key: cord-022070-soqeje4z
authors: Parry, Christopher M.; Peacock, Sharon J.
title: Microbiology
date: 2019-05-28
journal: Hunter's Tropical Medicine and Emerging Infectious Diseases
DOI: 10.1016/b978-0-323-55512-8.00021-1
sha: 
doc_id: 22070
cord_uid: soqeje4z

The management and containment of many treatable and preventable infectious diseases in resource-poor countries is limited by the failure to make an accurate diagnosis. Most of the world's population lacks access to accurate, affordable, easy-to-use, quality-assured, reliable, and accessible diagnostic tests and misdiagnosis of infectious diseases is common and compromises patient care. Laboratory diagnostics are also needed for the detection and surveillance of the increasing levels of antimicrobial resistance. Accurate clinical diagnosis in resource-poor settings relies strongly on the laboratory service, and the need to support the development of a quality-assured laboratory service in such settings is increasingly recognized. International organizations are actively working with local and national providers to improve laboratory services. The development of laboratory services will contribute to improved health for the local population, protection against emerging pathogens, and ensure better use of scarce health care resources.

Christopher M. Parry, Sharon J. Peacock support. Diagnostic algorithms have been developed for situations with no laboratory backup, an approach adopted, for example, in the Integrated Management of Childhood Illness (IMCI). Unfortunately, for many infections, clinical features lack sufficient specificity to allow them to be used to differentiate the possible diagnoses, and over-treatment to cover the various possibilities is common.

In the assessment of the febrile child in the tropics, for example, malaria and systemic bacterial infections often have an indistinguishable clinical picture. Malaria may be diagnosed by smear microscopy, but bloodstream infections require a blood culture service. It has become clear in recent years that bloodstream infections represent an underappreciated burden of disease and mortality. This was clearly demonstrated by a study conducted in Kenya in which bacterial bloodstream infections diagnosed by blood culture were responsible for 26% of deaths among children admitted to a rural district hospital. 1 Without an accurate diagnosis and specific treatment, bloodstream infections such as those due to Salmonella enterica, Staphylococcus aureus, Streptococcus pneumoniae, or Burkholderia pseudomallei can carry a high mortality. Distinguishing cerebral malaria, bacterial meningitis, and encephalopathic typhoid may be similarly difficult without laboratory support. Children in sub-Saharan Africa with clinical symptoms of pneumonia may have pneumococcal pneumonia but can equally have malaria or invasive salmonellosis. A child with dysentery may be suffering from amebic colitis, Shigella infection, or enterohemorrhagic Escherichia coli. In adults, syndromic management of sexually transmitted infections is widespread but needs to be informed by periodic surveillance of antimicrobial susceptibility patterns. Emerging and potentially epidemic viral infections such as severe acute respiratory syndrome (SARS), influenza (H5N1 and H1N1), Ebola, and Zika require relatively sophisticated tests to confirm the diagnosis. 2 Infections by pathogens that are resistant to multiple antimicrobials are common in many tropical countries where there is widespread availability of over-the-counter antimicrobials. Appropriate therapy of these infections requires isolation of the causative organism and antimicrobial susceptibility testing. 3 Laboratories also have an important public health role within the health care system. The ability to investigate outbreaks of disease as part of epidemic preparedness is a key function. These might include outbreaks of watery or bloody diarrhea, epidemics of meningitis, or clusters of patients with fever of unknown etiology. In addition, laboratories are a critical component of disease control programs such as the national programs for the control of tuberculosis, HIV, and malaria. The lack of laboratory capacity to support the expansion of diagnostic testing and antiretroviral therapy in HIV programs and in disease outbreaks such as Ebola has made many international organizations appreciate the desperate plight of the laboratory service for the first time. 2 Tuberculosis can be diagnosed in many patients with a Ziehl-Neelsen-stained smear of sputum, but to extend the diagnosis in those who are acid-fast bacilli (AFB) negative or have multi-drug-resistant (MDR) disease requires more developed laboratory support. Furthermore, laboratories have an increasing role in infection control in health care settings and congregate facilities and in the prevention of health care-associated infections.

Accurate disease surveillance requires a laboratory network and is vital to inform public health policy concerning allocation of resources and disease prevention. Laboratories can help to

• Accurate diagnosis in resource-poor settings is severely limited by the absence of good diagnostic laboratory services. • Laboratories in resource-restricted settings struggle with poor facilities, lack of reliable water and electricity, inadequate equipment and consumables, insufficient staff, poor training and low morale, absence of standard operating procedures and quality assurance programs, and inadequate levels of biosafety. • A country plan for the development of a laboratory network requires consideration of the needs at primary, district, provincial/regional, and national levels. • At the district hospital level, a quality-assured repertoire of essential laboratory tests can contribute to improved health care. • Surveillance by microbiology laboratories provides an understanding of the causes of infection in the local population and the levels of antimicrobial resistance in key pathogens, and informs public health policy on appropriate antimicrobial therapy and preventive strategies. • There is increasing recognition of the need to support the development of a quality-assured laboratory service in resource-restricted settings and develop simple and robust point-of-care diagnostics both for routine clinical care and outbreak response. • Point-of-care rapid diagnostic tests are changing our approach to the diagnosis of some infectious diseases, but care needs to be taken about their usage and interpretation of results.

The effective management and containment of many treatable and preventable infectious diseases in resource-restricted countries is limited by the failure to make an accurate diagnosis. Access to accurate, affordable, easy-to-use, quality-assured, reliable, and accessible diagnostic tests is severely lacking for most of the world's population, and misdiagnosis of infectious diseases is common. Disease identification, appropriate treatment choice, and implementing public health measures for the prevention and control of endemic and epidemic infections all require laboratory support. This lack of reliable diagnostics compromises patient care. Laboratory diagnosis also highlights the increasing levels of resistance to antimicrobials in many infections and the need for newer, possibly unaffordable, antimicrobials such as broad-spectrum antimicrobials in bacterial sepsis, or second-line combination therapy for AIDS, malaria, and tuberculosis. This issue is increasingly recognized and being addressed in many regions.

In most resource-restricted settings, individual patient diagnosis is based on clinical signs and symptoms with little or no laboratory be rudimentary so that specimens referred to the next level are not transported in a timely manner and results do not return in a time period that will influence clinical management. It is standard practice in tuberculosis programs that patients who fail treatment should have a sample cultured for tuberculosis so that susceptibility tests can be performed. In a study of the transport of such specimens to the central reference laboratory in Malawi, only 40% of specimens arrived in the reference laboratory and only 36% of those samples received were successfully cultured for susceptibility testing. 5 The shortage of staff with appropriate education and training is a further problem. Many laboratory workers have no formal training and are simply trained at the bench. At the peripheral level, there may be only one laboratory assistant, with no more than secondary school education. At the district level, there may be assistants and technicians (formally educated in laboratory medicine for 3 years). At the central level, technicians may work alongside technologists (with 2 years specialist post-technician training) and scientists (university science graduates). Regardless of qualifications, laboratory workers often have a lowly status within the health sector, and the attrition of health care personnel out of government service results in low morale among those who remain. Private or research laboratories may attract the best technicians from the government sector. Diagnostic laboratories frequently have no representation at the local, provincial, or national level, or, if they do, it is only as part of the support services. In many countries, the voice of the laboratory is rarely heard. These many problems contribute to a poor biosafety situation in laboratories. The lack of equipment, knowledge, and training means that laboratory workers are processing samples with hazardous pathogens in an unsafe manner. In a study of tuberculosis laboratories in Korea, before safety conditions had been upgraded, the relative risk of being diagnosed with tuberculosis for the technicians performing drug susceptibility tests was 21.5 (95% CI 4.5-102.5) compared with non-laboratory workers. 6 The true magnitude of this problem in laboratory workers is difficult to gauge because surveillance of infection in laboratory workers is rarely performed or reported.

At a national level, the important contribution of laboratories needs to be appreciated within the ministry of health, by national and local health care managers, and by funding organizations. A representative of the laboratory services should be present in the key decision-making committees. Support is also needed from clinicians, who often have disproportionate influence within the system. A plan for the laboratory network should become part of the overall health care development plan. There needs to be a priority list of core and essential services provided in a qualityassured manner. The laboratory plan should include the provision for a tiered laboratory network at the primary, district, regional/ provincial, and national levels. The plans should be realistic, affordable, and sustainable. At the Level I or primary level, perhaps in a health post or health center serving outpatients, microscopy for malaria and tuberculosis and testing for HIV with a same-day service would be essential. These laboratories can serve as a collection point for samples that need referral to the next level. The Level II facility in the local district hospital would have a dedicated laboratory space and a broader repertoire of tests serving inpatients and outpatients. The tests offered would depend on the spectrum of local diseases and resources available, and may be limited to microscopy, simple biochemistry and serology, and blood transfusion, or may include bacterial culture facilities. Laboratories can act as a hub for the primary-level laboratories, providing them with support, supplies of reagents, and QA activities. At the Level III, define clinical problems by sampling surveys. For example, determining the antimicrobial susceptibilities of bacterial pathogens such as S. aureus, S. pneumoniae, or S. enterica for a selection of isolates can inform the appropriate empiric therapy in a particular area. An understanding of the burden of disease in an area-drugresistant typhoid in an urban slum, for example-could lead to public health measures such as a vaccination program. Laboratory surveillance programs may produce the clue to the possibility of new organisms emerging, including both bacteria and viruses, most commonly at the animal-human interface.

At 

For many health care staff working in resource-restricted areas, the major problem is simply a lack of laboratory services. Hospital laboratories may be absent, or, if they are available, only offer a limited repertoire of tests. In other areas, particularly in Asia, a wide range of alternative services is offered by private diagnostic laboratories, typically outside the front gate of the hospital but with uncertain quality. Even when the tests are available, they may not be used or the results ignored. Lack of use may stem from a poor perception of the laboratory, and tests may not be available because the costs are prohibitive.

Even when laboratories are present, they face the many challenges that are familiar to all areas of the health care sector. Inadequate facilities are common, with laboratories that lack space and a secure supply of electricity and water. Appropriate equipment may be unavailable or poorly maintained. Even basic equipment required for a functioning laboratory can be in disrepair because of the absence of regular care and servicing. A functioning microscope is a key piece of equipment for a basic microbiology laboratory but is frequently found in poor condition. In a survey of 90 microscopes in laboratories in nine districts in Malawi, only 50% were in good condition. 4 There were 1.1 functioning microscopes per 100,000 population, and even microscopes in need of full servicing were still in daily use. The 90 microscopes were from 16 different manufacturers, illustrating the lack of standardization of laboratory equipment so frequently seen. The provision of biological safety cabinets is another area where equipment from multiple manufacturers and lack of spare parts and maintenance are common, and in this case may lead to unsafe and hazardous conditions for laboratory workers. Standardization of equipment and consumables with central ordering, maintenance contracts, and supplies of spare parts would seem a sensible response to this issue but is rarely seen. Tests may also be unavailable because of an inadequate supply route for consumables. This is another area where standardization of tests and central ordering and supply can lead not only to more reliable supply of quality-assured consumables but also to potential cost savings for the country.

The laboratory can generate results, but the quality may be poor. Standard operating procedures may be absent and quality control of routine procedures non-existent. The absence of national or regional laboratory guidelines or programs of external quality assurance (QA) by the laboratory network is common. Communications between different levels within the laboratory network may laboratories are further categorized into biosafety levels (BSL) so that the facilities available are matched to the pathogens handled. A standard diagnostic laboratory would be at BSL2, and the basic requirements for such a laboratory are outlined in Table 21 .2 and Box 21.1. More specialized laboratories such as tuberculosis reference laboratories where culture and susceptibility testing are performed require BSL3 facilities. BSL3 laboratories have particular design features to reduce the hazard of airborne transmission and incorporate directional airflows and the use of biological safety cabinets. They are particularly appropriate for laboratories handling pathogens such as tuberculosis and influenza. However, BSL3 facilities are very expensive and difficult to build and maintain. The WHO has recently indicated that in some circumstances, slightly less rigorous guidelines, so-called BSL2+ as outlined in Table 21 .2, may be appropriate for selected laboratories, for example, processing samples for tuberculosis culture. 8

Health care staff working at the district hospital level may be asked to advise on what would constitute an appropriate laboratory service for the hospital and district. The provision of an extensive range of tests is likely to be unaffordable and impractical. In a study evaluating the role of the laboratory in a district hospital in Malawi, the services considered essential were blood transfusion (including blood grouping and compatibility testing and screening for HIV, hepatitis B, and syphilis), hemoglobin estimation, and the microscopic diagnosis of malaria and tuberculosis. 9 This list will vary in different areas, and the services of the laboratory should be orientated to the requirements of the district and the available resources. Other tests that require relatively little investment and can be done where there are limited resources include microscopy of urine and stool samples for ova, cysts, and parasites; Gram stain and cell count in cerebrospinal fluid and other sterile fluids; and Gram stains of pus samples. The microscopic appearance of some typical bacterial pathogens is shown in Fig. 21 .1A-F. Guidelines for standard laboratory methods appropriate for resource-restricted areas are available. 10 A checklist of issues that should be considered when evaluating a diagnostic laboratory is in Box 21.2. provincial or regional level, laboratories will be located in larger referral hospitals. Laboratories at this level should be performing a more sophisticated range of tests with higher throughput. For example, facilities for tuberculosis culture might be available, together with molecular techniques for specific diseases and the ability to investigate disease outbreaks. Support for the Level II laboratories would be an important function, including periodic visits and laboratory assessment as part of a QA program.

National reference laboratories at Level IV are likely to be located in the capital and serve specialized public health functions that may be linked to specific disease control programs such as the central reference laboratory for the National Tuberculosis Programme. It is important that laboratories at the national level have links to regional supranational reference laboratories for advice and quality assurance. Level III and IV laboratories would conduct surveillance and monitoring of infections using laboratory data collected throughout the network, establish standard operating procedures and protocols, conduct training and quality improvement, and plan for equipment needs and maintenance throughout the network.

Biosafety is an essential consideration at all levels of the laboratory network and depends on three principles. 7 Good laboratory practice and technique are fundamental and require established standard operating procedures and appropriate induction and training of staff. Safety equipment provides a primary barrier, and this includes appropriate, properly maintained and used equipment (e.g., centrifuges, biological safety cabinets) and personal protective equipment (e.g., gloves, respirators). Finally, facility design and construction are a secondary barrier providing, for example, appropriate workflows (from clean to dirty areas) and directional airflows and containment if required.

Microorganisms are categorized into four hazard groups according to their risk to individuals and society and the availability of treatment and preventive measures (Table 21. The diagnosis of infection depends on detection of the pathogen or the host response to the pathogen. Direct pathogen detection is traditionally performed by light microscopy, although antigen detection and nucleic acid amplification tests (such as polymerase chain reaction [PCR]) are increasingly used. Pathogen detection may also be carried out by isolation of the microorganism by culture of relevant clinical samples, and this allows susceptibility testing to be performed. Methods based on detecting the immune response mainly rely on detecting pathogen-specific IgM or IgG antibodies. Technological advances in the design of testing methods have simplified antigen and antibody detection to the point that simple point-of-care test kits are now widely available. The rapid kits for HIV antibody detection have an established place in the voluntary counseling and testing framework being established in many countries. Rapid malaria detection tests have been recommended as a replacement for malaria microscopy in some guidelines and need to be positive before antimalarial treatment is given.

In recent years, organizations such as the UNICEF/United Nations Development Programme/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), and the Foundation for Innovative New Diagnostics (FIND) have played an important role in developing and evaluating new diagnostic tests for many tropical diseases. 11 The WHO Sexually Transmitted Diagnostics Initiative has developed an approach to the characteristics of an ideal diagnostic test in the developingcountry context. "ASSURED" tests should be affordable by those Resistance Surveillance System manual (E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, S. aureus, S. pneumoniae, Salmonella spp., Shigella spp., and Neisseria gonorrhoeae), as well as other pathogens of local or national importance.

There have also been considerable advances in the format and ease of use of molecular tests. This is exemplified by the increasing use in tuberculosis laboratories of nucleic acid amplification tests directly from AFB smear-positive sputum, or from culture isolates. Line probe assays (LPAs) use a multiplex PCR amplification followed by reverse hybridization to identify Mycobacterium tuberculosis complex and mutations in the genes associated with at risk of infection, sensitive and specific, user friendly (simple to perform and requiring minimal training), rapid (to enable treatment at the first visit), robust (does not require refrigerated storage), equipment-free, and able to be delivered to those who need it. There has been increased attention on the problem of antimicrobial resistance for many important pathogens and the critical role that the laboratory plays in the management of this. Initiatives have focused on methods and systems of surveillance of antimicrobial resistance in bacterial infections that countries can readily implement. 3, 12 The WHO guideline has recommended a focus on eight priority pathogens as described in the Global Antimicrobial rifampicin and isoniazid resistance. LPA can be performed with results in 1 to 2 days, which is considerably quicker than the weeks required for traditional culture methods, and the overall agreement for the diagnosis of MDR between these tests and conventional methods is 99%. The format of these tests is being simplified so that the feasibility of their routine use in tuberculosis reference laboratories in developing countries is becoming a reality. These methods are an important component of the roll-out of the programmatic management of MDR tuberculosis globally.

Quality assurance is defined as "planned and systematic activities to provide adequate confidence that requirements for quality will be met." The QA system is the basis for a guaranteed result. If this system is not followed, patients may get the wrong results, with important consequences for their health-such as receiving inadequate treatment. A program of QA in diagnostic laboratories involves not only internal quality control and external QA but also attention to appropriate staffing, training and supervision, and maintenance of equipment and facilities. International guidelines are now available and increasingly implemented for QA in many areas of laboratory practice such as AFB smear microscopy and HIV testing.

Accurate clinical diagnosis in resource-restricted settings relies strongly on the laboratory service. The increasing recognition of the need to support the development of a quality-assured laboratory service in such settings is therefore welcome. In many regions, international organizations are actively working with local providers to improve laboratory services. The development of laboratory services will contribute to improved health for the local population and ensure better use of scarce health care resources.

Bacteremia among children admitted to a rural hospital in Kenya

Diagnostic preparedness for infectious disease outbreaks

AMR Surveillance in low and middle-income settings -A roadmap for participation in the Global Antimicrobial Surveillance System (GLASS)

Evaluation of microscope condition in Malawi

Using a bus service for transporting sputum specimens to the Central Reference Laboratory: effect on the routine TB culture service in Malawi

Risk of occupational tuberculosis in National Tuberculosis Programme laboratories in Korea

World Health Organization (WHO)

Guidance on bio-safety related to TB laboratory diagnostic procedures

The operation, quality and costs of a district hospital laboratory service in Malawi

Medical laboratory manual for tropical countries

Diagnostics for the developing world

World Health Organization: Global Antimicrobial Resistance Surveillance System: manual for early implementation