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Health Surveillance and Disease Management / Communicable Diseases / Tuberculosis

Manual for the Bacteriological Diagnosis of Tuberculosis:

Standards and Technical Guidelines—Part 1, Smear Tests

Manual para el diagnóstico bacteriológico de la tuberculosis

Full text (in Spanish, 66 pp, PDF, 1540 Kb; chapter headings translated below for user orientation)
- Introduction (translated to right)
- Sample
- Sputum
- Other Samples
- Receiving, Conserving, and Transporting Samples
- The Smear Test
- Workspace and Materials
- Preparing and Fixing
- Staining
- Microscopic Observation and Readings
- Reporting Results
- Decontamination and Waste
- Deriving Samples for the Culture
- Registry System
- Smear Test Quality Control
- Internal Quality Control
- External Quality Control
- Selected Bibliography
- Annexes
Annex I: Normas mínimas de bioseguridad
Annex II: Preparación de colarantes y reactivos
Annex IV: Modelos de formularios

Editorial Team
Authors from the "Dr. Carlos G. Malbrán" National Laboratory and Health Institutes Administration (Administración Nacional de Laboratorios e Institutos de Salud / ANLIS), Argentina:
M.D. Sequeira de Latini, National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias / INER)
L. Barrera, National Institute of Infectious Diseases (Instituto Nacional de Enfermedades Infecciosas / INEI)
Technical Review:
S. Balandrano, Epidemiological Diagnositic and Reference Institute (Instituto de Diagnóstico y Referencia Epidemiológicos / InDRE), Mexico
M.C. Riquelme y M. Velazco, Institute of Public Health (Instituto de Salud Pública / ISP), Chile
E. Montoro, "Pedro Kourí" Institute of Tropical Medicine (Instituto de Medicina Tropical "Pedro Kourí" / IPK), Cuba
M.C. Garzón Torres, National Institute of Health (Instituto Nacional de Salud / INS), Colombia
Special Review by Experts:
I. Narvaiz de Kantor and A. Laszlo

Manual Part 2, Cultures

Stop TB / Alto a la TB

PAHO Links
- Tuberculosis Page

WHO Links Stop TB
- New Technologies for TB Control: A Framework for Their Adoption, Introduction, and Implementation:
Executive Summary
- WHO TB Page

transmisión de la TB

Handbook produced especially for use in Latin America for strengthening laboratory capacity

It is estimated that around a third of the world's population—about two billion people—is infected with Mycobacterium tuberculosis, the bacillus that causes TB; approximately 8 million of them annually become ill and nearly two million die from the disease, despite the fact that there are simple and precise diagnostic techniques and effective treatments.

Transmission of TB bacilli occurs almost exclusively by means of nuclei suspended in small droplets expelled with the expectoration of those affected by pulmonary tuberculosis. These minute droplets can remain infectious in air for enough time for them to be inhaled by other people. Infecting one's contacts is more probable when they live with or stay for a prolonged period near the patient who is expectorating bacilli and in an environment with scant ventilation.

The greater the number of patients expectorating bacilli in the community, the greater the spread of tuberculosis. Identifying infectious cases is the main solution to this problem, both for patients and in terms of public health.

Not all infected people will become ill, only about one out of every ten who are most susceptible. Tuberculosis can be manifested in any organ, because M. tuberculosis spreads throughout the entire body; however, pulmonary disease is most frequent (80-85% of all diagnosed cases) since the bacillus needs abundant oxygen in order to multiply. In patients' lungs, cavities can be formed that harbor large populations of bacilli, which can be detected in sputum samples. The symptoms most characteristic of pulmonary tuberculosis are a persistent cough and expectoration for more than two weeks. People with these symptoms are referred to as Respiratory Symptomatic (RS) patients. Other manifestations might be weight loss, a slight fever, night sweats, physical fatigue, and pain in the thorax area.

A definitive diagnosis of tuberculosis can be made reliably in the laboratory by demonstrating the presence of bacilli in a sample from the injured area, by means of sputum-smear microscopy (microscopic examination, commonly called the smear test) or through a culture.

For a sputum smear to be positive, the sample must contain a minimum of between 5,000 and 10,000 bacilli per milliliter of sample. This high bacillus content can be found in patients with pulmonary tuberculosis, especially in those at an advanced stage of the disease who have cavity-type lesions. These patients are the ones who can transmit the bacilli by keeping the disease in the community.

The main purpose of a National Tuberculosis Program (NTP) is breaking the chain of transmission, diagnosing infectious cases early on and treating them with effective schemes that will bring about a cure. The strategy recommended internationally to reach this goal is DOTSDirectly Observed Treatment, Short-course. This strategy requires political commitment to guarantee the resources needed to control tuberculosis, access to sputum-smear microscopy with quality assurance for case-finding, control of patient evolution, ensuring uninterrupted access and availability of the drugs integrating standardized treatment regimens that cure patients, and a recording and information system that allows for evaluation of treatment results and program performance.

Sputum-smear microscopy is the technique of choice for rapid diagnosis and control and treatment of pulmonary tuberculosis in adults

Smear tests are simple, economic, and efficient when it comes to detecting infectious cases. Hence, it is the fundamental tool of tuberculosis control programs. Sufficient laboratories are needed to ensure patients a timely, accurate, and accessible diagnosis. Laboratory services are more efficient and powerful when integrated into a national tuberculosis laboratory network that should involve laboratories from the public health system of all jurisdictions, including to those providing services to prisons, the health insurance system, private health systems, and those of nongovernmental organizations (NGOs). Network functions should be integrated at the level of the NTP for program formulation and decision-making, which in turn should make the necessary arrangements for sustaining network organization and operation.

All network components have a shared responsibility, complementing one another in ensuring access to rapid, reliable diagnosis through sputum-smear tests. All health units should receive RS samples that bear investigation. In addition, laboratories located in primary health care centers should carry out smear tests and be integrated into quality assurance programs. Intermediate laboratories have additional responsibilities, i.e. training laboratory personnel in their jurisdiction and ensuring the quality of the smear tests. Central or national reference laboratories must be capable of organizing quality assurance throughout the country, performing continuous evaluation of current offers and carrying out sputum-smear test, providing training tools for laboratory staff at all levels, planning, and providing input when centralized procurement is advisable. The remaining components of the NTP should make adequate use of available smear tests and the results produced by the laboratory network.

For sputum-smear microscopy to function as a good control tool, technical quality by itself is not enough. Also critical are the quality of the registries, laboratory reports, and information analysis produced by the laboratory.

Standardizing the procedures involved in sputum-smear microscopy is based on technical standards based on broad experience, periodically reviewed by such international organizations as PAHO/WHO and the International Union Against Tuberculosis and Lung Disease (IUATLD).

The first TB Microscopy Manual to be implemented by PAHO for Latin America was written by Dr. Luis Herrera Malmsten, Chief of the Tuberculosis Department at the Bacteriological Institute of Chile, reviewed and approved by an Advisory Committee on Tuberculosis Bacteriology and published as CD/TBST/LAB in 1973. The manual was used until 1983, when a PAHO/WHO Advisory Committee updated the standards in (PAHO/WHO) CEPANZO Technical Note No. 26. The committee was made up of Dr. Lamberto Blancarte of the Central Tuberculosis Laboratory of Mexico, Omar Latini of the National Epidemiology Institute (Instituto Nacional of Epidemiology / INE) of Argentina, Adalbert Laszlo of the Laboratory Centre for Disease Control of Canada, and Pedro Valenzuela of the Institute of Public Health of Chile, under the coordination of PAHO Drs. Isabel N. de Kantor and Álvaro Yáñez.

Although technological progress has promoted innovations in bacteriology, sputum-smear microscopy has not been subject to substantial technical modifications. However, new epidemiological situations—especially the incidence of tuberculosis among people living with HIV and the need to more rigorously guarantee the quality of results and human and environmental safety—have promoted a new update of the standards seen in this book.