A guide to aid the selection of diagnostic tests.

In recent years, a wide range of diagnostic tests has become available for use in resource-constrained settings. Accordingly, a huge number of guidelines, performance evaluations and implementation reports have been produced. However, this wealth of information is unstructured and of uneven quality, which has made it difficult for end-users, such as clinics, laboratories and health ministries, to determine which test would be best for improving clinical care and patient outcomes in a specific context. This paper outlines a six-step guide to the selection and implementation of in vitro diagnostic tests based on Médecins Sans Frontières' practical experience: (i) define the test's purpose; (ii) review the market; (iii) ascertain regulatory approval; (iv) determine the test's diagnostic accuracy under ideal conditions; (v) determine the test's diagnostic accuracy in clinical practice; and (vi) monitor the test's performance in routine use. Gaps in the information needed to complete these six steps and gaps in regulatory systems are highlighted. Finally, ways of improving the quality of diagnostic tests are suggested, such as establishing a model list of essential diagnostics, establishing a repository of information on the design of diagnostic studies and improving quality control and postmarketing surveillance.

Depuis quelques années, de multiples tests diagnostiques sont disponibles dans les lieux de soins disposant de ressources limitées. En conséquence, un nombre considérable de directives, d'évaluations des performances et de rapports de mise en œuvre ont été élaborés. Cette masse d'informations manque néanmoins de structure et est de qualité inégale, raisons pour lesquelles les utilisateurs finaux, tels que les centres de santé, les laboratoires et les ministères de la Santé, ont eu des difficultés à déterminer quel test conviendrait le mieux pour améliorer les soins cliniques et l'état de santé des patients dans un contexte donné. Ce document présente un guide en six étapes pour faciliter la sélection et la mise en œuvre de tests diagnostiques in vitro, en s'appuyant sur l'expérience pratique de Médecins Sans Frontières: (i) définir l'objectif du test; (ii) analyser le marché; (iii) obtenir l'approbation réglementaire; (iv) déterminer la précision du test diagnostique dans des conditions idéales; (v) déterminer la précision du test diagnostique dans le cadre d'une pratique clinique; et (vi) suivre les performances du test dans le cadre d'une utilisation courante. Ce document met en avant les informations manquantes nécessaires pour accomplir ces six étapes et les lacunes des systèmes de réglementation. Enfin, il propose des moyens d'améliorer la qualité des tests diagnostiques, à travers l'établissement d'une liste modèle des diagnostics essentiels, l'élaboration d'un référentiel d'informations sur la réalisation des études diagnostiques, et l'amélioration du contrôle de la qualité et de la pharmacovigilance.

En los últimos años se ha desarrollado una amplia variedad de pruebas de diagnóstico para el uso en entornos con recursos limitados. Como consecuencia, se ha producido una gran cantidad de manuales, evaluaciones de rendimiento e informes de implementación. No obstante, esta abundancia de información está desestructurada y tiene una calidad irregular. Esto ha dificultado que los usuarios finales, como clínicas, laboratorios y ministerios de salud, puedan determinar qué prueba es la más indicada para mejorar la atención sanitaria y los resultados de los pacientes en un contexto específico. En el presente informe se describe un manual de seis pasos para la selección y la implementación de pruebas de diagnóstico in vitro sobre la base de la experiencia práctica de Mèdecins Sans Frontières: (i) definir el propósito de la prueba; (ii) revisar el mercado; (iii) verificar la aprobación normativa; (iv) determinar la precisión del diagnóstico de la prueba en condiciones ideales; (v) determinar la precisión del diagnóstico de la prueba en la práctica clínica; y (vi) controlar el rendimiento de la prueba en su uso habitual. Se destacan los vacíos en la información necesarios para completar estos seis pasos y los vacíos del sistema normativo. Finalmente, se sugieren maneras para mejorar la calidad de las pruebas de diagnóstico, como establecer una lista modelo de los diagnósticos esenciales, establecer un repositorio de información sobre el diseño de los estudios de diagnósticos y mejorar el control de calidad y el control del postmarketing.

Critical review of the Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA): suggestions for harmonization, implementation and improvement.

OBJECTIVE: Clinical laboratories in low- and middle-income countries (LMIC) need fundamental improvement because quality laboratory services are essential for the decision-making capacity of clinicians, health workers and public health authorities. To this end, a tiered accreditation scheme Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA) was developed by WHO-AFRO, CDC and others for clinical laboratories in LMIC. One to five stars are accredited to laboratories based on the level of compliance with a checklist. Our aim was to evaluate the quality and applicability of this accreditation scheme compared with international quality standards. METHODS: We performed a critical review of this scheme to formulate recommendations for implementation, harmonization and improvement. Two analyses were performed: one assessing its coverage of the ISO 15189:2007 standard and one to identify and evaluate priorities of the accreditation checklist. RESULTS: Although the content of the checklist covers all aspects of total quality management, it strongly prioritizes resource management activities. We recommend identifying critical requirements for each tier of accreditation to assure a certain level of quality for each tier or instead using a pass/fail approach towards accreditation. In addition, the checklist should include more questions for assessing proper management, ethics and continuous improvement to meet ISO 15189. CONCLUSION: Launching accreditation schemes for laboratories in LMIC should be encouraged. After further optimization of SLIPTA, clinical laboratories may certainly benefit, leading to more correctly diagnosed patients and less waste of resources.

The realistic performance achievable with mycobacterial automated culture systems in high and low prevalence settings.

BACKGROUND: Diagnostic tests are generally used in situations with similar pre-test probability of disease to where they were developed. When these tests are applied in situations with very different pre-test probabilities of disease, it is informative to model the likely implications of known characteristics of test performance in the new situation. This is the case for automated Mycobacterium tuberculosis (MTB) liquid culture systems for tuberculosis case detection which were developed and are widely used in low burden settings but are only beginning to be applied on a large scale in high burden settings. METHODS: Here we model the performance of MTB liquid culture systems in high and low tuberculosis (TB) prevalence settings using detailed published data concentrating on the likely frequency of cross-contamination events. RESULTS: Our model predicts that as the TB prevalence in the suspect population increases there is an exponential increase in the risk of MTB cross-contamination events expected in otherwise negative samples, even with equivalent technical performance of the laboratories. Quality control and strict cross-contamination measures become increasingly critical as the burden of MTB infection among TB suspects increases. Even under optimal conditions the realistically achievable specificity of these systems in high burden settings will likely be significantly below that obtained in low TB burden laboratories. CONCLUSIONS: Liquid culture systems can play a valuable role in TB case detection in laboratories in high burden settings, but laboratory workers, policy makers and clinicians should be aware of the increased risks, independent of laboratory proficiency, of cross-contamination events in high burden settings.

Review of the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) version 2:2015.

BACKGROUND: In 2011 the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) was launched, aimed at strengthening the quality and competence of African clinical, public health and reference laboratories. We reviewed the first version of the SLIPTA checklist in 2011. The continued development and publication of a new version of the International Organization for Standardization (ISO) 15189 standard demands a renewed review. OBJECTIVE: This study aimed to determine the suitability of SLIPTA in guiding laboratories towards ISO 15189:2012 compliance and accreditation and provide recommendations for further SLIPTA improvement. METHODS: The study was conducted between September 2018 and April 2019. Coverage of ISO 15189:2012 by SLIPTA checklist version 2:2015 was determined and the point distribution of the scoring system over the different sections of the SLIPTA checklist was re-investigated. These findings were compared with the review of the first version of the SLIPTA checklist (based on ISO 15189:2007) and with findings published on SLIPTA implementation and roll-out. RESULTS: The coverage of ISO 15189 by the SLIPTA checklist has increased, even though ISO 15189:012 is more extensive than ISO 15189:2007. The point distribution is still skewed towards sections related to quality planning rather than quality control and improvement. Although to date 314 laboratories have been assessed, barriers for laboratories to participate in SLIPTA are high. Sustainability of SLIPTA results is insufficiently studied. CONCLUSION: SLIPTA checklist version 2:2015 has improved compared to earlier versions. We recommend increasing accessibility for laboratories to participate and increasing guidance for ISO-based quality management system implementation.

Resistant mutants of Mycobacterium tuberculosis selected in vitro do not reflect the in vivo mechanism of isoniazid resistance.

OBJECTIVES: The high prevalence of isoniazid-resistant Mycobacterium tuberculosis is often explained by a high mutation rate for this trait, although detailed information to support this theory is absent. We studied the development of isoniazid resistance in vitro, making use of a laboratory strain of M. tuberculosis. METHODS: Spontaneous isoniazid-resistant mutants were characterized by molecular methods allowing identification of the most commonly encountered resistance-conferring mutations. Additionally, we determined the in vitro mutation rates for isoniazid and rifampicin resistance, and characterized the genome of a triple-resistant strain. RESULTS: Results confirm that the in vitro mutation rate for isoniazid resistance (3.2 x 10(-7) mutations/cell division) is much higher than the rate for rifampicin resistance (9.8 x 10(-9) mutations/cell division). However, in the majority of the in vitro mutants katG was partially or completely deleted and neither of the two most common in vivo mutations, katG-S315T or inhA-C(-)15T, were found in 120 isogenic mutants. This implies that clinically prevalent resistance mutations were present in <0.8% of isoniazid-resistant strains selected in vitro (95% CI 0%-2.5%). The triple-resistant strain had acquired isoniazid resistance via a 49 kbp deletion, which included katG. Apart from previously identified resistance-conferring mutations, three additional point mutations were acquired during sequential selection steps. CONCLUSIONS: These outcomes demonstrate that the in vivo mechanism of isoniazid resistance is not reflected by in vitro experiments. We therefore conclude that the high in vitro mutation rate for isoniazid resistance is not a satisfactory explanation for the fact that isoniazid monoresistance is significantly more widespread than monoresistance to rifampicin.

Specific mutations in the Mycobacterium tuberculosis rpoB gene are associated with increased dnaE2 expression.

In Mycobacterium tuberculosis (MTB), rifampicin resistance is almost invariably due to mutations in the rpoB gene, whose function is critical for cell viability. Most of these mutations, at least initially, impair the fitness of the bacteria but confer a selective advantage when antibiotic pressure is exerted. Subsequent adaptation may be critical to restore fitness. The possibility was considered that MTB with mutations in the rpoB gene elicits a constitutive stress response, increasing the probability of subsequent adaptation. In order to test this hypothesis, the expression of recA and dnaE2, an inducible putative error-prone DNA polymerase, was determined in six different isogenic laboratory-generated rpoB-mutants of MTB. Expression levels were determined with real-time PCR and the data obtained were compared with those of the wild-type parent. In four of the six rpoB mutants, a two- to fivefold induction of dnaE2 was detected (P<0.05). Thus, the presence of specific mutations in rpoB is not only associated with impaired fitness but also results in a detectable, moderate yet persistent increase in the expression of dnaE2 but not recA.

The ML flow test as a point of care test for leprosy control programmes: potential effects on classification of leprosy patients.

OBJECTIVE: To evaluate the use of the ML Flow test as an additional, serological, tool for the classification of new leprosy patients. DESIGN: In Brazil, Nepal and Nigeria, 2632 leprosy patients were classified by three METHODS: : (1) as multibacillary (MB) or paucibacillary (PB) according to the number of skin lesions (WHO classification), (2) by slit skin smear examination, and (3) by serology using the ML Flow test detecting IgM antibodies to Mycobacterium leprae-specific phenolic glycolipid-I. RESULTS: The proportion of MB leprosy patients was 39.5, 35.6 and 19.4% in Brazil, Nepal and Nigeria, respectively. The highest seropositivity in patients was observed in Nigeria (62.9%), followed by Brazil (50.8%) and Nepal (35.6%). ML Flow test results and smears were negative in 69.1 and 82.7% of PB patients, while smears were positive in 58.6% of MB patients in Brazil and 28.3% in Nepal. In MB patients, both smears and ML Flow tests were negative in 15.6% in Brazil and 38.3%, in Nepal. Testing all PB patients with the ML Flow test to prevent under-treatment would increase the MB group by 18, 11 and 46.2% for Brazil, Nepal and Nigeria, respectively. Using the ML Flow test as the sole criterion for classification would result in an increase of 11.3 and 43.5% of patients requiring treatment for MB leprosy in Brazil and Nigeria, respectively, and a decrease of 3.7% for Nepal. CONCLUSIONS: The ML Flow test could be used to strengthen classification, reduce the risk of under-treatment and minimize the need for slit skin smears.

Risk factors for developing leprosy--a population-based cohort study in Indonesia.

We identified risk factors associated with increased yearly incidence rates of leprosy in five island populations. Age, sex, household size and Mycobacterium leprae-specific antibodies as well as contact factors were studied. Of 94 index patients (patients diagnosed in 2000), 43 (46%) were classified as multibacillary (MB), 17 (19%) were seropositive for PGL-1 [corrected] antibodies and 6 (7%) had M. leprae DNA in nasal swabs as determined by polymerase chain reaction (PCR) testing. All PCR positive patients were also seropositive. Forty-four of 4903 initially symptom free persons developed leprosy within 4 years, giving an incidence rate of 298 per 1000 person-years. Men had a 22 times higher risk [95% confidence interval (CI): 1.2-4.1] of developing leprosy than women. People living in households with more than 7 members had a 3.1 times higher risk (95% CI: 1.3-7.3) than households of 1-4 members. Persons who were seropositive in 2000 had a 3.8 times higher risk (95% CI: 1.1-12.6) than seronegative persons. Household contacts of MB patients had an adjusted hazard ratio (aHR) of 4.6 (95% CI: 1.6-12.9) and household contacts of PCR positive patients an aHR of 9.36 (95% CI: 2.5-34.9) compared with non-contacts. Patients with PCR positive nasal swabs, suggesting nasal excretion of M. leprae, are probably the patients with the highest transmission potential. Since all index patients who were PCR positive were also seropositive, serology seems an adequate tool to identify these patients. Preventing seropositive persons from becoming seropositive and infectious patients might break the chain of transmission.

Genetic, household and spatial clustering of leprosy on an island in Indonesia: a population-based study.

BACKGROUND: It is generally accepted that genetic factors play a role in susceptibility to both leprosy per se and leprosy type, but only few studies have tempted to quantify this. Estimating the contribution of genetic factors to clustering of leprosy within families is difficult since these persons often share the same environment. The first aim of this study was to test which correlation structure (genetic, household or spatial) gives the best explanation for the distribution of leprosy patients and seropositive persons and second to quantify the role of genetic factors in the occurrence of leprosy and seropositivity. METHODS: The three correlation structures were proposed for population data (n = 560), collected on a geographically isolated island highly endemic for leprosy, to explain the distribution of leprosy per se, leprosy type and persons harbouring Mycobacterium leprae-specific antibodies. Heritability estimates and risk ratios for siblings were calculated to quantify the genetic effect. Leprosy was clinically diagnosed and specific anti-M. leprae antibodies were measured using ELISA. RESULTS: For leprosy per se in the total population the genetic correlation structure fitted best. In the population with relative stable household status (persons under 21 years and above 39 years) all structures were significant. For multibacillary leprosy (MB) genetic factors seemed more important than for paucibacillary leprosy. Seropositivity could be explained best by the spatial model, but the genetic model was also significant. Heritability was 57% for leprosy per se and 31% for seropositivity. CONCLUSION: Genetic factors seem to play an important role in the clustering of patients with a more advanced form of leprosy, and they could explain more than half of the total phenotypic variance.

Prevention of leprosy using rifampicin as chemoprophylaxis.

An intervention study was implemented on five Indonesian islands highly endemic for leprosy to determine whether rifampicin can be used as chemoprophylaxis to prevent leprosy. The population was actively screened before the intervention and subsequently once a year for three years. In the control group, no chemoprophylaxis was given. In the contact group, chemoprophylaxis was only given to contacts of leprosy patients and in the blanket group to all eligible persons. The cohort consisted of 3,965 persons. The yearly incidence rate in the control group was 39/10,000; the cumulative incidence after three years was significantly lower in the blanket group (P = 0.031). No difference was found between the contact and the control groups (P = 0.93). Whether this apparent reduced leprosy incidence in the first three years in the blanket group is due to a delayed development of leprosy or a complete clearance of infection needs to be determined.

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency.

BACKGROUND: Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations. METHODS: Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 microg/ml but remained sensitive to rifabutin (MIC<0.8 microg/ml). This mutant was subjected to a second round of selection with rifabutin. RESULTS: All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain. CONCLUSION: First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.

Population survey to determine risk factors for Mycobacterium leprae transmission and infection.

BACKGROUND: Not every leprosy patient is equally effective in transmitting Mycobacterium leprae. We studied the spatial distribution of infection (using seropositivity as a marker) in the population to identify which disease characteristics of leprosy patients are important in transmission. METHODS: Clinical data and blood samples for anti-M. leprae ELISA were collected during a cross-sectional survey on five Indonesian islands highly endemic for leprosy. A geographic information system (GIS) was used to define contacts of patients. We investigated spatial clustering of patients and seropositive people and used logistic regression to determine risk factors for seropositivity. RESULTS: Of the 3986 people examined for leprosy, 3271 gave blood. Seroprevalence varied between islands (1.7-8.7%) and correlated significantly with leprosy prevalence. Five clusters of patients and two clusters of seropositives were detected. In multivariate analysis, seropositivity significantly differed by leprosy status, age, sex, and island. Serological status of patients appeared to be the best discriminator of contact groups with higher seroprevalence: contacts of seropositive patients had an adjusted odds ratio (aOR) of 1.75 (95% CI 0.922-3.31). This increased seroprevalence was strongest for contact groups living < or =75 m of two seropositive patients (aOR = 3.07; 95% CI 1.74-5.42). CONCLUSIONS: In this highly endemic area for leprosy, not only household contacts of seropositive patients, but also people living in the vicinity of a seropositive patient were more likely to harbour antibodies against M. leprae. Through measuring the serological status of patients and using a broader definition of contacts, higher risk groups can be more specifically identified.

A comprehensive comparison of Ziehl-Neelsen and fluorescence microscopy for the diagnosis of tuberculosis in a resource-poor urban setting.

SETTING: Nairobi City Council Chest Clinic, Kenya. OBJECTIVES: To establish the efficiency, costs and cost-effectiveness of six diagnostic strategies using Ziehl-Neelsen (ZN) and fluorescence microscopy (FM). DESIGN: A cross-sectional study of 1398 TB suspects attending a specialised chest clinic in Nairobi subjected to three sputum examinations by ZN and FM. Lowenstein-Jensen culture was used as the gold standard. Cost analysis included health service and patient costs. RESULTS: Of 1398 suspects enrolled, 993 (71%) had a complete diagnostic work-up involving three sputum specimens for ZN and FM, culture and chest X-ray (CXR). Irrespective of whether ZN or FM was used on one, two or three smears, the overall diagnostic process detected 92% culture-positive cases. Different strategies affected the ratio of smear-positive to smear-negative TB; however, FM was more sensitive than ZN (P < 0.001). FM performance was not affected by the patient's HIV status. The cost per correctly diagnosed smear-positive case, including savings, was 40.30 US dollars for FM on two specimens compared to 57.70 US dollars for ZN on three specimens. CONCLUSION: The FM method used on one or two specimens is more cost-effective and shortens the diagnostic process. Consequently, more patients can be put on a regimen for smear-positive TB, contributing to improved treatment and reducing transmission.

Early specific host response associated with starting effective tuberculosis treatment in an infection controlled placebo controlled mouse study.

Recently we proposed exploring the potential of treatment stimulated testing as diagnostic method for tuberculosis (TB). An infection controlled placebo controlled mouse study was performed to investigate whether serum cytokine levels changed measurably during the early phase of TB chemotherapy. Serum was collected prior to and during the first 3 weeks of isoniazid (INH) and rifampicin (RIF) chemotherapy, and levels of 23 selected cytokines/chemokines were measured using a liquid bead array. The serum levels of IFNγ, IP-10, MIG, MCP-1, IL-17 and IL-6 were elevated in the TB infected mice compared to non-infected mice at least at 1 time point measured. In infected mice, IFNγ, IP-10, MIG and MCP-1 levels decreased within 7 days of treatment with RIF+INH compared to placebo. Treatment of non-infected mice in the absence of tuberculosis infection had no effect on these cytokines. IL-17 and IL-6 had decreased to baseline in all infected mice prior to the initiation of treatment. This study demonstrates that systemic levels of some cytokines, more specifically IFNγ, IP-10, MIG and MCP-1, rapidly and specifically change upon starting TB chemotherapy only in the presence of infection in a mouse model. Thus, IFNγ, IP-10, MIG and MCP-1 are promising 'Treat-to-Test' targets for the diagnosis of TB and deserve further investigation in a study on human TB suspects.

Prospective evaluation of three rapid diagnostic tests for diagnosis of human leptospirosis.

BACKGROUND: Diagnosis of leptospirosis by the microscopic agglutination test (MAT) or by culture is confined to specialized laboratories. Although ELISA techniques are more common, they still require laboratory facilities. Rapid Diagnostic Tests (RDTs) can be used for easy point-of-care diagnosis. This study aims to evaluate the diagnostic performance of the RDTs LeptoTek Dri Dot, LeptoTek Lateral Flow, and Leptocheck-WB, prospectively. METHODOLOGY: During 2001 to 2012, one or two of the RDTs at the same time have been applied prior to routine diagnostics (MAT, ELISA and culture) on serum specimens from participants sent in for leptospirosis diagnosis. The case definition was based on MAT, ELISA and culture results. Participants not fulfilling the case definition were considered not to have leptospirosis. The diagnostic accuracy was determined based on the 1(st) submitted sample and paired samples, either in an overall analysis or stratified according to days post onset of illness. RESULTS: The overall sensitivity and specificity for the LeptoTek Dri Dot was 75% respectively 96%, for the LeptoTek Lateral Flow 78% respectively 95%, and for the Leptocheck-WB 78% respectively 98%. Based on the 1(st) submitted sample the sensitivity was low (51% for LeptoTek Dri Dot, 69% for LeptoTek Lateral Flow, and 55% for Leptocheck-WB), but substantially increased when the results of paired samples were combined, although accompanied by a lower specificity (82% respectively 91% for LeptoTek Dri Dot, 86% respectively 84% for LeptoTek Lateral Flow, and 80% respectively 93% for Leptocheck-WB). CONCLUSIONS: All three tests present antibody tests contributing to the diagnosis of leptospirosis, thus supporting clinical suspicion and contributing to awareness. Since the overall sensitivity of the tested RDTs did not exceed 80%, one should be cautious to rely only on an RDT result, and confirmation by reference tests is strongly recommended.

Review and comparison of quality standards, guidelines and regulations for laboratories.

BACKGROUND: The variety and number of laboratory quality standards, guidelines and regulations (hereafter: quality documents) makes it difficult to choose the most suitable one for establishing and maintaining a laboratory quality management system. OBJECTIVES: There is a need to compare the characteristics, suitability and applicability of quality documents in view of the increasing efforts to introduce quality management in laboratories, especially in clinical diagnostic laboratories in low income and middle income countries. This may provide valuable insights for policy makers developing national laboratory policies, and for laboratory managers and quality officers in choosing the most appropriate quality document for upgrading their laboratories. METHOD: We reviewed the history of quality document development and then selected a subset based on their current use. We analysed these documents following a framework for comparison of quality documents that was adapted from the Clinical Laboratory Standards Institute guideline GP26 Quality management system model for clinical laboratory services. RESULTS: Differences were identified between national and international, and non-clinical and clinical quality documents. The most salient findings were the absence of provisions on occurrence management and customer service in almost all non-clinical quality documents, a low number of safety requirements aimed at protecting laboratory personnel in international quality documents and no requirements regarding ethical behaviour in almost all quality documents. CONCLUSION: Each laboratory needs to investigate whether national regulatory standards are present. These are preferred as they most closely suit the needs of laboratories in the country. A laboratory should always use both a standard and a guideline: a standard sums up the requirements to a quality management system, a guideline describes how quality management can be integrated in the laboratory processes.

Survey of the diagnostic retooling process in national TB reference laboratories, with special focus on rapid speciation tests endorsed by WHO in 2007.

BACKGROUND: Successful integration of new diagnostics in national tuberculosis (TB) control programs, also called 'retooling', is highly dependent on operational aspects related to test availability, accessibility and affordability. This survey aimed to find out whether recommendations to use new diagnostics lead to successful retooling in high TB endemic countries, using immunochromatographic tests (ICTs) for TB culture speciation as a case study. ICTs are recommended to accurately confirm the presence of bacteria of the Mycobacterium tuberculosis complex in liquid culture isolates. METHODS AND FINDINGS: Questionnaires were sent to national TB reference laboratories (NRLs) in 42 high TB endemic countries to address their access to information on ICT implementation, logistics related to availability, accessibility and affordability of ICTs, and testing algorithms. Results from 16 responding countries indicated that half of the NRLs were aware of the contents of WHO guidance documents on liquid culture and ICT implementation, as well as their eligibility for a negotiated pricing agreement for ICT procurement. No major issues with availability and accessibility of ICTs were raised. When asked about testing algorithms, ICTs were not used as stand-alone or first test for TB culture identification as recommended by WHO. CONCLUSIONS: The low response rate was a limitation of this survey and together with NRLs managers' unawareness of global guidance, suggests a lack of effective communication between partners of the global laboratory network and NRLs. TB tests could become more affordable to high TB endemic countries, if the possibility to negotiate lower prices for commercial products is communicated to them more successfully. NRLs need additional guidance to identify where available technologies can be most usefully implemented and in what order, taking into account long-term laboratory strategies.

Bead array direct rRNA capture assay (rCapA) for amplification free speciation of Mycobacterium cultures.

Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay.

Retooling national TB control programmes (NTPs) with new diagnostics: the NTP perspective.

BACKGROUND: A delay is evident between the development of new policies on TB diagnostics and their implementation at country level. The Stop TB Partnership would benefit from information from national TB program (NTP) managers on progress towards implementation of new recommendations as well as the opportunities and challenges encountered in the process. METHODS AND FINDINGS: To solicit information on the introduction of new TB diagnostics at country level, questionnaires were sent out to NTP managers of high-burden TB countries and a subset of managers was interviewed. The results indicate that about 50% of high-burden TB countries are using the TB diagnostic tools newly recommended by the World Health Organization (WHO). Most NTP managers reported that new diagnostics would only be implemented when officially endorsed by the WHO. All countries have plans to adopt newly endorsed diagnostics at reference laboratory level, while approaches to optimize smear microscopy at lower levels of the health service are given less attention. NTP managers reported diverse challenges to the implementation of new diagnostics. CONCLUSIONS: More information on the obstacles and advantages of introducing new diagnostic tools should be provided to NTP managers to ensure the rational adoption of new diagnostics. A single recommendation covering the introduction of a package of diagnostic tools might be preferable to NTP managers and facilitate implementation in high-burden TB countries.