A Review of the Diagnosis and Treatment of

Smear-Negative Pulmonary Tuberculosis

R. Colebunders, I. Bastian

Institute of Tropical Medicine, Antwerp, Belgium

Running heading: Smear-negative pulmonary TB

Word Count: 5,038 (text excluding summary and references)

Keywords: tuberculosis; smear-negative; diagnosis; review

Correspondence: Dr. Ivan Bastian

Mycobacteriology Unit, Department of Microbiology

Institute of Tropical Medicine

Nationalestraat 155, B-2000 Antwerp, Belgium

Tel: + 32 3 247-6548; Fax: + 32 3 247-6333

E-mail: ibastian@microbiol.itg.be

Summary

Recommendations on the management of smear-negative pulmonary

tuberculosis (TB) are still based on the behaviour of this disease in

populations unaffected by the human immunodeficiency virus (HIV). Studies

prior to the HIV epidemic estimated that there were 1.22 cases of smear-

negative and extra-pulmonary TB for each smear-positive case. Patients with

smear-negative pulmonary TB were found to be less infectious and to have a

lower mortality but a significant proportion (ie. 50-71%) progressed to active

disease justifying treatment. Moreover, a wide variety of regimens also

proved effective in the treatment of smear-negative disease in HIV-negative

patients.

The advent of HIV has changed many of these parameters. Countries affected

by both HIV and TB have experienced a disproportionate increase in smear-

negative disease. While apparently remaining less infectious than smear-

positive cases, HIV-positive patients with smear-negative pulmonary TB are

generally more immunocompromised, have more adverse drug reactions, and

suffer higher mortality rates on treatment. Clinical decision making has also

been complicated because HIV co-infection broadens the differential

diagnoses of smear-negative pulmonary TB to include diseases such as

Pneumocystis carinii pneumonia (PCP), pulmonary Kaposi’s sarcoma, and

Gram-negative bacteraemia.

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Our approach to smear-negative pulmonary TB must therefore adapt to these

changed parameters. Management algorithms based on several features (eg.

clinical symptoms, response to antibiotic trials, smear investigations, and

chest radiography) have been developed to improve case detection. These

algorithms must be validated in each locale because their performance will

vary depending on numerous local factors such as the regional prevalence of

PCP. Alternative methods of specimen collection (eg. sputum induction) and

processing must be evaluated. National tuberculosis programmes should also

consider extending the use of rifampicin-based short-course chemotherapy

(SCC) to new patients with smear-negative disease. This latter intervention,

and the much-needed establishment of additional microscopy and culture

facilities, will depend on increased financial and technical support from the

international community.

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Introduction

The detection and management of smear-positive pulmonary disease are

quite rightly the principal aims of national and international tuberculosis (TB)

control programs.1 However, smear-negative disease (ie. patients with

clinical and radiological evidence of pulmonary TB but repeatedly negative

sputum investigations) is a common clinical problem,2 particularly in countries

affected by the dual TB/human immunodeficiency virus (HIV) epidemics.3,4

Despite the high and increasing frequency of smear-negative pulmonary TB, a

search of the medical literature has found only two editorials and two reviews

on this topic in the last 20 years.4-7 This article will describe the incidence,

natural history and differential diagnoses of smear-negative pulmonary TB in

HIV-negative and HIV-positive patients. The various strategies that have

attempted to address smear-negative TB will then be reviewed highlighting

plausible interventions for developing countries and areas for future research.

Incidence and natural history in HIV-negative patients

Our understanding of smear-negative pulmonary TB is based on experience

from the pre-HIV era (Table 1). Murray et al extrapolated from data collected

in the United States to estimate that there are 1.22 cases of smear-negative

and extra-pulmonary TB for every case of smear-positive TB in developing

countries.2 In HIV-negative populations, smear-negative pulmonary TB is

more common among children and the elderly.2,6,8,9 For example, less than

10% of cases aged 0-14 years in the US (1985-1987) and Norway (1951-

1972) were smear-positive.2 The low rate of smear-positive disease among

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children may be explained by the fact that children generally have primary

disease without extensive cavitation. Difficulties in collecting adequate

specimens from children and misdiagnosis of other paediatric illnesses as TB

may also be contributing factors.

About 5,000-10,000 acid-fast bacilli (AFB) per millilitre of sputum must be

present for detection by smear whereas culture requires only 10-100 viable

organisms.10 Hence, smear-negative culture-positive patients generally have

minimal disease with low bacillary counts rather than far-advanced cavitary

TB with heavy bacillary burdens (Table 1).5,6,11 For example, a US study of

HIV-negative culture-positive patients estimated that negative smears are

obtained from 60%-80% of patients with minimal disease, from 30%-40% of

cases with more extensive disease, but from only 5%-10% of patients with

extensive cavitary lesions.11

The association of AFB-negative smears with lower bacillary burdens and

minimal pulmonary lesions would imply that the infectivity and the mortality

of smear-negative disease should be lower, and that less-intensive

chemotherapy may adequately treat this condition (Table 1). Rouillon et al

reviewed five studies comparing the bacteriological status of the source case

with the prevalence of infection (as measured by tuberculin reactivity) among

household contacts aged less than 15 years.10 The prevalence of infection

among children exposed to smear-positive cases varied between 39%-65%

whereas the tuberculin reactivity rate was only 4.7%-26.8% among contacts

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of smear-negative patients, whose TB diagnoses were based on positive

cultures or radiography. However, in studies in high incidence environments

in India and Africa, the infection rate among young contacts of smear-

negative cases was similar to that in the general population or in households

without documented TB.10 Nonetheless, a recent DNA-fingerprinting study

from San Francisco attributed 17% of tuberculosis transmission in this low-

prevalence setting to patients with smear-negative culture-positive pulmonary

TB.12

The supposition that mortality is lower among HIV-negative smear-negative

cases has also been verified. Longitudinal surveys conducted between 1961

and 1963 among untreated TB patients in Bangalore District, South India,

found that the mortality rate during the first 18 months of follow-up was

34.7% for smear-positive patients, 14.1% for smear-negative culture-positive

cases, and about 5.0% for smear-negative culture-negative cases diagnosed

radiologically.5,13

The natural progression of smear-negative disease was studied in a

chemotherapeutic trial in Hong Kong.14 Table 2 describes the outcome after

30-months follow-up for a sub-group of 283 smear-negative patients who

were randomly assigned to receive chemotherapy only when active disease

was confirmed by culture, radiography, or clinical deterioration. At least 71%

of these patients initially had or developed active disease requiring treatment.

Importantly, nearly half of the smear-negative cases who required treatment

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developed active disease within the first three months. Follow-up of untreated

smear-negative patients must therefore concentrate on this early period after

initial presentation. Surveys in Bangalore also followed the progression of

smear-negative patients to active disease and found similar results to the

Hong Kong study. Over 50% of 457 patients with radiographic suspicion of TB

but one negative smear progressed to active disease within 12 months (cited

in reference 5).

Finally, smear-negative pulmonary TB in HIV-negative patients can be

successfully treated with a wide variety of regimens.5,15 For example, a study

in Hong Kong found that treatment with streptomycin, isoniazid, rifampicin,

and pyrazinamide for four months cured all 293 patients with smear-negative

pulmonary TB and had a relapse rate of only 2% after five years. Longer,

cheaper, less-intensive therapies are also effective. The World Health

Organisation (WHO) therefore recommends one regimen (ie. 2 EHRZ/4 HR)

for Category I smear-positive patients and a different treatment (eg. 2 HRZ/6

HE) for Category III smear-negative cases who are not severely ill and do not

have extensive parenchymal involvement.1 African studies from the pre-HIV

era have justified the treatment of smear-negative cases with even cheaper

less-aggressive 12-month regimens (eg. 2 STH/10 TH, 2 EHZ/10 HT).4,16

The impact of the HIV epidemic

Since the advent of HIV, the annual incidence of TB has more than doubled in

some African countries,3,4,17,18 and there has been a disproportionate increase

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in the reported rate of smear-negative disease (Table 1).19-21 For example,

the number of notified TB cases in Blantyre, Malawi, tripled from just over

400 cases in 1986 to more than 1200 in 1991; this increase was almost

entirely attributable to an ‘additional’ 800 smear-negative cases in 1991

compared with 1986.20 This apparent predominance of smear-negative

disease may be partly due to (i) heavy workloads increasing the likelihood of

false-negative laboratory errors, and (ii) misdiagnosis of other HIV-related

pulmonary conditions as smear-negative TB, but several studies have found

that smear-negative disease is actually more common among HIV-positive

patients.20,22-26 For example, Elliot et al found that 43% of 72 HIV-positive

patients with culture-proven pulmonary TB in Lusaka, Zambia, were smear-

negative compared with 24% of 37 HIV-negative cases (p=0.003).24

Other researchers have not detected a difference in smear positivity rates

between HIV-positive and HIV-negative cases.27-30 The apparent discrepancies

between these studies may be due to differences in the study populations.

Some studies were conducted among patients seen at specialist centres who

may be more or less likely to be smear-positive depending on the referral

procedure. The level of immunosuppression among the HIV-positive patients

in the various studies may also have differed. Less severely

immunocompromised HIV-positive patients tend to have classic cavitary TB

which is smear-positive.3,19,25 As the level of immunocompromise increases

with advancing HIV disease, atypical pulmonary features predominate and

smear examinations prove less sensitive (Table 1).

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Overall, population trends and most clinical-based studies do suggest that

HIV-positive patients have a higher rate of smear-negative disease.20,22-26

Furthermore, while having no apparent affect on the infectiousness of smear-

negative pulmonary TB,31,32 HIV infection does seem to alter some other

features of this disease (Table 1). For example, adverse drug reactions are

more common among HIV-positive patients.33-35 These side effects include

life-threatening cutaneous reactions to thioacetazone,33 which is commonly

used in ‘standard’ chemotherapy regimens for smear-negative pulmonary TB.

In developing countries, treatment outcomes in the presence of HIV infection

have been studied almost exclusively in patients with smear-positive TB.

These studies have found that HIV-positive TB patients who survive and

complete treatment have a similar response to therapy as HIV-negative

populations.36-38 However, mortality while on treatment is consistently higher

in the HIV-positive group. This excess mortality is largely attributed to

diseases other than TB (eg. gram-negative bacteraemia).3,4,36,37 Furthermore,

a recent study from Malawi has confirmed the clinical impression that HIV-

positive patients with smear-negative pulmonary disease (which is an

indicator of advanced immunosuppression) do even worse than HIV-positive

patients with smear-positive disease.39 The mortality rate was 3.9 times

greater among smear-negative patients (who received ‘standard’

chemotherapy with 1 STH/11TH) than in smear-positive patients (who

received more intensive treatment with 2 SHRZ/6 HT); the prevalence of HIV

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in this TB patient population was 77%. ‘Standard’ chemotherapy regimens

containing streptomycin, isoniazid and thioacetazone have also been

associated with higher relapse rates among HIV-positive patients.38

In summary, the HIV epidemic has been associated with an increased

incidence of smear-negative pulmonary TB, more adverse drug reactions,

higher mortality rates on treatment, and perhaps higher relapse rates on

‘standard’ chemotherapy. The clinical management of patients suspected of

having smear-negative pulmonary TB has also been complicated because the

differential diagnoses of this condition are broadened by HIV co-infection.

Differential diagnoses of smear-negative disease

Microscopic examination of sputum smears for AFB forms the basis of TB

diagnosis in developing countries.1 AFB microscopy is rapid, simple and

cheap.2,40-43 Murray et al estimated that a single smear examination in

Tanzania costs less than US$ 0.25.2 Unfortunately, AFB microscopy lacks

sensitivity compared with culture. In patients with culture-confirmed

pulmonary TB, the sensitivity of AFB microscopy ranges from 22 to 80%.5,11

Table 3 outlines the factors that may produce false-negative smear results in

patients with pulmonary tuberculosis. In under-resourced over-worked TB

control programs, laboratories cannot cope with the influx of diagnostic and

follow-up smear examinations. Smears may not be done at all! For example,

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in Botswana in 1992, 48% of patients reported with pulmonary tuberculosis

had not had any smears performed.44

Alternatively, the sputum specimens collected may be inadequate in quality or

number. Similar incremental yields from serial smear examinations have been

reported from industrialised and developing countries. Ipuge et al found that

83.4 % of smear-positive cases were detected on the first specimen, 12.2%

on the second, and 4.4% on the third, by Ziehl-Neelsen (Z-N) staining under

routine program conditions in Tanzania.53 Using an auramine-rhodamine

stain in a diagnostic laboratory in the US, Nelson et al reported that 73% of

smear-positive cases were found on the first specimen, 14% on the second,

7% on the third, and 6% on the fourth or later.54 At least two good-quality

specimens must therefore be examined to reliably detect smear-positive

pulmonary TB.

Finally, the performance of the smears may be technically inadequate.

Declining quality of smear examination is a particular problem in

overburdened laboratories in HIV-endemic countries. For example, as part of

an epidemiological study of TB and HIV in Tanzania, Chum et al compared the

sputum microscopy results obtained in local and reference laboratories.55

Twenty-nine per cent of new smear-negative cases (on the basis of local

microscopy) were found to be smear-positive by the reference laboratory.

False-negative results can be due to inadequate staining, under- or over-

decolourisation, or inspection of too few fields (ie. a minimum of 100 fields of

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a Z-N smear must be examined before reporting a negative result and this

examination takes about 5-10 minutes),56,57 Finally, overly thick smears can

obscure the presence of AFB or may fall off the slide.41-43

Table 3 also describes the other medical conditions that may be misdiagnosed

as smear-negative pulmonary TB, and highlights the differential diagnoses of

increased importance in HIV-positive patients. Bacterial pneumonia is the

main differential diagnosis in HIV-positive and HIV-negative individuals while

Pneumocystis carinii pneumonia (PCP), cryptococcosis, and nocardiosis are of

increased importance in HIV-positive subjects.30,45-52 The reported rates of

PCP in African HIV-positive patients with respiratory symptoms vary between

0-33%.30,45-48,50 This variation has not been fully explained but has been

attributed to differences in patient selection, the level of immunodeficiency of

HIV-positive patients in Africa, the limited availability of specialised laboratory

diagnostics, the failure to diagnose PCP in the presence of multiple other

infections, and geographic differences in the prevalence of PCP.48,50

HIV-associated nocardiosis may also be under-diagnosed. Lucas et al

conducted an autopsy study of 247 HIV-positive cadavers in Abidjan, Ivory

Coast, and found one case of nocardiosis for each nine TB cases.49 While

nocardiosis is less common than TB, a simple Gram stain of AFB-negative

sputa may prove a worthwhile investigation. Surprisingly, a recent study of

HIV-positive patients in a respiratory medicine unit in Abidjan reported that

9% had a Gram-negative bacteraemia (usually due to non-typhoid

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salmonellae).52 Kamanfu et al found a similar percentage (ie. 10.4%) of HIV-

positive patients hospitalised with acute respiratory disease in Burundi had

Salmonella bacteraemia, usually with S. typhimurium.46 Blood culture facilities

are required to make this diagnosis but are rarely available. These two studies

highlight that Gram-negative bacteraemia must be considered in patients

presenting with pulmonary disease but negative sputum smears, particularly if

they are HIV-positive.

These medical conditions account for significant morbidity and mortality in

patients presenting with ‘smear-negative pulmonary disease’ in HIV- and TB-

endemic developing countries (Table 3). However, the pre-eminent position of

TB as the major pathogen in these circumstances must be emphasised. The

lowest rate of TB reported in the studies cited in table 3 was still 22.5% (in a

study that selected smear-negative patients for bronchoscopy)48 and the

highest rate was 64%.52

Addressing the problem of smear-negative pulmonary TB

Improving selection of patients with TB

Various management algorithms have been proposed to optimise the number

of patients correctly treated for smear-negative tuberculosis while minimising

over-treatment of patients who do not have the disease.1,50,58-60 Samb et al

investigated which symptoms could be effectively included in these diagnostic

algorithms.59 They studied 182 smear-negative patients with respiratory

disease (41 with culture-confirmed TB and 141 with non-TB disease) in

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Burundi and Tanzania; 71% of the patients were HIV-positive. Four

symptoms were associated with TB: cough >21 days, chest pain >15 days,

absence of expectoration, and absence of dyspnoea. Presence of any two of

these symptoms diagnosed TB with 85% sensitivity but only 67% specificity.

If at least three of these symptoms were required for a diagnosis of TB, the

specificity improved to 86% but the sensitivity fell to 49%. Presence of

lymphadenopathy and haematocrit <30% aided discrimination.

An additional indicator of smear-negative pulmonary TB could be failure to

respond to a trial of antibiotics. Wilkinson et al studied 237 South African

patients with suspected TB, including 56 smear-negative culture-positive

cases. Smear-negative patients were given a course of ampicillin (500mg qid

for 7-10 days).60 A final diagnosis of TB was correctly made in 28 (50%) of

the smear-negative culture-positive cases (ie. the sensitivity of Z-N smear

alone was 61% and rose to 80% when combined with a failed clinical

response to an antibiotic trial). However, the remaining 28 culture-positive

patients appeared to respond to antibiotic therapy, either because of

unrelated fluctuations in disease severity or successful treatment of a

superimposed bacterial infection, and were incorrectly discharged.

Furthermore, 32 culture-negative patients failed to respond to antibiotics and

were misdiagnosed with TB (ie. specificity of Z-N alone was 94% but fell to

78% when combined with the outcome of an antibiotic trial).

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Failure of non-TB patients to respond to a trial of antibiotics may be due to

infection with a resistant organism. Unfortunately, little data is available on

the prevalence of antibiotic resistance in developing countries. For example,

the serotypes and resistance profiles of 5,000 invasive isolates of

Streptococcus pneumoniae from the United States were published in 1996 but

similar information was available for <500 strains from the entire African

continent.61 Reported levels of antibiotic resistance in developing countries

have also varied widely due to study design and the in vitro susceptibility

tests employed.62 Nonetheless, the few published African studies have failed

to detect penicillin-resistant S. pneumoniae (ie. minimum inhibitory

concentration ≥ 2.0 μg/mL) that would adversely affect successful treatment

of pneumococcal pneumonia with penicillin but have found pneumococci with

reduced susceptibility (ie. MIC 0.1-2.0 μg/mL).61-63

Improving selection of patients with other conditions

Failure of non-TB patients to respond to a trial of antibiotics could also be due

to the presence of a disease other than bacterial pneumonia. In some

countries, PCP, nocardiosis, Salmonella bacteraemia, and pulmonary Kaposi’s

sarcoma are important differential diagnoses in HIV-positive patients

presenting with respiratory disease (Table 3). Various clinical predictors of

PCP, bacterial pneumonia, and TB have been proposed in HIV-positive

patients.4,50,64 Selwyn et al studied 229 cases of pulmonary disease in HIV-

positive patients in the US.64 The independent predictors were exertional

dyspnoea, an interstitial infiltrate, and the presence of oral thrush for PCP; a

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‘toxic’ appearance, a lobar infiltrate, and a fever ≤ 7 days for bacterial

pneumonia; and cavitary infiltrate, fever > 7 days, and weight loss for TB. In

Zimbabwe, Malin et al found similar clinical indicators for PCP: respiratory rate

> 40/min, fine reticulonodular shadowing on the chest radiograph, and severe

hypoxia.50

Unfortunately, many features of these pulmonary syndromes overlap hence

syndromic diagnoses lack both sensitivity and specificity. For example, Selwyn

et al found that 81% of PCP patients complained of exertional dyspnoea but

so did 33% of TB and 43% of pneumonia patients.64 The specificity of these

syndromic diagnoses was improved by combining indicators but with an

invariable reduction in sensitivity (eg. a combination of exertional dyspnoea

and an interstitial infiltrate diagnosed PCP with 92% specificity but the

sensitivity fell to 58%)64 Multiple pathologies can also co-exist in HIV-positive

patients further complicating the clinical diagnosis of pulmonary syndromes.

For example, Malin et al found that 6 of 21 HIV-positive Zimbabwean patients

with PCP also had TB.50

In an attempt to further differentiate patients with TB from those with other

pulmonary diseases, Harries et al have recently recommended that smear-

negative TB suspects receive a second trial of antibiotics.58 Ideally, this

second drug should be unrelated to the β-lactams (eg. ampicillin) which are

generally used in the initial antibiotic trial. Cotrimoxazole is a cheap, widely-

available antibiotic that could be reasonably used in this second treatment

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trial. Cotrimoxazole is a recognised treatment for pneumonia,62,63 and has

activity against Salmonellae, Pneumocystis carinii, and nocardia.65

Unfortunately, high-dose cotrimoxazole (ie. trimethoprim 15-20 mg/kg/day-

sulfamethoxazole 75-100 mg/kg/day) is required for effective treatment of

PCP and nocardiosis, and side effects, such as rash and neutropenia, are not

uncommon.65 High-dose cotrimoxazole would therefore be problematic as the

‘second antibiotic trial’ in a diagnostic algorithm and could only be justified by

documenting significant rates of PCP and nocardiosis in a locale.

Chest radiography has also been included in algorithms for selecting patients

with smear-negative pulmonary tuberculosis. However, CXR changes may be

atypical or may be due to other infections, particularly in HIV-positive

patients.6 Harries et al have also shown that using CXR, rather than AFB

microscopy, as the initial screen in a diagnostic algorithm is less sensitive and

more expensive.66 Hence, collection of 2-3 sputum specimens for microscopy

should precede CXR in any diagnostic algorithm. In developing countries,

tuberculin skin testing (TST) is confounded by the high coverage of BCG

vaccination, asymptomatic TB infection, the presence of nontuberculous

mycobacteria, and anergy due to HIV or malnutrition.67 TST therefore has no

place in diagnostic algorithms in these settings.

Figure 1 outlines an algorithm for managing TB suspects which is based on

several studies.1,6,30,58-60,66,68 The sensitivity and specificity of this algorithm

will vary depending on the acumen of the local clinicians, and the prevalence

07/11/08 17

of HIV, of antibiotic-resistant S. pneumoniae and H. influenzae that may

cause the treatment trials to fail, and of the diseases that can mimic smear-

negative pulmonary tuberculosis (Table 3). Such algorithms must therefore be

validated and optimised in each country.

While most steps in this algorithm are broadly accepted, controversy

surrounds the correct management of smear-negative patients who have

failed two courses of antibiotics. The physician has several choices: (i) to

make a definitive diagnosis of smear-negative TB and to treat accordingly, (ii)

to consider other diagnoses, or (iii) to observe and re-investigate for TB over

the next three months. Some authors have also suggested instituting a two-

month ‘therapeutic trial’ of antituberculosis treatment.6,68 However,

inappropriate implementation of ‘therapeutic trials’ for TB could lead to

treatment failures, acquired drug resistance, and subsequent difficulties in

correctly categorising patients into treatment and re-treatment programs.

These ‘therapeutic trials’ may also lack specificity because patients with other

infections may respond to the broad antibacterial effect of rifampicin-

containing regimens. Definitive antituberculosis treatment is therefore

recommended for patients who complete the management algorithm (Figure

1) and are still suspected of having TB.

Improving specimen collection

Two or three high-quality sputum specimens are required to reliably detect

smear-positive pulmonary TB.53,54 Occasionally patients cannot produce

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adequate specimens. Simple chest physiotherapy may be worthwhile.6 Pithie

and Chicksen have shown that Z-N staining of a fine-needle aspirate from a

palpable extra-thoracic lymph node is another worthwhile investigation for

HIV-positive patients with suspected smear-negative TB in developing

countries.69

In industrialised countries, bronchoscopy would be the preferred investigation

for such patients. Rao has demonstrated the usefulness of bronchoscopy

among smear-negative patients in India.70 Of 55 sputum smear-negative

patients, 15 (27.3%) had AFB-positive bronchial washings (cultures were not

performed); bronchial carcinoma was diagnosed in another 5 patients.

However, bronchoscopes are expensive and require on-going disinfection and

maintenance. Bronchoscopy is also an invasive procedure with recognised

complications. In fact, Daley et al evaluated the role of bronchoscopy among

237 patients in Tanzania, and found that, if an algorithm such as figure 1 was

used, only 17 (7%) patients required bronchoscopy and a treatable disease

was found in only three.30 They concluded that bronchoscopy had little place

in the investigation of smear-negative TB patients in resource-poor areas.

Sputum induction with nebulised hypertonic saline is a realistic alternative

investigation in such settings.71 The technique has been used widely in

industrialised countries for diagnosing PCP in patients with AIDS. However, it

was originally used in the 1960s to obtain adequate sputum samples for

cytological examination and TB investigations.71,72 Anderson et al found that

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sputum induction performed as well as bronchoscopy in the diagnosis of 101

smear-negative Canadian patients.71 Smear microscopy on bronchoscopic and

induced sputum specimens had sensitivities of only 12% and 19%,

respectively, but culture of the bronchoscopic and induced sputum specimens

had similar improved sensitivities (ie. 73% and 77%, respectively).

Culture is generally not available in resource-poor countries. Nonetheless,

Parry et al have demonstrated the usefulness of sputum induction in Malawi.20

Among 82 patients who could not produce sputum or were smear-negative,

induced sputum was smear-positive in 18 (22%); positive cultures were

obtained from these 18 induced sputum specimens and from an additional 12

smear-negative samples. A nurse or physiotherapist could perform the

procedure on 7-8 patients in one morning. To prevent the spread of TB to

other patients or staff, the procedure was done in a well-ventilated room and

contaminated equipment was washed then soaked in glutaraldehyde. In fact,

infection control measures are the problematic factor in using sputum

induction in developing countries. For example, Parry et al could not establish

this technique as a routine procedure after their study because of an

unreliable supply of glutaraldehyde for decontamination. Nonetheless, sputum

induction appears the best procedure for obtaining better specimens from

smear-negative patients in resource-poor countries.

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Improving laboratory diagnosis

Before the HIV epidemic, two well-performed sputum examinations were

considered as sensitive as a single culture.5 With the increasing rate of smear-

negative disease and the rising prevalence of drug resistance, culture facilities

may need to be more widely available in developing countries and new

convenient TB diagnostics are urgently required.73,74

In the meantime, can sputum microscopy be improved? The carbol fuchsin

staining procedures (ie. Z-N and Kinyoun stains) generally used in developing

countries are less sensitive than fluorochrome-stained smears, which take

only 1-2 minutes to read.41-43,73 The cost-effectiveness of fluorescence

microscopy largely depends on local labour costs but may become worthwhile

when a laboratory processes more than 30 smears per day.43 The major

disadvantages of fluorescence microscopy are the need for a reliable

electricity supply, the extra capital outlay (ie. a fluorescence microscope is 4-5

times as expensive as a light microscope), and the additional maintenance

(eg. halogen lamps must be replaced after 200 hours of use).41-43,73

Standard light microscopy may be improved by homogenisation and

concentration of the sputum specimen. Various methods have been described

(eg. N-acetyl-L-cysteine plus 2% NaOH, 4% NaOH, dithiothreitol plus 2%

NaOH) and are generally followed by concentration by centrifugation.40-43, The

efficiency of these techniques depends on the toxicity of the digestant-

decontaminant solution, heat build-up in the centrifuge, and the centrifugal

07/11/08 21

force applied. This final factor is of most importance.40 The mycobacterial cell

wall has a high lipid content and is therefore naturally buoyant. Hence, the

specific gravity of the suspending fluid must be minimised and the relative

centrifugal force (RCF, measured in g) maximised to optimise the recovery of

AFB. Adequate sedimentation efficiency (95%) can be achieved by

centrifugation at 3,000 x g for 15 minutes. Unfortunately, centrifuges in many

laboratories, particularly in developing countries, cannot attain this RCF.

Acceptable recovery rates (ie. >70%) are only attainable at lower RFC if the

centrifugation time is prolonged (eg. 2,000 x g for 20 minutes).75

Gebre et al have reported using household bleach (sodium hypochlorite,

NaOCl) to liquefy sputa and then centrifugation to concentrate the

mycobacteria.76 Initial homogenisation with NaOCl produced better recovery

of AFB than did digestion with either NaOH or dithiothreitol, and this

improved efficiency was seen following centrifugation at different RCFs. In

practical terms, the sensitivity of AFB microscopy increased from 30.8% for a

direct smear to 69.2% using the NaOCl technique. Miörner et al simplified the

technique by showing that AFB could be concentrated as effectively by

overnight sedimentation as by centrifugation.77 Household bleach is readily

available and is mycobactericidal thereby reducing the risk of laboratory-

acquired infections. Microscopy examinations are easier because liquefaction

with NaOCl removes background debris and a higher density of AFB per

microscope field is obtained by concentration. The main disadvantage of

these techniques is the additional processing time.

07/11/08 22

Unfortunately, these techniques have not been widely applied in the field.73 In

the only published evaluation, Wilkinson and Sturm found that NaOCl

liquefaction and subsequent centrifugation did not increase the overall

diagnostic sensitivity of smear microscopy.78 While investigating 166

consecutive TB suspects in Hlabisa Hospital, South Africa, an extra 12 smear-

positive specimens were detected after processing. However, 13 specimens

that were positive by direct smear were negative after processing,

presumably because AFB were not effectively pelleted during centrifugation.

Of the diseases that mimic smear-negative pulmonary TB (Table 3), only

nocardiosis could be detected by a simple investigation. In direct sputum

smears, nocardia appear as gram-positive beaded branching filaments and

are acid-fast using a modified Z-N stain.79 Gram and modified Z-N stains of

sputum may therefore be worthwhile examinations if nocardiosis is prevalent

in a particular locale, such as in Abidjan, Ivory Coast.49

Improving treatment

The recommended management of smear-negative pulmonary TB is still

based on the characteristics of this disease in HIV-negative populations (Table

1).1,15,16 Developing countries are encouraged to concentrate their limited

resources on the effective treatment of smear-positive cases, who are more

infectious and who were considered to have a higher mortality. There is also

a fear that inclusion of ‘smear-negative’ cases in TB programs may lead to

07/11/08 23

over-treatment of patients who do not have TB. Treatment of smear-negative

disease is therefore restricted in many developing countries, such as Rwanda

and the DRC,80,81 or prolonged inferior regimens (eg. 2 STH/10 TH, 2 EHZ/10

HT) are used.82,83

Can inferior regimens continue to be recommended for smear-negative TB in

the face of the HIV/TB dual epidemic? Firstly, local diagnostic facilities have

been so over-burdened that smear microscopy may be either unavailable or

unreliable.44,55 Remember our earlier example that nearly one-third of patients

in Tanzania defined as smear-negative based on local microscopy were found

to be smear-positive by a reference laboratory.55 Classification of patients as

smear-positive and –negative (and, more importantly, prescription of

treatment based on this differentiation) becomes almost arbitrary in these

circumstances.

Secondly, the HIV epidemic has changed some basic characteristics of smear-

negative pulmonary TB (Table 1). HIV-positive patients with smear-negative

TB may actually have a higher mortality rate than smear-positive cases.39

‘Standard’ 12-month regimens containing thioacetazone have also been

associated with higher rates of adverse reactions and with higher relapse

rates in HIV-positive patients.33-35

In countries affected by the dual HIV/TB epidemic, De Cock and Wilkinson

have therefore suggested that short-course chemotherapy (SCC) based on

07/11/08 24

rifampicin (with DOT supervision) be recommended for all new cases of TB

irrespective of smear status.44 Harries et al have made a similar suggestion.4

Short-course regimens have been associated with improved survival in HIV-

positive TB patients.4,34,36,37,39 For example, in a Ugandan study of 191 HIV-

positive patients with smear-positive TB receiving standard (2 STH/10 TH) or

short-course (2 HRZ/7 HR) chemotherapy, the relative risk of death for

standard compared with short-course chemotherapy was 1.57 (95% CI 1.0-

2.48).34 This improved survival has been partly attributed to the

prevention/treatment of other intercurrent infections by the broad

antibacterial activity of rifampicin.4 The survival advantage of SCC has not

been studied specifically in smear-negative HIV-positive patients but they

should benefit equally from the broad activity of rifampicin.

A single SCC regimen for all new TB cases would also avoid confusion,

simplify guidelines, increase completion and cure rates, and reduce non-

adherence and side effects.44 Fears of over-treating non-TB patients should

be assuaged if validated management algorithms (eg. figure 1) are followed.

In the end, cost is the only prohibitive factor to universal SCC for new TB

cases. In 1995, Chaulet calculated that the WHO Category I regimen for

smear-positive cases (eg. 2 EHRZ/4 HR) cost US$ 32.34 while the Category

III regimen (ie. 2 HRZ/6 HE) for smear-negative patients cost US$ 21.40.83 A

similar US$ 10-15 price differential existed between various Category I and III

regimens that contained streptomycin, ethambutol or thiocetazone.83 This

additional cost is significant but some savings could come from the

07/11/08 25

streamlined approach of a universal regimen, from bulk purchasing of a single

regimen, and from the reduced number of patients requiring re-treatment or

developing resistance. The additional cost of using SCC for all new cases must

be weighed against the definite benefits, and the international community

must facilitate the introduction of a universal SCC regimen for new TB cases if

it proves cost-effective.

Conclusion

Smear-negative pulmonary TB is an increasing clinical problem in developing

countries affected by the dual HIV/TB epidemic. Management algorithms that

have been validated by local studies should improve case detection. Wider

use of sputum induction and evaluation of novel sputum processing

techniques may also improve the investigation of these patients. Some

authors have argued for the wider availability of TB culture facilities in

developing countries,74 and for universal access to SCC for all TB patients

irrespective of smear status.4,44 However, these Utopian interventions will

require increased financial and technical support from the international

community.

07/11/08 26

Acknowledgements

We thank Prof. Françoise Portaels and Dr. Armand Van Deun for their helpful

comments during the preparation of this manuscript. Financial support was

obtained from the National Fund for Scientific Research, Belgium. IB is

supported by a Neil Hamilton Fairley Fellowship (987069) awarded by the

National Health and Medical Research Council of Australia.

07/11/08 27

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Table 1 Characteristics of smear-negative pulmonary tuberculosis in

HIV-negative and HIV–positive populations

Classic features of smear-negative pulmonary tuberculosis

1.22 cases of SNP and EP TB for each SPP TB case

Low bacillary burden with minimal disease and no cavitation

Less infectious than smear-positive cases

Lower mortality than smear-positive cases

Effectively treated with a variety of regimens

Effect of HIV on smear-negative pulmonary tuberculosis

Disproportionate increase in incidence of SNP TB

SNP TB becomes a marker of advanced immunosuppression

Infectivity of smear-negative cases probably unchanged

HIV-positive patients with SNP TB have higher mortality rates

‘Standard’ chemotherapy associated with more side effects,

increased mortality rates, and higher relapse rates

SNP TB, smear-negative pulmonary tuberculosis; EP, extra-pulmonary; SPP,

smear-positive pulmonary; ‘standard’ chemotherapy denotes 12-month

regimens employing streptomycin, isoniazid, and thioacetazone

07/11/08 39

Table 2 Outcome at 30 months for 283 Hong Kong patients initially

diagnosed with pulmonary tuberculosis despite five negative smears*

Patients (%)

Required treatment 200 (71%)

Initial specimens proved culture positive 107 (38%)

Subsequent progress demanded treatment† 93 (33%)

Evidence of active disease not treated‡ 49 (17%)

No evidence of active disease 34 (12%)

* Adapted from reference 14

† Developed active disease requiring treatment based on bacteriological,

radiological or clinical findings

‡ Had one or more isolated positive cultures and/or radiological evidence of

active disease

07/11/08 40

Table 3 Important differential diagnoses of smear-negative pulmonary

tuberculosis in developing countries

Diagnoses Frequency* Reference False-negative smear results No smear examination performed 44 Technical problems 41-43 Inadequate specimen quality Inadequate number of specimens Poor staining technique Overly thick smears Other medical conditions Bacterial pneumonia 14.0%-41.2% 30, 45-47 Empyema 2% 30 Pulmonary nocardiosis‡ 0%-4% 46-51 Pneumocystis carinii pneumonia‡ 1%-33% 30, 45-48, 50 Cryptococcal pneumonia‡ 0%-13% 30, 45-48, 50 Histoplasmosis 3% 47 Pulmonary Kaposi’s sarcoma‡ 1.3%-9.3% 30, 45-48, 50 Interstitial pneumonitis 38% 48,51 Cytomegalovirus pneumonitis 1.5% 50 Gram-negative bacteraemia‡ 9%-10.4% 46,52 Carcinoma 1% 52 Lymphoma Congestive cardiac failure Asthma, chronic obstructive lung disease Allergic bronchopulmonary aspergillosis Occupational lung diseases (eg. silicosis) Extrinsic allergic alveolitis Psittacosis

07/11/08 41

Legend to Table 3

* Bronchoalveolar lavage and autopsy studies have been performed in

developing countries to determine the cause of respiratory disease in HIV-

positive and HIV-negative patients. The prevalences of non-tuberculous

disease shown are from a compilation of these studies and may be affected

by geographic differences in the rates of some diseases, such as

cryptococcosis,45,48 by patient selection, by the range of laboratory

investigations performed, and by the local prevalence of HIV.

‡ Conditions of increased importance in HIV-positive patients

07/11/08 42

Initial screen based on symptoms:eg. cough > 3 weeks; loss of weight;

no response to ampicillin 500 mg qid x 7-10 days

↓ Microscopy of 2-3 sputum specimens

07/11/08 43

AFB + ↓ ↓ AFB -

Registration and Treatment

for Pulmonary TB CXR and

← Clinical Consultation

*additional ↓ investigations

Second Antibiotic Trial (eg. cotrimoxazole)

and Physician Review

↓ if no response

Antituberculosis Treatment to be considered by Physician†

Figure 1

Algorithm for managing TB suspects in developing countries. Adapted from

references 1, 6, 30, 58-60, 66, and 68. * Additional investigations may include

lymph-node aspiration and sputum induction.24,69,71 † See text for discussion

of the management of smear-negative patients who fail to respond to the

second course of antibiotics. AFB, acid-fast bacilli.