Introduction of the rapid MTBDRplus diagnostic led to a significant improvement in time to multidrug-resistant tuberculosis treatment initiation. However, delays in laboratory processing, result reporting, and therapy initiation require reduction to have maximum impact on treatment outcomes and transmission interruption.
Keywords: multidrug-resistant tuberculosis, MTBDRplus, rapid molecular diagnostic
Abstract
Background. Diagnosis of drug resistance and timely initiation of multidrug-resistant (MDR) tuberculosis therapy are essential to reduce transmission and improve patient outcomes. We sought to determine whether implementation of the rapid MTBDRplus diagnostic shortened the time from specimen collection to patient MDR tuberculosis therapy initiation.
Methods. We conducted a retrospective cohort analysis of 197 MDR tuberculosis patients treated at Brewelskloof, a rural tuberculosis hospital in Western Cape Province, South Africa, between 2007 and 2011.
Results. Eighty-nine patients (45%) were tested using conventional liquid culture and drug susceptibility testing (DST) on solid medium and 108 (55%) were tested using the MTBDRplus assay after positive acid-fast bacilli or culture. Median time from sample taken to therapy initiation was reduced from 80 days (interquartile range [IQR] 62–100) for conventional DST to 55 days (IQR 37.5–78) with the MTBDRplus. Although the laboratory processing time declined significantly, operational delays persisted both in the laboratory and the clinical infrastructure for getting patients started on treatment. In multivariate analysis, patients tested using the MTBDRplus test had a reduced risk of starting treatment 60 days or more after sputum collection of 0.52 (P < .0001) compared with patients tested with culture-based DST, after adjustment for smear status and site of disease.
Conclusions. Use of MTBDRplus significantly reduced time to MDR tuberculosis treatment initiation. However, DST reporting to clinics was delayed by more than 1 week due, in part, to laboratory operational delays, including dependence on smear and culture positivity prior to MTBDRplus performance. In addition, once MDR tuberculosis was reported, delays in contacting patients and initiating therapy require improvements in clinical infrastructure.
Successful tuberculosis control depends on early diagnosis and effective treatment of active cases in order to stop spread to new individuals. Globally, the most widely used test to detect active tuberculosis has been smear microscopy, which routinely misses half of all cases and does not determine whether the patient is infected with a drug-resistant strain of Mycobacterium tuberculosis [1]. Culture-based detection followed by culture-based drug susceptibility testing (DST) delays the diagnosis by weeks to months. Recognition of drug resistance and timely initiation of effective therapy are essential to reducing transmission and improving tuberculosis patient outcomes. Patients with multidrug-resistant (MDR) tuberculosis, defined as resistance to isoniazid (INH) and rifampin (RIF), and extensively drug-resistant (XDR) tuberculosis, defined as MDR tuberculosis plus resistance to a fluoroquinolone and an injectable agent, have significantly worse treatment outcomes than patients infected with drug-susceptible disease, especially when patients are treated with standard first-line regimens [2–4].
Recognizing this urgent need to improve laboratory capacity to detect drug resistance at time of tuberculosis diagnosis, the World Health Organization (WHO) has endorsed the use of new, rapid molecular diagnostics, including the Genotype MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) and Xpert MTB/RIF (Cepheid, Sunnyvale, CA) [5, 6]. MTBDRplus is a molecular line probe assay that detects mutations in the rpoB gene, which is associated with RIF resistance, and in the katG gene and the inhA promoter region, which are associated with INH resistance. Initial evaluation studies of MTBDRplus focused on the test's accuracy in identifying MDR tuberculosis, reporting high sensitivity and specificity compared with culture-based DST [7, 8]. In 2007, researchers performed a large demonstration project in the high-volume laboratory of the National Health Laboratory Services (NHLS) in Cape Town, South Africa, and reported interpretable results from the molecular assay in 1–2 days [9]. In addition, they estimated that total turnaround time from sample collection to diagnosis should be <7 days, including specimen transport, time to perform smear microscopy, and reporting of results.
Based on these findings and the WHO endorsement, MTBDRplus replaced traditional culture-based DST for the first-line drugs INH and RIF for all smear-positive and culture-positive specimens in the Western Cape Province, South Africa, in mid-2008. In this study, we sought to determine whether implementation of this test shortened the time from specimen collection to patient MDR treatment initiation in a rural district in South Africa.
METHODS
Study Population
We conducted a retrospective cohort study of all patients who initiated a first MDR tuberculosis treatment regimen at Brewelskloof Hospital, the tuberculosis referral hospital for the rural Cape Winelands East and Overberg districts, Western Cape Province, South Africa, between 1 January 2007 and 1 January 2011. In these districts, patients initially present to their closest primary health clinic and referral hospital and are transferred to Brewelskloof once the diagnosis of MDR tuberculosis is made. The population in this region lives in small towns, where clinics are usually within walking distance, or more remotely on farms. We enrolled patients who had culture-confirmed tuberculosis with documented resistance to INH and RIF and were subsequently started on a standardized MDR tuberculosis treatment regimen (including second-line drugs such as a fluoroquinolone and an injectable agent). We excluded patients if they had been previously treated for MDR or RIF-monoresistant tuberculosis because they were started on MDR therapy prior to DST result availability. We also excluded patients if the type of DST performed was not recorded. Hospital records were abstracted using a standardized form for patient age, gender, race, and body mass index (BMI); human immunodeficiency virus (HIV) status, CD4 count, and antiretroviral therapy (ART) use; substance abuse history; marital status; town vs farm address; prior tuberculosis history; date of sputum collection; acid-fast bacilli (AFB) sputum grade; site of disease; and MDR treatment start date. The study protocol was approved by the Health Research Ethics Committee, Stellenbosch University, and the Office of Human Research Administration, Harvard School of Public Health (exemption granted of additional review).
Drug Susceptibility Testing
Patients’ specimens were collected at their primary health clinics and referral hospitals and then transported approximately 110 km for testing at the NHLS in Green Point, Cape Town, South Africa. NHLS is the tuberculosis reference laboratory for Western Cape Province and is accredited by the South African National Accreditation System to ISO15189 compliance. In South Africa during this study period, DSTs for INH and RIF resistance were ordered by clinicians or nurses if patients had previously been treated for tuberculosis, had persistently positive smears or cultures after 5 months of therapy, were in contact with confirmed drug-resistant cases, or based on clinical discretion.
For all patient specimens, a concentrated auramine smear was prepared and examined followed by culture using the BACTEC MGIT 960 system (BD Diagnostics Systems, Sparks, MD), including mycobacterial growth indicator tubes with PANTA and OADC [9]. Positive cultures were those that were positive for AFB using Ziehl-Neelsen staining and subsequently confirmed as M. tuberculosis complex using p-nitrobenzoic acid testing or the Capilia tuberculosis immunochromatographic assay post-2009. Prior to mid-2008, DST was performed indirectly using the proportion method on Middlebrook 7h11 agar slants with 1.0 µg/mL RIF and 0.2 µg/mL INH. After mid-2008, the rapid diagnostic MTBDRplus replaced culture-based DST. DNA was extracted from a portion of decontaminated sediment, followed by multiplex polymerase chain reaction amplification and reverse hybridization using the MTBDRplus test, according to the manufacturer's instructions [9]. We abstracted NHLS records using a standardized form for type of DST performed; date specimen logged at laboratory; date auramine smear reported; and date when genotypic or culture DST result was reported via fax to the facility from which the patient initially had the sample collected.
Statistical Methods
We looked for a linear trend in decreasing time to reporting results in the time period before mid-2008 in order to rule out the decrease in time being due to a secular trend rather than the rapid diagnostic. We dichotomized time from specimen collection to MDR treatment initiation at ≥60 days or less, which is the time when patients on first-line empiric therapy are first evaluated for response to treatment with repeat AFB smear and culture. We used Poisson regression with robust variance estimators to estimate the univariable relative risk of initiation of MDR treatment ≥60 days after specimen collection and potential risk factors including type of DST performed [10]. Characteristics associated with initiation of MDR treatment ≥60 days after sputum collection at a P < .20 in univariate analysis were assessed in multivariate analyses. To account for missing data, we performed multivariate regression analyses on datasets that were multiply imputed using covariate and outcome data. We conducted the imputation using Markov chain Monte Carlo methods (SAS MI procedure; SAS Institute) [11] and pooled effect estimates across datasets. Characteristics that were significant at P < .05 or changed the effect size of the primary variable, that is, DST method, by more than 10% were kept in the multivariate model. We performed statistical analyses using SAS, version 9.2.
RESULTS
Characteristics of the Study Population
We identified 227 patients who initiated MDR tuberculosis treatment at Brewelskloof Hospital between 1 January 2007 and 1 January 2011. We excluded 19 patients with previous drug-resistant tuberculosis diagnoses: 6 had previously demonstrated RIF monoresistance and 13 had previously demonstrated or presumed MDR tuberculosis. An additional 11 patients were excluded due to lack of documentation of method of DST performed. Of the remaining 197 patients, mean age was 37.1 years (SD 12.2) and 89 (45%) were female (Table 1). Mean BMI was 18.9 (SD 3.9). Sixty patients (30%) were HIV positive and 3 (1.5%) were not tested for HIV. Among the 56 HIV-positive patients with available CD4 counts, the median was 183 (interquartile range [IQR] 66.5–281). Sixty-nine patients (38%) were smear positive. One hundred seventy-one patients (88%) had only pulmonary disease, and 23 (12%) had extrapulmonary or both pulmonary and extrapulmonary disease. Eighty-nine patients (45%) received culture-based DST and 108 (55%) received the MTBDRplus test.
Table 1.
Demographic and Clinical Characteristics of Multidrug-Resistant Tuberculosis Patients Tested With a Culture-Based Drug Susceptibility Test or MTBDRplus
| Characteristic | Culture-based DST, N = 89 | MTBDRplus, N = 108 |
|---|---|---|
| Female | 37/89 (41.6) | 52/108 (48.2) |
| Mean age, years (SD) | 34.7 (12) | 39.1 (12) |
| Race | ||
| Cape Colored | 75/89 (84.3) | 78/108 (72.2) |
| Black | 13/89 (14.6) | 29/108 (26.9) |
| White | 1/89 (1.1) | 1/108 (0.9) |
| Mean BMI, N = 161 (SD) | 18.7 (3.6) | 18.4 (4.1) |
| HIV positive | 21/87 (24.1) | 39/107 (36.5) |
| Median CD4, N = 56 (IQR) | 209 (85–289) | 179 (61–273) |
| ART at start MDR tuberculosis episode | 2/21 (9.5) | 6/39 (15.4) |
| New tuberculosis case | 16/88 (18.2) | 17/105 (16.2) |
| Positive AFB smear | 36/87 (41.4) | 33/95 (34.7) |
| Site of disease | ||
| Pulmonary | 80/89 (89.9) | 91/105 (86.7) |
| Extrapulmonary | 1/89 (1.1) | 2/105 (1.9) |
| Both pulmonary + extrapulmonary | 8/89 (9) | 12/105 (11.4) |
| Married | 19/87 (21.8) | 32/104 (30.8) |
| Alcohol use | 56/85 (65.9) | 57/102 (55.9) |
| Tobacco use | 71/88 (80.7) | 68/100 (68) |
| Drugs use | 11/80 (13.8) | 11/99 (11.1) |
| Town address | 57/89 (64) | 76/106 (71.7) |
Data are proportion of patients (%), unless otherwise indicated.
Abbreviations: AFB, acid fast bacilli; ART, antiretroviral therapy; BMI, body mass index (calculated as the weight in kilograms divided by the square of the height in meters); DST, drug susceptibility test; HIV, human immunodeficiency virus; IQR, interquartile range; MDR, multidrug resistant; SD, standard deviation.
Median Time to MDR Treatment Initiation
The median time to MDR treatment initiation was 80 days (IQR 62–100) in patients tested with culture-based DST in contrast to 55 days (IQR 37.5–78) in patients tested with MTBDRplus (Table 2). This reduction in time was restricted to the laboratory processing phase. Laboratory processing time declined from a median of 55 days (IQR 46–66) for culture-based testing to a median of 27 days (IQR 20–34) when using MTBDRplus. Among those patients who were tested with the MTBDRplus, smear-positive cases were processed by the laboratory a median of 22 days (IQR 13–26) before resistance was reported compared with a median of 29.5 days (IQR 24–38.5) for smear negatives. The time required to transport the sample from regional facilities to NHLS remained a median of 2 days for both DST groups. From the day that the laboratory reported resistance, patients with extrapulmonary disease were started on treatment a median of 7 days (IQR 2–17) later, whereas patients with pulmonary-only disease were started a median of 21 days (IQR 11–45) later. There was no linear trend in decreasing time to reporting results in the time period before mid-2008.
Table 2.
Median Time Delay by Drug Susceptibility Testing Method, Days (Interquartile Range)
| Variable | Culture-Based DST | MTBDRplus | Total N |
|---|---|---|---|
| Specimen collection to start MDR treatment | 80 (62–100) | 55 (37.5–78) | 197 |
| Specimen collection to specimen arrived at laboratory | 2 (1–3) | 2 (1–4) | 192 |
| Laboratory processing time | 55 (46–66) | 27 (20–34) | 160 |
| Clinic notification of DST result to start MDR treatment | 19 (9–38) | 20 (9–38) | 161 |
Abbreviations: DST, drug susceptibility test; MDR, multidrug resistant.
Risk Factors for Treatment Initiation Delay
Sixty-four (59%) patients who were tested with MTBDRplus started treatment before 60 days vs 19 MTBDRplus (21%) patients who were tested with culture-based DST. In univariate regression, receipt of the MTBDRplus test (risk ratio [RR] = 0.52; 95% CI, .40–.67; P < .0001), a positive AFB smear (RR = 0.59; 95% CI, .44–.80; P = .0007), and having a town address (RR = 0.8; 95% CI, .63–1.00; P = .06) were associated with starting MDR treatment in less than 60 days (Table 3). Patients who had pulmonary disease only (RR = 2.02; 95% CI, 1.08–3.79; P = .029) and who smoked or had smoked in the past (RR = 1.35; CI, .98–1.86; P = .07) were more likely to start MDR treatment at 60 days or greater. In multivariate analysis, patients tested with MTBDRplus had a reduced risk of starting treatment 60 days or more after sputum collection of 0.52 (95% CI, .41–.67; P < .0001) compared with patients tested with culture-based DST, after adjustment for smear status and site of disease. Tobacco use and town address did not meet criteria to remain in the final multivariate model.
Table 3.
Predictors of Multidrug-Resistant Treatment Initiation ≥60 Days After Specimen Collection
| Univariate Analysis |
Multivariate Analysisa |
||||
|---|---|---|---|---|---|
| Variable | N | Risk Ratio (95% CI) | P Value | Risk Ratio (95% CI) | P Value |
| MTBDRplus | 197 | 0.52 (.40–.67) | <.0001 | 0.52 (.41–.67) | <.0001 |
| Female | 197 | 0.88 (.69–1.13) | .32 | … | |
| Age, years | 197 | 1.00 (.99–1.01) | .97 | … | |
| Cape Colored | 197 | 1.14 (.83–1.55) | .42 | … | |
| BMI | 161 | 1.01 (.98–1.04) | .56 | … | |
| HIV positive | 194 | 0.82 (.61–1.09) | .17 | … | |
| New tuberculosis case | 193 | 1.05 (.78–1.43) | .74 | … | |
| Positive AFB smear | 182 | 0.59 (.44–.80) | .0007 | 0.61 (.46–.81) | .0006 |
| Pulmonary | 194 | 2.02 (1.08–3.79) | .029 | 1.91 (1.00–3.61) | .048 |
| Married | 191 | 0.97 (.74–1.28) | .83 | … | |
| Alcohol use | 187 | 1.13 (.87–1.46) | .35 | … | |
| Tobacco use | 188 | 1.35 (.98–1.86) | .07 | … | |
| Drugs use | 179 | 1.03 (.71–1.50) | .87 | … | |
| Town address | 195 | 0.80 (.63–1.00) | .06 | … | |
Abbreviations: AFB, acid fast bacilli; BMI, body mass index (calculated as the weight in kilograms divided by the square of the height in meters); CI, confidence interval; HIV, human immunodeficiency virus.
a N = 197 for multivariate analysis after multiple imputation.
DISCUSSION
The introduction of the rapid molecular diagnostic, MTBDRplus, significantly reduced the time from specimen collection to initiation of MDR tuberculosis treatment from a median of 80 days to 55 days for patients treated at this rural tuberculosis hospital in Western Cape Province, South Africa. Patients who were tested with the rapid test were half as likely to start MDR tuberculosis treatment more than 60 days after specimen collection compared with patients who were tested with culture-based DST. All reduction in time occurred in the laboratory, supporting the hypothesis that the rapid diagnostic itself is responsible for decreasing the time to MDR tuberculosis treatment start. In addition to knowledge of the sensitivity and specificity of these new molecular diagnostics, it is critical to have these real results of time delays in order to predict any rapid diagnostic's impact on disease diagnosis, management, and transmission.
Despite this improvement in the time to appropriate treatment, the delays in initiating patients on therapy still far exceeded the 1 week anticipated based on initial demonstration projects of MTBDRplus [9]. These persistent delays in time to treatment initiation are due to multiple operational issues: sample transportation, laboratory-based diagnostic, and patient notification and admission to hospital. In addition, some of this delay is due to the protocol for processing smear-negative samples. Smear-positive samples tested with MTBDRplus spent less time in the laboratory, a median of 22 days, compared with smear-negative samples, which took a median of 29.5 days for laboratory processing. When samples are smear positive, the laboratory technician performs the MTBDRplus immediately, whereas with smear-negative samples, the technician first confirms tuberculosis presence by culture prior to use of MTBDRplus. WHO has endorsed a testing strategy whereby culture or smear positivity is required prior to use of MTBDRplus because the rapid test performs best under these conditions, with higher sensitivity in smear-positive samples than in smear-negative samples [5, 12]. However, only a minority of patients in this study sample had smear-positive disease (38%), and patients with HIV/tuberculosis coinfection are more likely to have smear-negative disease [13, 14]. The low smear-positive rate in this cohort may be due, in part, to the fact that patients infected with MDR tuberculosis often receive first-line therapy prior to DST and, therefore, are more likely to be smear negative because the partially active regimen can decrease the bacillary load initially, which may, in turn, induce time delays in diagnosis. A rapid DST must perform well on initial decontaminated specimens for both smear-positive and smear-negative cases in order to avoid delays and to have its fullest impact. Use of rapid molecular diagnostics, including Xpert MTB/RIF, is the current strategy adopted to identify MDR tuberculosis patients early in order to get them on effective therapy more rapidly [5, 6].
Even for smear-positive cases, time in the laboratory took a median of 22 days, much longer than expected for a test that can be read within 48 hours, despite the NHLS subscribing to external quality control and proficiency testing programs for the MTBDRplus assay. Since the inception of the program in 2008, the NHLS has been in compliance of having interpretable MTBDRplus test results in less than 7 days (Marinus Barnard, personal communication). Therefore, further investigations are needed to understand the complex factors contributing to total laboratory reporting delay, particularly in the context of high-volume settings where up to 50 000 first-line DSTs are performed annually. Elimination of these laboratory delays would potentially reduce DST reporting by an additional 2 weeks.
The other patient characteristic that significantly predicted treatment delay was the site of disease. After we adjusted for smear status and type of DST, we found that patients who had extrapulmonary disease, either alone or in addition to pulmonary tuberculosis, were started on treatment significantly sooner than patients with pulmonary disease alone. This difference may reflect that patients with extrapulmonary disease are sicker and more likely to be hospitalized prior to diagnosis of MDR, thus more easily started on treatment once the laboratory reports the diagnosis.
After the MDR result was transmitted from the laboratory to the clinic or hospital, MDR tuberculosis treatment initiation was still delayed by a median of 20 days. In this rural population, patients must be contacted once a result returns; patients do not always have telephones or other mechanisms that facilitate direct communication. In addition, the first 6 months of MDR treatment have been most often administered in a hospital in South Africa because injectables can be difficult to deliver in the outpatient setting and for infection control purposes [15]. Treatment initiation time could be shortened if patients are given the option of receiving injections at their local clinics; ambulatory MDR treatment is being piloted in townships in Cape Town and other resource-limited settings globally [16–19].
We note limitations to our study. First, patients were enrolled at time of treatment initiation rather than when specimens were collected. This means that we did not include any patients who tested positive for MDR but were lost to follow-up prior to starting treatment. Given that longer treatment delays are likely to increase loss to follow-up, we believe that including those patients would likely have only increased the difference we detected here. Second, it is not clear if our findings can be generalized to urban or even other rural settings because our results reflect the specific infrastructure for MDR diagnosis and treatment initiation in this setting. However, we believe the lessons learned from this project do have implications for other settings by identifying operational steps where delays can occur even with a rapid diagnostic in use. Third, we considered the possibility that the 2 cohorts were from different time periods and that laboratory practices may have differed between these 2 periods. To check for this possibility, we looked for a linear trend in decreasing time to reporting results prior to mid-2008 but did not identify one. The only formal change in the laboratory after mid-2008 was the introduction of the rapid diagnostic. Also, the only changes in time to treatment initiation occurred in the laboratory between these 2 time periods (not during transportation or from time of results reporting to patients’ first MDR treatment dose). For these reasons, we felt this was a reasonable approach to evaluating the association of time to MDR tuberculosis treatment initiation and type of DST performed.
Overall, our study shows that the introduction of the MTBDRplus test led to a significant reduction in time to MDR treatment initiation and supports the importance of rapid diagnostic tests for the improvement of MDR tuberculosis detection and treatment. The delays we found in initiating patient treatment compared with expectations based on the initial demonstration studies emphasize the importance of research on operational effectiveness in real clinical settings for all new technologies, such as Xpert MTB/RIF, which is currently being extensively implemented and could run into similar delays. Furthermore, a rapid test for detection of both INH and RIF is critically needed, particularly one that will perform well on both smear-positive and smear-negative samples. In addition, once MDR tuberculosis is detected, a clinical infrastructure that supports rapid transmission of that information to patients and initiation of therapy is necessary in order to have the greatest impact on improving outcomes and decreasing transmission.
Notes
Financial support. This work was supported by a Burroughs Wellcome Fund/American Society of Tropical Medicine and Hygiene Postdoctoral Fellowship in Tropical Infectious Diseases (K. R. J.), the National Institutes of Health/Fogarty International Center (1K01 TW009213; K. R. J.), and a Partner Center of Excellence Travel Grant (E. A. K.).
Potential conflicts of interest. D. T. has received an honorarium for a lecture from BD Diagnostics. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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