guinea pigs. This research has for the first time (to our knowledge) demonstrated that amongst HIV-positive patients TB infectiousness is extremely variable, that a few HIV-positive patients were highly infectious, and that inadequately treated MDR TB patients accounted for the great majority of TB transmission. In contrast to seminal studies of TB transmission using a similar guinea-pig method of detection half a century ago, this study was conducted in a real-life busy ward in a low-resource setting with unselected patients, composed of a heterogeneous mix of new and established diagnoses of drug-susceptible and drug-resistant TB, with varying treatment regimens. These results therefore have important implications for TB infection control, especially in the era of increasingly integrated TB and HIV care and the emergence of XDR TB strains. Average patient infectiousness over the whole study period for these HIV-positive TB patients with high rates of MDR TB was up to six times greater than that calculated for the heterogeneous mix of patients in the 1950s studies (q = 1.25) [4,27]. However, this average masks considerable variability in infectiousness between patients. Three highly infectious patients were observed, all with MDR TB, with q-values of 40, 52, and 226. It should be noted that these q-values reflect TB transmission from humans to guinea pigs. The infectious dose of M. tuberculosis for humans is unknown; hence the concept of infectious quanta used in airborne infection models [27]. For fully virulent M. tuberculosis strains just one droplet nucleus can establish infection and disease in guinea pigs, but for strains of reduced virulence for guinea pigs, up to four aerosolised colony-forming units may be required to establish a single pulmonary primary focus [30]. Thus some caution is needed in comparing the infectiousness of patients in this study with published q-values calculated for human-to-human transmission, such as q = 13 for an untreated office worker who infected 27 coworkers over 4 wk prior to diagnosis [31], and q = 250 for an outbreak associated with intubation and bronchoscopy of a TB patient [11,27]. However, comparisons can be drawn with q-values calculated for Riley's study: q = 1.25 average for all patients; q = 60 for the most infectious case, with laryngeal TB [27]. Direct comparisons should, however, be made cautiously owing to methodological differences between the studies such as the type of guinea pig used and the cutoff for a positive skin test [32]. This model for identifying infectious patients required TB strains to be not only transmissible to guinea pigs, but also sufficiently virulent to cause disease from which a strain could be recovered with corresponding spoligotype. Animals with positive PPD skin test conversions but culture negative for TB were observed throughout the study. Due to the continuous exposure to ward air, mixed infections might be expected in the animals, but in fact were not observed despite the culture of separately dissected lung foci. Because they are relatively uncommon, mixed infections were not specifically sought in patients [33]. The relative infectiousness of patients in this study was highly variable, and a patient with MDR TB was found to be highly infectious, producing 226 infectious quanta/h. The TB strain responsible, not a Beijing strain, was also seen in a second highly infectious MDR TB patient, producing 52 infectious quanta/h. This observation suggests a potentially strain-related factor involved in transmissibility, perhaps an enhanced ability to survive aerosolisation and the physical stresses of being airborne. An alternative explanation might be an effect on disease phenotype. It is interesting to note that both patients had fever and cough and produced large volumes of sputum, between 10 and 90 ml daily. Neither patient had cavitation on chest X-ray. Although no formal ear-nose-throat assessment was documented, neither was diagnosed with laryngeal TB. Both patients also spent time on the ward untreated. The first was untreated for the first 11 d of a 32 d admission before second-line drugs were commenced, due to difficulties in access to medications. The second patient had recently been commenced on suboptimal treatment (standard first-line therapy plus streptomycin) and this was suspended for ten of 26 ward days because of adverse effects. The finding that sputum smear positivity was associated with TB transmission concurs with previous studies [5–7]. The effect of treatment on the infectiousness of TB patients is also well known [2,5], with numbers of viable bacteria falling precipitously following initiation of effective chemotherapy [34,35]. Whilst some data suggest that apparent cure of MDR TB may be achieved with first-line drugs [36,37], other studies have shown poor outcomes for such patients [38]. The current study shows how suboptimal treatment of MDR TB patients is likely to facilitate ongoing TB transmission. There is conflicting evidence concerning the relative transmissibility of MDR versus drug-susceptible TB strains [14–20]. In this study, patients with MDR TB were significantly more likely than those without MDR TB to transmit TB to guinea pigs. However, this finding should be interpreted with caution because of colinearity with suboptimal treatment (5 of 6 identified infectious MDR TB patients were on suboptimal regimens, and the other had treatment initiation delayed for 11 days whilst suitable medications were acquired). Regardless, the high relative infectiousness of inadequately treated MDR TB patients demonstrated in this study underscores the importance of prompt specific treatment guided by rapid drug-susceptibility testing, rather than restricting MDR TB testing and specific therapy to patients who survive failing empiric first-line therapy, as currently happens in most low-resource settings. It also has important implications for hospital policies that allow suboptimally treated MDR TB cases to be cared for in multi-bedded rooms. The highly infectious nature of some of the HIV-positive MDR TB patients identified in this study has important implications for TB infection control. Administrative control measures that facilitate the rapid diagnosis, isolation, and prompt treatment of such patients are paramount. With increasing congregation of infectious and susceptible individuals not only in hospitals but also in such settings as antiretroviral therapy roll-out, HIV antenatal care, and voluntary counselling and testing facilities [39], environmental control measures are also of great importance. As the infectiousness of a TB source increases, the relative protection provided by dilutional room ventilation decreases [31] and may become inadequate at the relatively low levels of air exchange usually provided by mechanical ventilation. High rates of ventilation would be required to provide protection from the extremely infectious newly diagnosed MDR TB case observed in this study. Achieving this through mechanical means is an expensive solution for much of the world where TB is most prevalent. In contrast, well-designed natural ventilation [40] provides high ventilation rates for little cost, and furthermore is highly applicable to areas such as crowded waiting rooms where infectious, untreated TB patients are most likely to be encountered. TB infection control must be a priority in the current roll-out of enhanced HIV care, and should be carefully considered in the design and construction of any new infrastructure for such programmes. The need for strengthened TB infection control is also highlighted by the recent outbreak of XDR TB amongst HIV-coinfected patients in South Africa; this outbreak was predominantly nosocomial and resulted in extremely high mortality [41]. The variability of infectiousness of patients demonstrated in this study highlights the usefulness of a potential test for TB infectiousness that would allow targeted isolation of the most infectious patients in the settings where isolation facilities are sparse, as is unfortunately the case in much of the world where TB is most prevalent. One patient in our study, with MDR TB, infected over half of the guinea pig colony. The development of tests that allow early identification and isolation of such patients in a clinical setting is a research priority. There are some limitations to this study. The first is the incomplete set of spoligotyping data, with results in only 49 of 118 pulmonary TB admissions. Despite this deficiency, ten of at least 12 infectious patients were identified. It is possible that transmission occurred from other patients for whom spoligotyping was unavailable, but certain factors suggest that this is not the case. Most patients without spoligotype results had negative sputum cultures, and whilst smear-negative TB transmission occurs [8], smear-positive patients account for the majority [5–7]. In this study, 16 culture-positive patients, of whom four were smear positive, had no spoligotype result. Fortuitously, these patients were either temporally or phenotypically (drug susceptibility pattern) unrelated to the ten guinea pig clusters linked with infectious patients, excluding them as coinfectors. Indeed the two clusters of MDR TB guinea pigs with unidentified infectious sources became infected at times corresponding to the ward residency of smear-positive MDR TB patients without spoligotype results. We cannot exclude the possibility that guinea pig infections occurred from staff or visitors with TB, however all staff and visitors wore particulate respirators. It is possible that the large monoclonal outbreak observed in the guinea pigs was in fact made up of more than one strain. However, the concordant drug susceptibility data and epidemiological match with a patient on the ward at an appropriate time prior to the infections obviates the need for secondary typing of strains. The Wells-Riley model has inherent limitations [42], but these do not influence evaluations of relative infectiousness, and it allows comparison with published values of TB infectiousness calculated using the same model. The design of this study did not permit determination of the duration of patient infectiousness because of the interval of one month between skin tests, and the variability in the period required for these guinea pigs to become PPD positive following TB infection. It is possible that values for patient infectiousness are underestimates, because the entire period of a patient's hospital admission was used for the exposure duration variable in calculations, and it would normally be expected for patient infectiousness to tail off once treatment was initiated, although this would not be the case with suboptimal treatment. However, in univariate analyses patient days on the ward was not significantly associated with TB transmission. The relatively narrow age range and the small number of women amongst the patients is a further limitation of this study, because both young age and male sex have been associated with TB transmission to contacts [15,43]. Because all patients were HIV positive, our study was unable to yield evidence concerning the infectiousness of HIV-positive versus HIV-negative MDR TB patients, and this could be a future area of study using our airborne infection facility. In conclusion, this study has demonstrated the potential of HIV-positive patients with MDR TB to be highly infectious. With the great majority of TB transmission in this study occurring from inadequately treated MDR TB patients, these results identify the importance of early drug susceptibility testing and initiation of effective chemotherapy for drug-resistant TB to prevent ongoing transmission and facilitate TB control. Furthermore, this study highlights the importance of environmental control measures to prevent airborne TB transmission in crowded health care settings, especially in areas with a high prevalence of HIV infection and drug-resistant TB, and in today's era of emerging XDR TB. HIV-positive patients with unrecognised or inadequately treated MDR TB coinfection may be highly infectious, and effective TB infection control measures are essential to prevent health care facilities from disseminating drug-resistant TB. Supporting Information Text S1 Click here for additional data file.
