Tuberculosis

Laboratory Diagnosis and Treatment Strategies

Timothy McHugh, Timothy D McHugh

• Publisher: CAB International
• Year: 2013
• Print ISBN: 9781845938079
• eBook ISBN: 9781789244670
• Topic: Medicine
• Subtopic: Medical Microbiology & Parasitology

Tuberculosis is a global health threat and the unique features of Mycobacterium tuberculosis and emergence of drug-resistant strains highlight the challenge it presents. Covering a wealth of state-of-the-art knowledge from active international experts, this book captures the latest developments in the advent of bacteriological, immunological and molecular tools for diagnosis and the development of new drugs. It shows how the challenge of tuberculosis is currently being met, providing insight into the evidence base underlying new developments in diagnosis, drug development and treatment.

Part I

Diagnosis

1 Improving on Sputum Smear Microscopy for Diagnosis of Tuberculosis in Resource-poor Settings

Andrew Ramsay,¹ Karen R. Steingart² and Madhukar Pai³

¹UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, Geneva, Switzerland; ²University of Washington School of Pubiic Health, Seattie, USA; ³Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada

1.1 Introduction

In 2011, despite the long-established tradition of solid culture for Mycobacterium tuberculosis and the numerous more recent advances in tuberculosis diagnosis, direct sputum smear microscopy remains the cornerstone of global tuberculosis control. However, direct sputum smear microscopy has major limitations. It is associated with low and variable sensitivity, particularly in HIV-associated tuberculosis. It gives no information regarding the drug resistance of the infecting tuberculosis bacilli. It is, however, to date, the only diagnostic method for tuberculosis that has been shown to be implementable and sustainable in resource-poor health-care facilities in low-and middle-income countries (LMICs). The dire situation is well recognized by national tuberculosis programmes, technical agencies and donors. The World Health Organization (WHO), in an effort to improve diagnosis, has made more than 15 global recommendations related to tuberculosis diagnosis in the past 4 years, and yet little has changed on the ground (WHO, 2011a). This chapter will discuss the limitations of direct sputum smear microscopy as practised currently, review the recent WHO recommendations and discuss how the performance of sputum smear microscopy may be improved, explore how sputum smear microscopy services may be complemented by add-on tests and identify which tests (now and in the future) may replace smear microscopy in certain defined situations.

1.2 Direct Sputum Smear Microscopy

Direct sputum smear microscopy is inherently insensitive since the typical acid-fast bacilli (AFB) can only be detected when large numbers of them are present in the sputum (>10⁵ organisms/ml). This impacts particularly on the diagnosis of HIV-associated tuberculosis, which is often paucibacillary, as well as the diagnosis of tuberculosis in children, who are often unable to produce a sputum specimen. Moreover, well-trained and dedicated microscopists are needed to ensure a good quality service. Ziehl-Neelsen (ZN) stained smears require examination under the microscope for 5-10 min before being declared negative (IUATLD, 2000). To counter the inherent lack of sensitivity of the method, the WHO recommended that three sputum specimens (including an early morning specimen) be collected for examination from each suspected tuberculosis patient over 2 days. This clearly has implications for the laboratory workload. A microscopist can only examine some 20-25 smears/day (IUATLD, 2000). Smear microscopy workloads heavier than that impact adversely on service quality. The requirement for three sputum specimens also places a burden on the suspected tuberculosis patient, since they have to make repeated visits to the health-care facility to submit specimens, receive results and start any treatment indicated. This is particularly burdensome if the patient is poor, weak or has travelled a long distance to the healthcare facility. Patient dropout during the diagnostic process is common (Kemp, 2007; Botha, 2008).

The three-specimen, multi-day standard approach above, which has been devised to improve the sensitivity of sputum smear microscopy, also includes, paradoxically, a requirement that reduces the sensitivity of the technique further. A patient must have two positive smears to be defined as a smearpositive tuberculosis case, and one of these smears must contain at least 10 bacilli/100 high-power microscopic fields (HPF). These last requirements were introduced to maximize the specificity of direct sputum smear microscopy. The requirement dates back to the times when treatment regimens were longer and rifampicin was very expensive and was likely a measure to reduce costs and inconvenience to patients resulting from unnecessary treatment. Today, first-line treatment for tuberculosis is shorter, but 6-month regimens are still a considerable undertaking for patients. The cost of all the drugs for the standard 6-month first-line regimen is now approximately US$10-30 (Management Sciences for Health, 2005).

Sputum smear microscopy gives no information on the drug susceptibility of tuberculosis bacilli detected in the sputum smear of new tuberculosis patients and therefore cannot diagnose multidrug-resistant tuberculosis (MDR-TB) or extremely drug-resistant tuberculosis (XDR-TB).

In addition to the diagnosis of new cases, direct sputum smear microscopy repeated at different time points following initiation of anti-tuberculosis treatment is used to monitor patients’ response (Gilpin et al., 2007). Continued presence of AFB in sputum may indicate treatment failure, but this may be due to causes other than drug resistance, such as failure to take medication as indicated.

The recommended system for external quality assessment (EQA) of smear microscopy services requires the blinded rechecking of a sample of the slides examined by each laboratory. Usually, the national tuberculosis reference laboratory is expected to take on this monumental task. It is recommended that blinded rechecking is complemented by regular visits to microscopy laboratories to ensure a proper staining technique is used and to check the condition of microscopes (Association of Public Health Laboratories, 2002). Microscopes can be damaged or impaired by rough handling. In hot, humid climates, the lenses of microscopes are prone to colonization by algae or mould, which can render them worse than useless. A fully functional national EQA scheme based on this system is rarely achieved or sustained.

Nevertheless, most district hospitals and large health centres in countries with a high prevalence of tuberculosis can offer a sputum smear microscopy service of some kind. It is estimated that more than 50 million smear microscopy examinations are conducted globally each year (TDR/FIND, 2006). The only equipment required is a microscope (relatively inexpensive as laboratory equipment goes) and a gas, or spirit, burner. Microscopes generally need an electricity supply, but they can accommodate some fluctuation in power without damage. Microscopes may also be powered off car batteries or solar cells or, with a mirror and some experience, can be operated using directed daylight. The only consumables and reagents required are low cost and include specimen pots, applicator sticks for making smears, glass slides, a simple set of heat-stable stains with very long shelf lives and small amounts of microscope immersion oil to use with the ×100 oil objective lens. Training of staff is straightforward and relatively short and, importantly, is a well-established module in laboratory assistant and laboratory technician training courses and given importance in all countries where tuberculosis is a public health problem.

1.3 Optimized Sputum Smear Microscopy

In the absence of simple, low-cost alternatives to sputum smear microscopy much effort has been expended in recent years in developing optimized smear microscopy procedures. Much of this effort was catalysed by a series of systematic reviews of smear microscopy conducted in 2005-2006 and which led to the development of a prioritized international research agenda and to five global policy changes by the WHO in 2007-2009 (Steingart, 2006a,b, 2007a; Mase, 2007; WHO, 2007b,c, 2010b,c).

These systematic reviews focused on methods of improving sputum microscopy services in LMICs through more efficient and patient-friendly specimen collection, the use of fluorescence microscopy and the chemical digestion of sputum followed by a physical sputum concentration method.

The main findings of the systematic reviews were as follows.

1.3.1 Serial sputum smear examination (Mase, 2007; Steingart, 2007a)

The average increase in sensitivity/incremental yield of tuberculosis cases through examination of the third specimen was only 2–5%. Thus, evaluating 1000 persons suspected of having tuberculosis in a setting with 10% prevalence of the disease would require examination of 900 third specimens to yield an additional two–five cases. However, the analysis defined a smear-positive case as one with only one or more positive smears (≥3 AFB/100 HPF). The examination of only two specimens decreased the number of sputum smear-positive cases defined as having two positive smears (≥10 AFB/100 HPF). A service that examined only two specimens might be more efficient and less onerous for laboratory staff. However, such a policy decision would require reassessment of the standard definition of a smear-positive case. If both specimens could be collected (and preferably examined) on the same day, this could reduce the number of patient visits required, reduce patient expenditure and possibly reduce default during the diagnostic process.

1.3.2 Fluorescence microscopy (Steingart, 2006a, 2007a)

Fluorescence microscopy (FM) was, on average, 10% more sensitive than ZN microscopy, with similar (98%) specificity. FM was also much quicker than ZN microscopy, with FM examination for 1 min being associated with a higher sensitivity and similar specificity to ZN examination for 4 min. The systematic review was conducted prior to the recent work on FM based on light-emitting diodes (LEDs). Conventional FM was dependent on very expensive, sophisticated equipment considered inappropriate for use outside of high throughput reference laboratories in LMICs. However, the authors noted that simple, low-cost LED-FM systems were being developed and should be evaluated in resource-poor settings (Gilpin, 2007).

1.3.3 Specimen processing (Steingart, 2006b, 2007a)

The systematic review indicated that, in comparison with direct smears, centrifugation preceded by digestion with any one of several different chemicals (including household bleach) was more sensitive. The review also revealed that overmight sedimentation preceded by chemical processing (including with household bleach) was more sensitive and specificity was similar. The latter method, since it did not require an expensive centrifuge, was considered to be more appropriate to basic laboratories in resource-poor settings.

A 5-year long, multi-partner, internationally coordinated programme of research followed, which provided evidence that (Ramsay, 2011):

1. ‘Scanty’ smears (i.e. with less than 10 AFB/100 HPF) were ‘true positive’ smears and considering these as positives increased the sensitivity of sputum smear microscopy without reducing specificity.

2. A single positive smear (including scanty smears) was sufficient to confirm a patient as a smear-positive tuberculosis case and increased the sensitivity of sputum smear microscopy without reducing specificity.

3. Reducing the number of specimens examined from three to two resulted in more efficient detection of cases, with only marginal reductions in sensitivity (2-5%).

4. Examining two specimens collected 1 h apart on the same day was equivalent to examination of two or three specimens collected over 2 days (Ramsay, 2009; Cuevas, 2012a).

5. Sensitivity and specificity of LED-FM was comparable to that of conventional FM.^21,22 The use of LED-FM was more sensitive than ZN microscopy, and if sufficient attention was paid to proper training of microscopists, it had similar specificity (including when used with a same-day specimen collection approach) (Cuevas, 2012b).

6. Bleach digestion followed by overnight sedimentation led to only small increases in sensitivity (9%), with associated small decreases in specificity (-3%).

7. LED-FM combined with bleach digestion and over night sedimentation had the same sensitivity and slightly lower specificity than direct LED-FM alone (Bonnet, 2011).

Between 2007 and 2010, the WHO endorsed a new definition of a positive smear, a new definition of a smear-positive case, a reduction in the number of sputum specimens to be examined in the investigation of pulmonary tuberculosis, same-day microscopy and direct LED-FM (Ramsay, 2011).

A pragmatic randomized trial in Pakistan showed that simple standardized instructions on how to produce good quality sputum specimens increased significantly the number of smear-positive cases detected among women (Khan, 2007).

Thus, an optimized sputum smear microscopy service would incorporate simple standardized instruction on sputum specimen production, collection (and examination) of two specimens on the same day, use of LED-FM and new definitions of a positive smear and a smear-positive case. Wherever possible, smear results should be reported on the same day and treatment initiated to reduce initial default.

1.4 Serological Tests as Add-on or Replacement Tests

A series of systematic reviews have evaluated the performance of various serological (antibody detection) tests for the diagnosis of tuberculosis (Steingart, 2007b,c,d, 2009, 2011a; WHO, 2008). These tests used formats that required laboratory infrastructure such as enzyme-linked immunosorbent assays (ELISAs), as well as rapid immuno-chromatographic test platforms. The latter tests, if they could perform as well as smear microscopy, potentially could replace it, making diagnosis simpler and quicker. Moreover, the tests, being simple and robust, could be performed by non-specialized staff in health centres without laboratories and closer to the community. If performance did not compare with smear microscopy, there was the possibility that their use might complement smear microscopy and serological testing of smear-negative patients might result in an incremental gain in tuberculosis cases detected. A laboratory-based head-to-head evaluation of 19 commercially available rapid antibody detection tests for tuberculosis was included in the systematic review (WHO, 2008a).

The systematic reviews and metaanalyses concluded that diagnostic accuracy data indicated that none of the tests could replace sputum smear microscopy, nor could they be used as an add-o...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Contents
• Contributors
• Abbreviations
• Introduction
• Part I – Diagnosis
• Part II – Measuring Resistance
• Part III – Understanding Treatment
• Part IV – Treatment Strategies
• Index