PROLONGED POSITIVE SPUTUM SMEARS WITH NEGATIVE CULTURES

DURING TREATMENT OF PULMONARY TUBERCULOSIS.

By: Frits van der Kuyp and C. Scott Mahan (or visa versa)

(IJTLD: Major article- 2500 words, 35 references, max 7 figures/tables)

Summary (max of 200))

SETTING: Sputum smears for acid fast bacilli (AFB) are the most widely used tool for

both diagnosing and monitoring response to treatment in pulmonary tuberculosis (PTB).

It is not uncommon for patients who are otherwise clinically improving to have

prolonged positive sputum smears (S+) often with corresponding negative cultures (C-).

OBJECTIVE: To assess treatment outcomes and characteristics associated with

prolonged S+/C- status.

DESIGN: A retrospective chart review was performed on all patients seen by the

Cuyahoga County TB Program from 2000-2009. There were 162 consecutive cases of

S+, drug susceptible pulmonary tuberculosis with sufficient analyzable bacteriologic,

clinical and radiographic data.

RESULTS: A S+/C- pattern was seen in 51 patients (31.5%) ≥ 2 months after initiation

of treatment There was no significant correlation of the frequency of a late S+ (≥60 days

after initiation of treatment) with either the gender or the age group < 50 or ≥ 50 years of

age. There was, however, a statistically significant correlation of the late S+/C- pattern

with the radiographic extent at the start of treatment and especially with the presence of

cavitation.. There were no S+/C+ patients after > 2 months. There were 4 deaths (median

of 5 months from the initiation of TB therapy, none attributed to TB), and no confirmed

TB relapses.
CONCLUSION: We conclude that in our population of patients, in the absence of

clinical or radiographic evidence of deterioration, a late S+ usually has no clinical

significance and requires no specific action.

INTRODUCTION (text up to 2500 words).

Direct sputum smear microscopy for acid fast bacilli (AFB) remains the most frequently

used method for diagnosing pulmonary tuberculosis (PTB). It is also widely used for

monitoring patients on treatment for the disease. Diminishing numbers of AFB during

treatment, followed by smear negative status are the hallmark of successful therapy.

Increasing numbers of AFB in the later phase of treatment are a harbinger of failure.

While sputum smear microscopy is frequently the only diagnostic modality for

the diagnosis and monitoring of PTB patients worldwide, culture on solid or in liquid

media is the recognized “gold standard. Reliance solely on the AFB smear has a number

of limitations. The poor sensitivity of the AFB smear, particularly in patients with

limited pulmonary involvement or immunosuppression, leads to under diagnosis of the

disease(D). Additionally, sputum smear lacks specificity for MTB disease, non

tuberculous mycobacterial and various other bacteria can stain AFB positive. Moreover,

specimens with positive AFB smears (S+) are sometimes negative on culture (S+/C-

pattern), indicating that these positive AFB smears represent the excretion of dead

bacilli(Ref A5).

Delayed sputum smear conversions may be mistakenly considered treatment

failures pending their negative culture reports, leading to unnecessary change in the

treatment regimen as well as unnecessary public health action such as isolation of the
patient and expanded contact investigation. In order to determine the frequency and

significance of this late S+/C- pattern (≥2 months after the initiation of treatment), we

reviewed the medical records of our patients.

STUDY POPULATION AND METHODS

We conducted a retrospective review chart review of 162 consecutive patients with drug

susceptible pulmonary TB reported from 2000-2009 to the Cuyahoga County TB Control

Service, located at MetroHealth Medical Center in Cleveland, Ohio. All patients had

received directly observed treatment (DOT) usually consisting of an initial intensive

phase of daily isoniazid (H), rifampin(R), ethambutol (E), and pyrazinamide (Z) for two

months, followed by daily H and R for 4-7 months. Sputum smear and culture results at

the time of diagnosis and during treatment were correlated with the chest radiographs and

clinical features. Sputum for AFB and culture was routinely obtained at baseline , every

1 to 2 weeks after the initiation of therapy including at the two month visit and thereafter

until unable to produce sputum or at least two consecutive negative cultures were

confirmed. Sputums were either morning expectorated samples or induced sputums

performed by the TB clinic staff. The majority of patients were seen periodically

throughout there treatment course, including a two month visit, an end of treatment visit,

and in follow-up 6 months after completion of the TB therapy. Additionally, TB records

were reviewed for evidence of relapse or recurrent TB disease. The average interval

between completion of treatment and the time of this review was 51.2 months with a

median of 53 months. The disease at the time of diagnosis was quantified by a single

experienced chest physician using the National Tuberculosis Association classification of

minimal, moderately advanced and far advanced with the sub classification of cavitary
and non-cavitary for advanced disease (Ref). This study was approved by the

MetroHealth Medical Center Institutional Review Board (IRB07-00409). Consent was

waived due to the retrospective design.

Statistical Analysis

( will need brief description of statistical methods used) Commented [csm1]: Will need full description of stastical
methods.

RESULTS

Baseline characteristics

At total of 162 consecutive patients had complete analyzable clinical radiographic and

mycobacteriologic data for the time period studied. The median age was ---, there were Commented [csm2]: What was the median ageand IQR of the
entire group studied (all 162 patients)?

117 (72%) males, extent of disease based on chest radiograph was 19 (12%) with

minimal disease, 41 (25%) with moderately advanced non-cavitary disease, 29 (18%)

with moderately advanced cavitary disease, 14 (9%) with far advanced non-cavitary

disease, and 59 (35%) with far advanced cavitary disease. Commented [csm3]: Frits- did we collect the baseline sputum
smear grading: rare, 1+, 2+, 3+? If available this would be nice to
include in the baseline characteristics and to correlate with S+/C-
numbers.

Longitudinal sputum smear and culture results

We found that the late S+/C- pattern occurred in 51 (31.5%). These 51 patients ranged in

age from 16 to 83 years with a median of 50 years. Among these patients the first

reported S+/C- result occurred a median of 54 days (range 13 to 188 days) after initiation

of TB therapy. The last reported S+/C- occurred a median of 86 days (range from 62 to

208 days) after treatment initiation. The interval from start of treatment to the last S+/C-

was less than 2 months in 16 (9.9%) of the 162 patients, 2-3 months in 23 (13.6%), and
more than 3 months in 29 (17.9%) while one patient still produced S+/C- specimens after

188 days.The frequency of repeated prolonged S+/C- specimens ranged from once to

nine times. In 65% of the 51 patients with this phenomenon the frequency was three

times or less. In no instance was there either radiographic or clinical evidence of

deterioration of the disease at the time of the late S+ report, hence our decision not to take

special therapeutic or public health action, except for prolongation of the duration of

treatment in nine instances. None of the 162 patients had S+/C+ (for MTB) at >2

months. Among the 51 patients with the late S+/C- pattern there we 8 instances among 6

patients where either mycobacterium avium-complex (5) or mycobacterium gordonae (3)

was grown in culture. These represented usually 1 or at most 2 episodes of culture growth

among many subsequent late S+/C- samples and were felt to be clinically non-significant.

Four patients died of unrelated medical problems while on treatment at 5, 5 5, and 6

months of TB therapy, respectively. The remaining 47 patients with prolonged S+/C-

pattern had a favorable outcome. . While in most instances active surveillance of the

patients has been limited to one visit six months following completion of treatment, the

TB Control Service was not aware of any reactivations among the prolonged S+/C- cases

presented. Table 1 show that there was no significant correlation between the late S+/C-

pattern and either the age or gender of the patient, but that cavitatary disease was

associated with an increased likelihood of having prolonged S+/C- results. Commented [csm4]: In table 1: who did the statistical analysis?
What type of analysis was done? Are we sure the extent of disease
was not associated with S+/C-? what about baseline sputum smear
grading?

DISCUSSION

The “gold standard” for the bacteriologic diagnosis of tuberculosis (TB) is the culture of

MTB from an appropriate clinical specimen, most often sputum. However, in resource
limited settings diagnosis is often based solely on sputum smear results. Even within the

United States treatment is often initiated based positive smear reports before culture

results are available. Sputum microscopy for AFB is very helpful for estimating the

degree of infectiousness (Ref C 6-13, L) and has been used determine the response to

treatment. Reliance on sputum smears alone for the diagnosis of pulmonary TB and to

monitor one’s response to therapy has several pitfalls. The suboptimal sensitivity of the

test may lead to under diagnosis of active TB resulting in further transmission.

Moreover, its specificity is suboptimal; some cases of non-tuberculous mycobacterial

disease may be mistakenly considered TB and managed as such. Also, positive sputum

smear reports during the later phase of treatment may indicate continued excretion of

dead bacilli rather than failure of therapy. . Diminishing numbers of bacteria during the

early phase of treatment indicates response, while increasing numbers, especially during

the later phase, generally are an ominous sign, suggesting treatment failure.

Determining when a continued positive sputum smear represents treatment failure as

opposed to reflecting dead bacilli may present a challenge. Standard methodology does

not rapidly distinguish live from dead AFB’s. The same limitation is true for even more

advanced methods, including newer molecular procedures, e.g. the nucleic acid

amplification test. While a staining procedure for determining the viability of

mycobacteria was introduced by Kvach and Veras (A6) in 1982 and more recently been

confirmed by Salim et al (Ref A) to determine live versus dead bacilli it has not been

widely embraced.. A long interval between collection and processing of the sputum

specimen may conceivably account for some instances of S+/C- reports. Some

investigations, however, have concluded that a period of up to one week between

expectorating and processing of the culture should not adversely affect the reliability of

the culture report. (Ref AA6) In our study all specimens were promptly processed within

(? Time frame) . Dominguez and Vivas (Ref AA) reviewed 6415 S+ specimens, 6.5% of

which had a negative culture (S+/C-). They concluded that approximately one half of the

S+/C- instances involved patients on treatment, while the other half represented false-

positive reports.

A sharp increase in the S+/C- phenomenon since the introduction of Rifampicin

has been noted (Ref AA, AA8). This is apparently the result of the stronger bactericidal

effect of Rifampicin. Among a group of 606 patients Kim et al (Ref C-6) noted the S+/C-

phenomenon in 22.6% of those whose treatment regimen included Rifampicin, but only

9.1% in those who did not receive this drug.

Our review is based upon experience in a low TB incidence area, where virtually

all sputum specimens for TB are examined by both smear and culture. Smear positive

results with culture reports pending during the continuation phase of treatment may cause

concern for treatment failure with the possibility of emergence of drug resistance,

especially when they appear three months or later after initiation of treatment. This

concern may lead to unnecessary medical and public health actions, such as a change in

the treatment regimen, extensive and expensive investigations, which may include special

radiographic studies and serum drug levels, as well as prolongation of treatment. Public

health actions may range from requiring strict isolation to imposing restrictions on the

patient’s and social activities as well as expanded contact investigations.

In this review a late (≥2 months after treatment initiation) S+/C- pattern was

observed in 51 of the 162 (31.5%) patients with fully available radiographic, clinical and
mycobacteriologic data. The vast majority of the late S+/C- specimens contained small

numbers of AFB’s. Neither the patients’ age nor gender, were associated with an Commented [csm5]: Was there a correlation between smear
grading and the likilood of a positive culture?

increased frequency of this pattern. However, the extent of radiographic changes,

especially the presence of cavitation, did correlate with a late S+/C- pattern.

The increased prevalence of this phenomenon in cavitary disease has been previously

reported(C-6, AA-8). After receiving a late S+ report we relied primarily on clinical and

radiological parameters to determine whether to take immediate action (suspecting

treatment failure) or wait for the culture report (suspecting non-viable AFB). In all of the

51 instances we were reassured by the absence of either clinical or radiological evidence

of deterioration. The value of these two parameters in monitoring treatment for TB is

underscored by the fact that all our patients, except for the four who died of unrelated

medical problems, had a favorable outcome.

A change in the chemotherapy regimen has been advocated for patients with a

prolonged S+/C- phenomenon (Ref C 6-13). Others (Ref C) have questioned the need for

this. Our review supports the opinion that there is no need for changing the treatment in

the absence of additional evidence of deterioration. All instances of late positive sputum

smears among our patients represented delayed smear conversion, rather than treatment

failure. Six patients with several S+/C- specimens had either one or two sputum culture

yielding few colonies of non-tuberculous mycobacteria (four instances of M.avium

complex and two of M.gordonae). These cultures had no clinical significance and

apparently represented contaminants or colonizers. It should be noted that seven of our

patients (4.3%) still had a S+ for more than five months following initiation of treatment.

They met the WHO criteria of treatment failures (Ref), but were actually therapeutic
successes. The limitation of this WHO definition has been pointed out in the past (Ref

F). In 13 of our 51 (25.5) patients with a late S+/C- pattern the treatment was prolonged

beyond nine months because of persistent positive smears. The need for this practice, has

been questioned (C) and we realize that these 13 patients may have received excessive

treatment. Commented [csm6]: Need a paragraph on the study limitations.

Limitiations of our study include that we were evaluating a population in an area

of low endemicity for TB. Conclusions from our study should not be applied to TB

endemic areas where more cases of far advanced disease might be seen and the

significance of a positive smear beyond 2 months might have more dire implications.

Also the retrospective nature of our study might introduce inherent biases although we

attempted to limit these through standardized collection of data and blinding during

radiographic interperetation.

Our review leads to the following conclusions: (1) Late positive sputum smears

for AFB with negative cultures, well into the continuation phase during the treatment of

pulmonary TB are not unusual. These smears usually contain small numbers of AFB’s.

This phenomenon is most often seen in patients with advanced disease, especially in

those with cavitary lesions. For patients with documented drug susceptible

M.tuberculosis receiving an appropriate drug regimen, documented by directly observed

therapy (DOT), a late positive S+ report, particularly with only few organisms, usually

indicates late sputum smear conversion, rather than treatment failure. (2) In the absence

of either clinical or radiographic deterioration there is no need for changing the

chemotherapy regimen or to immediately embark upon extensive and expensive

additional tests (e.g. special radiographic studies, invasive procedures or serum drug

levels determination). (3) There is, likewise, no need for additional public health

measures, such as expanding contact investigation, isolating the patient or imposing

restrictions on their permission to work or their social activities. (4) All such unexpected

S+ specimens should be cultured. If the cultures are positive one may consider repeating

the susceptibility studies and taking other appropriate action as warranted.