Impact of introducing fluorescent microscopy on hospital tuberculosis control: A before-after study at a high caseload medical center in Taiwan.

BACKGROUND: Undiagnosed tuberculosis (TB) patients hospitalized because of comorbidities constitute a challenge to TB control in hospitals. We aimed to assess the impact of introducing highly sensitive fluorescent microscopy for examining sputum smear to replace conventional microscopy under a high TB risk setting.
METHODS: We measured the impact of switch to fluorescent microscopy on the smear detection rate of culture-confirmed pulmonary TB, timing of respiratory isolation, and total non-isolated infectious person-days in hospital at a high-caseload medical center (approximately 400 TB cases annually) in Taipei. Multivariable Cox regression was applied to adjust for effects of covariates. The effect attributable to the improved smear detection rate was determined using causal mediation analysis.
RESULTS: After switch to fluorescence microscopy, median non-isolated infectious duration decreased from 12.5 days to 3 days (P<0.001). Compared with conventional microscopy, fluorescence microscopy increased sputum smear detection rate by two-fold (for all patients: from 22.8% to 48.1%, P<0.001; for patients with cavitary lung lesion: from 43% to 82%, P = 0.029) and was associated with a 2-fold higher likelihood of prompt respiratory isolation (odds ratio mediated by the increase in sputum smear detection rate: 1.8, 95% CI 1.3-2.5). Total non-isolated infectious patient-days in hospital decreased by 69% (from 4,778 patient-days per year to 1,502 patient-days per year).
CONCLUSIONS: In a high TB caseload setting, highly sensitive rapid diagnostic tools could substantially improve timing of respiratory isolation and reduce the risk of nosocomial TB transmission.

Introduction

Pulmonary tuberculosis (TB) is an airborne disease [1]. Unless promptly isolated, hospitalized patients with active pulmonary TB can transmit to both healthcare workers (HCWs) and other patients [2]. HCWs could have an incidence rate of active TB 2- to 20-fold higher than that in general population [3]. The discovery of hospital-acquired extensively drug-resistant TB, with an extremely high mortality among HIV-positive patients, further highlights this deadly hazard [4, 5]. Rapid isolation of hospitalized patients with pulmonary TB is the pivotal step to prevent nosocomial transmission [6]. However, undiagnosed TB patients hospitalized for treatment of comorbidities constitute a challenge to TB control within hospitals.

Clinical predictive rules had been proposed to guide the decision to implement respiratory isolation [7]. An expanded isolation policy which pre-emptively isolated all patients with possibility of TB achieved immediate isolation of >95% of patients with TB in low TB risk settings [8]. Nevertheless, the same policy would be impractical in a high TB risk setting because there would be too many patients to be pre-emptively isolated [9, 10]. For a laboratory diagnosis-based respiratory isolation policy, an important barrier is the limited sensitivity of conventional sputum smear [11, 12]. Compared with conventional microscopy, fluorescence microscopy has a superior sensitivity for detecting TB bacilli [13]. In 2011, World Health Organization (WHO) recommends switching conventional to fluorescence microscopy [14], under the condition that the switch should be carefully planned at country level with training, quality assurance, and monitoring on TB detection rate [14]. WHO also endorsed highly sensitive TB nucleic acid amplification test (TB-PCR) such as Xpert® MTB/RIF (Cepheid, Sunnyvale, CA) [15]. Point-of-care Xpert® MTB/RIF reduces all-cause 12-month mortality in patients positive for TB symptoms at the time of HIV diagnosis [16]. Nevertheless, to our knowledge, the effect of introducing these more sensitive diagnostic tools on reducing risk of nosocomial TB transmission has not been documented.

Taiwan is a developed country with an incidence of TB at the range of approximately 70 per 100,000 general population in 2001 [17]. In 2003, severe acute respiratory syndrome (SARS)-related chest radiograph screenings led to the discovery of a large nosocomial TB outbreak at a rehabilitation facility in Taipei, involving 65 cases of active TB in HCWs [18]. The index case of this outbreak was an elderly patient hospitalized for stroke rehabilitation without suspicion of TB [18]. Subsequent investigations found that the problem of delayed isolation of undiagnosed TB patients also existed in other hospitals [19, 20]. In response, Taiwan Centers for Disease Control (CDC) issued guidance on nosocomial TB control [21]. To facilitate laboratory diagnosis-based isolation, Medical Center A started to roll out auramine-rhodamine staining with fluorescence microscopy since 2006 and completed the switch by early 2010s. We aimed to assess whether switching from conventional microscopy to a more sensitive rapid diagnostic tool improves early detection and prompt isolation of hospitalized patients with undiagnosed TB.

Methods

Setting

Medical Center A, a leading university-affiliated general hospital having the second-highest TB caseload (approximately 400 cases annually) in Taiwan, was chosen for this study. The center in Taipei had a 2,200-bed capacity and provided both primary and tertiary referral care reimbursed by National Health Insurance (NHI). The service amount steadily increased from 2001 to 2014. There were 3,454,724 outpatient visits and 91,645 admissions in 2014, nearly 2-fold than that in 2001. Medical Center A followed the guidance on hospital respiratory isolation policies issued by Taiwan CDC [21]. Contact investigation had been expanded to all HCWs who were exposed to TB patients since 2004. Sputum smear auramine-rhodamine staining with fluorescent microscopy started in 2006. The national laboratory personnel training programs for quality assurance of acid-fast staining [21], mandated by Taiwan CDC, helped to ensure the technical proficiency and performance quality of fluorescence microscopy (see details in S1 Table) [22].

Study design

This before-after retrospective cohort study included all hospitalized patients with culture-confirmed pulmonary TB in 25 wards/units in 2001 and those in 2014. We compared the duration from admission/arrival to respiratory isolation in 2001 (conventional microscopy with Ziehl-Neelsen staining, represented the baseline situation before 2003 SARS outbreak) with that in 2014 (after full switching to fluorescent microscopy with auramine-rhodamine staining and the quality assurance program). Cox regression was used to adjust for effects of covariates. the effect mediated by improved smear detection rate was precisely identity using causal mediation analysis. The study procedure and exemption of informed consent were reviewed and approved by Research Ethics Committee of National Taiwan University Hospital (Taipei, Taiwan).

Study procedure

For each included TB case, medical and administrative records were reviewed to determine the infectious duration. A computerized data collection form was used to systematically collect the following information from the medical records: demographic data, sputum smear and culture results, presentations, comorbidities, reasons of hospitalization, and other relevant data, with pre-defined criteria.

Definitions

Hospitalized patients had typical presentations of pulmonary TB if they had: (a) a prolonged cough for >3 weeks; (b) clinical suspicion of pulmonary TB based on chest radiography, such as cavitary pulmonary lesions, upper lobe diseases, or miliary lesions; or (c) already received a confirmed diagnosis of pulmonary TB by a positive sputum culture of Mycobacterium tuberculosis, positive acid-fast stain (AFS), or positive TB PCR, before the hospitalization. The hospitalization was considered as TB-related if the chief complaint suggested an infectious etiology or the admission was for inpatient TB treatment. The hospitalization was considered comorbidities-related when the patients was admitted for management of acute complications of non-infectious diseases, such as myocardial infarction, pulmonary edema, malignancy, or acute exacerbation of chronic lung diseases.

Identification of TB cases

We retrospectively identified all cases of culture-confirmed pulmonary TB patients in 2001 (January 1 to December 31, 2001) and in 2014 (January 1 to December 31, 2014), using a computerized registry of Mycobacteriology Laboratory. The diagnosis was verified in each case with review of medical records.

Time to respiratory isolation

For each included infectious TB case, the zero time was the date of admission to the hospital or the date of arrival to emergency department (ER). The end of follow-up was the date when the patients was sent to a respiratory isolation room (event), the date of discharge (from hospital or ER) before respiratory isolation can be implemented (censored), the date of completion 14-day anti-TB treatment (censored), or the date of mortality due to any cause (censored). For patients who had multiple admissions or multiple positive sputum cultures, only the admission with or following the first positive sputum culture (the index culture) was used to calculate the Kaplan-Meier estimates for time to respiratory isolation.

Total non-isolated infectious patient-days in hospital

To estimate the total non-isolated infectious patient-days in hospital, each TB case/patient was considered infectious from 3 months prior to the first positive sputum culture unless being put in a respiratory isolation room or had already received a 14-day course of at least two in vitro active anti-tuberculous agents after the last positive sputum culture. For those who had multiple hospitalization or had ever been transferred between wards/units before being diagnosed with pulmonary TB or adequately treated, all hospitalizations or stay in each ward/unit were counted in the calculation of total infectious patient-days.

Statistical analysis

Statistical analyses were performed using SPSS 21.0 (IBM, Armonk, New York, USA). Cox regression, with backward selection, was used to adjust for covariates. Causal mediation analyses were performed using proc causalmed (based on logistic regression for isolation status on day 7) in SAS 9.4 (SAS Institute, Cary, North Carolina, USA). All analyses were two-sided. P values less than 0.05 was considered statistically significant.

Results

Time to respiratory isolation: 2001 vs. 2014

In 2001 and 2014, 180 of 403 (45%) and 81 of 301 (27%) patients with culture-confirmed pulmonary TB were hospitalized, respectively (Table 1). The median non-isolated infectious duration decreased from 12.5 in 2001 to 3 days in 2014 (P<0.001) (Table 1). Improvement occurred over all subgroups of patients (S2 Table). Fig 1 shows Kaplan-Meier estimates for time to respiratory isolation (discharge of undiagnosed TB is counted as censored rather than as the end of infectiousness) in 2001 versus that in 2014 (median: 46 vs. 19 days, p = 0.028, log-rank test). Patients with cavitary lung lesions were more quickly isolated, while lack of typical clinical presentation and hospitalized due to comorbidities were associated with delayed respiratory isolation (Table 2). After adjusting for patient characteristics, TB patients in 2014 were more quickly isolated than those in 2001 (adjusted hazard ratio [aHR] 4.7, 95% confidence interval [CI] 2.7–8.2, P<0.001) (Table 2: Model 1).

Table 1

Characteristics of hospitalized patients with culture-confirmed pulmonary tuberculosis.

Variables	2001	2014	P value
Number of patients	180	81
Age (years), mean (SD)	63.0 (20.7)	66.1 (19.8)	0.271
Men, n (%)	126 (70.0)	60 (74.1)	0.501
Positive sputum smear, N (%)	41 (22.8)	39 (48.1)	<0.001
Sputum TB-PCR performed, n/N (%)	4/41 (9.8)	37/39 (94.9)	<0.001
Negative sputum smear, N (%)	139 (77.2)	42 (51.9)	<0.001
Sputum TB-PCR performed, n/N (%)	25/139 (18.0)	8/42 (19.0)	0.876
Available sputum TB PCR data, N (%)	29 (16.1)	45 (55.6)	<0.001
Positive TB-PCR, n/N (%)	14/29 (48.3)	40/45 (88.9)	<0.001
Patients with cavitary pulmonary lesions, n (%)	30 (16.8)a	11 (13.6)	0.515
Positive sputum smear, % (n/N)	43.3 (13/30)	81.8 (9/11)	0.029
Patients with non-cavitary pulmonary lesions, n (%)	149 (83.2)a	70 (86.4)	0.515
Positive sputum smear, % (n/N)	28 (18.8)	30 (42.9)	<0.001
Patients without typical presentations, n (%)	119 (66.1)	48 (59.3)	0.286
Positive sputum smear, % (n/N)	14.3 (17/119)	27.1 (13/48)	0.051
Hospitalization due to comorbidity, n (%)	69 (38.3)	31 (38.3)	0.992
Positive sputum smear, % (n/N)	14.5 (10/69)	25.8 (8/31)	0.173
Non-chest/ID specialty attending doctors, n (%)	124 (68.9)	57 (70.4)	0.810
Fluoroquinolone exposure within 6 months, n (%)	32 (17.8)	16 (19.8)	0.703
Underlying diseases, n (%)
Hypertension	52 (28.9)	28 (34.6)	0.357
Diabetes mellitus	39 (21.7)	26 (32.1)	0.071
Malignancy	45 (25.0)	17 (21.0)	0.481
Chronic lung disease	53 (29.4)	6 (7.4)	<0.001
Chronic kidney disease	9 (5.0)	3 (3.7)	0.886
Congestive heart failure	9 (5.0)	4 (4.9)	0.983
Liver cirrhosis	8 (4.4)	1 (1.2)	0.343
Transplantation	4 (2.2)	2 (2.5)	0.903
HIV infection	1 (0.6)	1 (1.2)	0.525
Immediate respiratory isolationb, n (%)	11 (6.1)	23 (28.4)	<0.001
Non-isolated infectious duration, median (IQR), days	12.5 (6.8–28.3) days	3.0 (0–8.0) days	. <0.001c

P values are based on chi-square test or Fisher exact test unless specified otherwise. ID: infectious diseases; NA: not available

aOne patient in 2001 did not have chest radiography available

bImmediate respiratory isolation was defined as respiratory isolation on the date of admission or arrival of the emergent room.

cLog-rank test

Fig 1

Kaplan-Meier estimates for time to respiratory isolation of hospitalized patients with tuberculosis, 2001 vs. 2014.

Table 2

Factors associated with prompt respiratory isolation (Model 1: 2014 vs. 2001; Model 2: The effects of positive sputum acid-fast smear and TB-PCR; Model 3: The effect of physician alertness).

	Univariable analysis		Multivariable analysis
Variables	HR (95% CI)	P value	aHR (95% CI)(Model 1)	P value	aHR (95% CI)(Model 2)	P value	aHR (95% CI)(Model 3)a	P value
2014 vs. 2001	2.4 (1.6–3.6)	<0.001	4.7 (2.7–8.2)	<0.001	2.0 (1.2–3.4)	0.006	2.7 (1.7–4.3)	<0.001
Men vs. Women	0.9 (0.6–1.3)	0.578
Cavitary lung lesions	2.8 (1.9–4.1)	<0.001	2.0 (1.3–3.1)	0.001	1.7 (1.1–2.6)	0.022	1.8 (1.2–2.9)	0.007
Positive sputum smear	5.5 (3.8–8.0)	<0.001			3.2 (2.1–4.9)	<0.001	3.6 (2.3–5.5)	<0.001
Sputum TB-PCR test
Done vs. Not done	0.3 (0.2–0.5)	<0.001			1.5 (0.9–2.4)	0.094
Duration from hospital visits to the date of index culture (days)	0.96 (0.94–0.98)	<0.001					0.98 (0.96–0.99)	0.004
Lack of typical clinical presentations	0.2 (0.2–0.3)	<0.001	0.4 (0.2–0.6)	<0.001	0.3 (0.2–0.5)	<0.001	0.3 (0.2–0.5)	<0.001
Fluoroquinolone use	0.8 (0.5–1.2)	0.239
Hospitalization for comorbidities	0.4 (0.3–0.6)	<0.001	0.5 (0.3–0.7)	<0.001	0.6 (0.4–0.9)	0.025
Physician speciality
Chest/ID vs. Others	1.4 (1.0–2.1)	0.055
Cancer	0.5 (0.3–0.9)	0.009
Transplant recipients	0.4 (0.1–1.5)	0.153
Chronic kidney disease	1.2 (0.5–2.8)	0.601
Diabetes mellitus	1.0 (0.7–1.5)	0.952
Chronic lung diseases	1.2 (0.8–1.8)	0.461
Liver cirrhosis	0.3 (0.1–1.2)	0.084
Congestive heart failure	0.5 (0.2–1.3)	0.177
Hypertension	1.1 (0.8–1.7)	0.484
HIV infection	1.6 (0.2–11.4)	0.644

HR, hazard ratio; aHR, adjusted hazard ratio

aBecause some patients already received medical order of smear/culture at outpatient clinics, Model 3 was restricted to those who had not been suspected to have TB at admission. Only patients who had their index cultures sent after hospital visits were included for Model 3. Thus, 169 (93.9%) patients in 2001 and 73 (90.1%) in 2014 had their index cultures sent after their hospital visits (p = 0.307).

Effect of fluorescent microscopy

Switching to auramine-rhodamine staining with fluorescent microscopy doubled the overall positive sputum smear rate from 22.8% (2001) to 48.1% (2014) (P<0.001), particularly in patients with non-cavitary lung lesions (18.8% to 42.9%, P<0.001) (Table 1). Cox regression analyses shows that a positive sputum smear was associated with an earlier respiratory isolation (aHR 3.2, 95% CI 2.1–4.9, P<0.001) (Table 2: Model 2). Causal mediation analyses show that the two-fold higher sputum smear detection rate of fluorescence microscopy doubled the likelihood of early respiratory isolation (odds ratio [OR] for natural indirect effect mediated by improved sputum smear detection rate: 1.8, 95%CI 1.3–2.5, P<0.001) (Fig 2).

Fig 2

Causal mediation analyses of the effect attributable to switching from conventional to fluorescence microscopy.

Abbreviations: Year of 2014, 2014 vs. 2001; Positive AFS, positive acid-fast smear results; Isolation, respiratory isolation within 7 days.

Effect of TB-PCR

In patients with positive sputum smear, the use of TB-PCR grew from 9.8% (2001) to 94.9% (2014) (P<0.001); however, in patients with negative sputum smear, the use of TB-PCR remained infrequent (18.0% [2001] vs. 19.0% [2014], P = 0.876) (Table 1). In Cox regression analysis, TB-PCR testing was also helpful for early respiratory isolation (aHR 1.5, 95% CI 0.9–2.4, P = 0.094) but the effect of TB-PCR did not reach statistical significance (Table 2: Model 2).

Physicians alertness

Alertness of physicians, measured by duration from patient arrival to physician’s ordering of smear or culture, also improved from 2001 to 2014 (median: 5 vs. 2 days, P<0.001) (S2 Table). Cox regression analysis shows that physician alertness was also associated with earlier respiratory isolation (aHR 0.98 for each additional day before physician ordering TB smear/culture, 95% CI 0.96–0.99, P = 0.004) (Table 2: Model 3). Causal mediation analyses showed that improved physician alertness increased the likelihood of early respiratory isolation by 1.3-fold (OR for natural indirect effect mediated by early ordering of smear/culture: 1.3, 95% CI 1.02–1.5, P<0.001) (S1 Fig).

Total non-isolated infectious patient-days per year

In 2001, there were a total of 4,778 infectious patient-days in hospital (582 from smear-positive patients, 4,196 from smear-negative patients). In 2014, the total non-isolated infectious patient-days in hospital decreased by 69%, to 1,502 infectious patient-days (229 from smear-positive patients and 1,273 from smear-negative patients). Improvement occurred over all types of wards/units, including ER, internal medicine wards, surgical wards, and intensive care units (Fig 3, S2 and S3 Figs).

Fig 3

Total non-isolated infectious patient-days from hospitalized patients with culture-confirmed tuberculosis, 2001 vs. 2014.

Abbreviations: AFS, acid-fast smear; ED, emergency department; ICUs, intensive care units. Internal medicine wards: general medicine, cardiovascular medicine, pulmonary medicine, endocrinology, gastro-enterology, hematology, infectious diseases, nephrology, oncology, and paediatrics. Surgical wards: cardiovascular surgery, neurosurgery, otolaryngology, general surgery, chest surgery, proctology, ophthalmology, orthopaedics, plastic surgery, and urology.

Discussion

Our study showed that introducing highly sensitive rapid diagnostic tools decreases the risk of nosocomial TB transmission from hospitalized patients with undiagnosed TB in a high TB risk setting. Switching from conventional to fluorescence microscopy doubles the sputum smear detection rate and was associated with a two-fold increase in likelihood of prompt respiratory isolation.

A genuine improvement in time-to-respiratory-isolation of hospitalized TB patients should reduce nosocomial TB transmission, especially to HWCs. This is precisely what we observed in Medical Center A. Our previous survey on age/sex-standardised TB incidence ratio of HCWs (using general population as reference)–––the excess TB risk that are attributable to nosocomial TB transmission–––in Medical Center A showed a drop of this risk, from 3.11 in 2006 to 1.37 in 2012 [23], and the decrease in time-to-isolation and total non-isolated infectious patient-days was in parallel in the present study.

Traditional Ziehl-Neelsen staining and conventional light microscopy have unsatisfactory sensitivity in detecting acid-fast bacilli [24]. A systematic review showed that, compared with conventional method, fluorescence microscopy has higher sensitivity and similar specificity [13]. This present study found that, after switch to fluorescent microscopy, the overall sputum smear detection rate doubled (23% vs. 48%, P<0.001), particularly in patients with non-cavitary lung lesions (18.8% to 42.9%, P<0.001) but also in patients with cavitary lesion (43% to 82%, P = 0.029). Furthermore, the superior detection rate of fluorescence microscopy translated to a more timely respiratory isolation.

The 20%–40% difference in sensitivity between fluorescence and conventional microscopy from our real life data is much larger than the 10% (on-average) reported in previous studies that compared the diagnostic performance of fluorescent versus conventional microscopy under the optimal conditions [13]. This highlights an often overlooked problem of traditional Ziehl-Neelsen staining, i.e. the majority of clinical laboratories just did not have the manpower to adequately check 300 high-power fields [13] (which takes around 4 minutes) per sample that are required for conventional microscopy [24], particularly in a busy, high clinical caseload settings. In contrast, it takes only 30–60 seconds per sample for an adequate check with fluorescent microscopy [24] which would have a decisive advantage in the real world implementation.

Although TB-PCR has been considered an important rapid diagnostic tool, this study did not show a significant role of TB-PCR in the reduction in time-to-respiratory isolation from 2001 to 2014. There were several probable reasons. First, the low adoption rate (due to cost issues) may not be enough to make an impact on shortening the overall infectious duration. Second, because of the lower sensitivity of TB-PCR in smear-negative respiratory specimens, TB-PCR was performed predominantly in specimens with positive AFS to distinguish M. tuberculosis from non-tuberculous mycobacteria. The negative result does not exclude the possibility that universal use of automatic TB-PCR, such as Xpert® MTB/RIF, may further shorten the time to respiratory isolation.

In keeping with previous observations (S3 Table), the absence of cough and other typical symptoms is a barrier to prompt respiratory isolation of TB patients (S2 Table). Another cause of delay is hospitalization due to comorbidities, a problem that was previously neglected or confused with the lack of typical clinical presentations. To distinguish these two different situations, we defined hospitalization due to comorbidities as the reason of admission being for the management of acute complications from non-infectious diseases, while lack of typical presentation was defined as the absence of prolonged cough for more than 3 weeks, clinical suspicion of pulmonary TB based on chest radiography, or already having a confirmed diagnosis. Multivariable regression established that hospitalization due to comorbidities was a risk factor independent from lack of typical presentations (Table 2).

The diverse distribution of hospitalized infectious TB patients across 25 medical/surgical sub-specialities wards/units (S2 and S3 Figs) further supports that hospitalization of patients with undiagnosed TB for treatment of comorbidities is an unrecognized but important issue requiring to be addressed. The HCWs in specialties units often concentrate on the management of acute complications of non-infectious chronic diseases and are not trained or prepared for diagnosing concomitant TB in their patients. The 2003 large nosocomial TB outbreak in Taipei [18] involving more than 65 HCWs, occurred exactly under such scenario—an elderly patient with acute stroke was hospitalized to a rehabilitation unit, without being suspected to also have active TB. The harm is two-way. First, the patients are at increased risk for morbidities and mortality from delayed diagnosis and treatment of TB. Second, the HCWs are at increased risk of nosocomial TB outbreak, which could be fatal if the strain is multidrug-resistant or extensively drug-resistant [25].

The interplay between TB and chronic diseases further complicates the clinical scenarios. Chronic non-communicable diseases increase the risk of TB—the associations have been well established for diabetes mellitus [26] and rheumatoid arthritis treated with anti-tumour necrosis factor (TNF) agents [27]. On the other hand, TB may increases the risk of complications from chronic diseases, e.g. hyperglycaemia in diabetes mellitus and ischemic stroke in people with atherosclerosis [28]. Moreover, certain risk factor, such as smoking, increases the risk of both TB and chronic diseases [29]. Therefore, hospitalization of undiagnosed TB patients for treatment of comorbidities is more than just a coincidence by chance. In low HIV prevalence countries, TB is a disease of elderly [30]. With population aging, concurrence of TB and comorbidities could be an increasing challenge to clinicians.

Currently, most hospitals in Taiwan still used traditional Ziehl-Neelsen staining (or Kinyoun staining, a similar method) and conventional light microscopy in laboratory diagnosis of TB. An analysis of 2003–2010 Taiwan National Health Insurance database revealed that frequent exposure to hospital environment is a risk factor for contracting TB in Taiwan (adjusted odds ratio: 1.77, for those with ≥ 30 outpatient care visit annually) [31]. Our findings on the impact of switching to auramine-rhodamine staining with fluorescence microscopy suggest that a nationwide adoption and roll-out might cut the risk of nosocomial TB transmission to both patients and healthcare workers.

To address the remaining barriers to respiratory isolation of hospitalized TB patients, “FAST (Find cases Actively by cough surveillance and rapid molecular sputum testing, Separate safely, and Treat effectively based on rapid drug susceptibility test)” is an option to decrease the time to respiratory isolation [32, 33] although the substantial cost of rapid molecular testing could be a barrier to NHI reimbursement. Alternatively, environmental controls—the second level in the hierarchy of TB control—with adequate ventilation (>6–12 air changes per hour) or upper room ultraviolet germicidal irradiation (to disinfect the air) can be applied to reduce the concentration of infectious droplet nuclei in hospital indoor air, and decrease risk of nosocomial transmission [6, 34].

The present study has several important limitations. First, it was not a randomized controlled trial (which cannot be performed in the context, due to ethical reasons). Physician education and expansion of respiratory isolation facilities were simultaneously implemented along with the switching to fluorescence microscopy during the same intervention period. Nevertheless, we applied multivariable regression and causal mediation analyses to estimate the effect attributable to switching to fluorescence microscopy. Second, the study hospital closely followed the national guidance on nosocomial TB control policies issued by Taiwan CDC. Therefore, our findings might not be generalizable to hospitals with less proficiency. Third, the study hospital is a medical center, and therefore might not represent situations in smaller hospitals. Nevertheless, under NHI in Taiwan, people can and did directly seek health care in medical centers, without the need of referral from general practitioners. The 3 million annual outpatient visits and more than 90 thousand hospitalizations per year in the study hospital were predominantly from primary care services, rather than tertiary referral care. Finally, since Taiwan has a low HIV prevalence [35], only two of TB patients had HIV coinfection in our study. Thus, our conclusions might not be generalized to countries with a high HIV prevalence.

In conclusion, highly sensitive rapid diagnostic tools could substantially improve timing of respiratory isolation and reduce risk of nosocomial tuberculosis transmission in high TB risk settings. Lack of typical presentations and hospitalization due to comorbidities continued to be main reasons of delayed isolation. Studies will be required to assess whether routine sputum smear or TB-PCR of all hospitalized patients with cough or abnormal chest radiograph is effective in overcoming these remaining barriers.

Tuberculosis control practices, 2001–2014.

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Non-isolated infectious duration of hospitalized patients (at the first admission after the index culture).

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Studies which investigating the factors associated with delayed respiratory isolation of hospitalized patients with pulmonary tuberculosis.

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Causal mediation analyses of the effect mediated by the higher rate of early ordering (less than 4 days after admission) of smear/culture in 2014.

(DOCX)

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Number of infectious tuberculosis patients in 25 medical/surgical subspecialty wards/units in 2001 and 2014.

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Total non-isolated infectious patient-days in 25 medical/surgical subspecialty wards/units in 2001 and 2014.

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(XLSX)

Click here for additional data file.

27 Jan 2020

PONE-D-19-32578

Impact of introducing fluorescent microscopy on hospital tuberculosis control: a before-after study at a high caseload medical center in Taiwan

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript had described the introduction of a novel fluorescent microscopy methodology in the diagnosis of TB. Globally, TB is still a major concern in public health, thus this manuscript contains importance and significance for clinicians. My suggestion is minor revision, with my comments as follows.

Firstly, the authors had mentioned the moderate to high case load several times in this manuscript, and I reckon the high cases in this manuscript is one remarkable highlight which would be more convincing and supportive. However, what is the definition of the moderate to high, or high case load in regards with TB?

Secondly, despite the large number of cases involved, this study had been performed in the Medical Center A as a single center study. Please provide some information about the routine/conventional diagnosis of TB in other medical settings and how this introduction of fluorescent microscopy methodology would improve or make difference in other hospitals in Taiwan.

Thirdly, the writing could be benefit from language editing. For example, a lot of the sentences begin with subjective tense like “we…”. Some description could be more concise, for example, the approval of ethics could be combined as one sentence. Also, “we provide the data… showing that introducing… helps to…” would be a little bit too oral, “was parallel to the decrease” should be “in parallel”.

Reviewer #2: I found this manuscript to be relevant, sound and well-written, apart from a few instances e.g.

(a) Paragraph 2 of the "Introduction" section, Page 3; Remove "Fluorescence microscopy is a more sensitive test to diagnose pulmonary TB than conventional microscopy [16]" as it is a repetition; it is mentioned in the aforementioned sentences.

(b) Still in the same section as above, the last sentence in the paragraph i.e. "Nevertheless, the effect of introducing these more sensitive diagnostic tools on risk of nosocomial TB transmission has not been studied" may not be entirely accurate as you claim. Perhaps in high-income countries with a low burden of TB (e.g. Taiwan). I suggest you revisit this.

(c) 1st paragraph on page 5, Methods section; 'strain' appears to be a typo. Did you mean 'stain'? i.e. "---already received a confirmed diagnosis of pulmonary TB by a positive sputum culture of Mycobacterium tuberculosis, positive acid-fast strain (AFS),---" should be "---already received a confirmed diagnosis of pulmonary TB by a positive sputum culture of Mycobacterium tuberculosis, positive acid-fast stain (AFS),---"

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Reviewer #1: No

Reviewer #2: Yes: David Patrick Kateete

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12 Feb 2020

Dear Prof. Quinn,

Thank you for your encouraging response to our work. We greatly appreciate the reviewers’ constructive comments. We have endeavored to incorporate the feedback and revised our manuscript accordingly, with alterations highlighted in red color. Responses to each point of the reviewer’s comments were listed below.

Thank you again for your kind consideration and look forward to hearing from you soon.

Sincerely yours,

Chi-Tai Fang, MD, PhD

Professor

Division of Infectious Diseases

National Taiwan University Hospital

7 Chun-Shan South Road., Taipei 100, Taiwan.

Phone: +886 2 3366-8035

E-mail: fangct@ntu.edu.tw

To Reviewer 1:

Thank you for your positive response to our work and the kind advice. We greatly appreciate your constructive comments that have helped us improve our paper. We have endeavored to incorporate the feedback and revised our manuscript accordingly. The itemized response (abbreviated as R) are as follows:

C1. Firstly, the authors had mentioned the moderate to high case load several times in this manuscript, and I reckon the high cases in this manuscript is one remarkable highlight which would be more convincing and supportive. However, what is the definition of the moderate to high, or high case load in regards with TB?

R1. There are approximately 400 culture-confirmed TB cases annually in medical center A (ll. 33-34, 94 and 172), which has the second-highest caseload in Taiwan. We admit that we did not have a definition for “moderate to high” or “high” case load. For consistency, we revised all “moderate-to-high” to “high”. (ll. 30, 45, 62, 223, 337)

C2. Secondly, despite the large number of cases involved, this study had been performed in the Medical Center A as a single center study. Please provide some information about the routine/conventional diagnosis of TB in other medical settings and how this introduction of fluorescent microscopy methodology would improve or make difference in other hospitals in Taiwan.

R2. We added a paragraph in Discussion section to address these important issues:

“Currently, most hospitals in Taiwan still used traditional Ziehl-Neelsen staining (or Kinyoun staining, a similar method) and conventional light microscopy in laboratory diagnosis of TB. An analysis of 2003-2010 Taiwan National Health Insurance database revealed that frequent exposure to hospital environment is a risk factor for contracting TB in Taiwan (adjusted odds ratio: 1.77, for those with ≥ 30 outpatient care visit annually) [31]. Our findings on the impact of switching to auramine-rhodamine staining with fluorescence microscopy suggest that a nationwide adoption and roll-out might cut the risk of nosocomial TB transmission to both patients and healthcare workers.” (ll. 301-308)

C3. Thirdly, the writing could be benefit from language editing. For example, a lot of the sentences begin with subjective tense like “we…”. Some description could be more concise, for example, the approval of ethics could be combined as one sentence. Also, “we provide the data… showing that introducing… helps to…” would be a little bit too oral, “was parallel to the decrease” should be “in parallel”.

R3. The manuscript has been edited to avoid repetitive use of sentence begin with subjective tense “We…”. The following sentences were also revised as recommended:

“The study procedure and exemption of informed consent were reviewed and approved by Research Ethics Committee of National Taiwan University Hospital (Taipei, Taiwan).” (ll. 114-115)

“Our study showed that introducing highly sensitive rapid diagnostic tools decreases the risk of nosocomial TB transmission from hospitalized patients with undiagnosed TB in a high TB risk setting.” (ll. 222-224)

“the decrease in time-to-isolation and total non-isolated infectious patient-days was in parallel in the present study.” (ll. 233-234)

To Reviewer #2:

Thank you for your positive response to our work and the kind advice. We greatly appreciate your constructive comments that have helped us improve our paper. We have endeavored to incorporate the feedback and revised our manuscript accordingly. The itemized response (abbreviated as R) are as follows:

C1. I found this manuscript to be relevant, sound and well-written, apart from a few instances e.g. (a) Paragraph 2 of the "Introduction" section, Page 3; Remove "Fluorescence microscopy is a more sensitive test to diagnose pulmonary TB than conventional microscopy [16]" as it is a repetition; it is mentioned in the aforementioned sentences.

R1. We thank the reviewer for the kind comments and deleted the repetitive sentence as suggested.

C2. (b) Still in the same section as above, the last sentence in the paragraph i.e. "Nevertheless, the effect of introducing these more sensitive diagnostic tools on risk of nosocomial TB transmission has not been studied" may not be entirely accurate as you claim. Perhaps in high-income countries with a low burden of TB (e.g. Taiwan). I suggest you revisit this.

R2. We searched PubMed but did not find any published study which assessed the effect of introducing more sensitive diagnostic tools on risk of nosocomial TB transmission in either high-income or low-income countries. However, we cannot exclude the possibility that such assessment may have been performed but not published in academic journals. We therefore revised the sentences as below:

“to our knowledge, the effect of introducing these more sensitive diagnostic tools on reducing risk of nosocomial TB transmission has not been documented.” (II. 72-74)

C3. (c) 1st paragraph on page 5, Methods section; 'strain' appears to be a typo. Did you mean 'stain'? i.e. "---already received a confirmed diagnosis of pulmonary TB by a positive sputum culture of Mycobacterium tuberculosis, positive acid-fast strain (AFS),---"

R3. We apologize for the typo. The correct spelling should be:

“positive acid-fast stain (AFS)” (ll. 129)

21 Feb 2020

Impact of introducing fluorescent microscopy on hospital tuberculosis control: a before-after study at a high caseload medical center in Taiwan

PONE-D-19-32578R1

Dear Dr. Fang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Authors have satisfactorily addressed all the comments. I recommend that the revised manuscript be published.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Zhenbo Xu

Reviewer #2: Yes: David Patrick Kateete

23 Mar 2020

PONE-D-19-32578R1

Impact of introducing fluorescent microscopy on hospital tuberculosis control: a before-after study at a high caseload medical center in Taiwan

Dear Dr. Fang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Frederick Quinn

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PLOS ONE