Evaluation of the WHO laboratory quality stepwise implementation tool.

OBJECTIVES: To introduce the Laboratory Quality Stepwise Implementation (LQSI) tool and provide data about its roll-out, usage and effectiveness in assisting laboratories with quality improvement. METHODS: The LQSI tool, a freely available stepwise guide, was developed by WHO to assist laboratories with efficiently implementing a quality management system. RESULTS: Since the tool's launch in 2014, it has been accessed by 130 986 unique users from 195 of 206 listed states. Of 35 respondents to a survey, 12 (34%) indicated that their laboratory had been able to achieve accreditation/certification/licensing as a result of using the tool. CONCLUSIONS: The LQSI tool, currently being used worldwide and available in English, French, Russian, Spanish, Arabic and Turkish, positively impacts the quality of services provided by clinical and public health laboratories, leading to improved clinical care and disease surveillance capacity as required by the IHR (2005) and envisioned by the Global Health Security Agenda.

Buoyant density of Mycobacterium tuberculosis: implications for sputum processing.

SETTING: A tuberculosis (TB) research laboratory in the Netherlands. OBJECTIVE: The concentration of Mycobacterium tuberculosis cells from sputum is almost universally performed by centrifugation after chemical liquefaction. These methods are thus dependent on the effective sedimentation of mycobacterial cells, and the buoyant density of these cells relative to sputum is therefore of critical importance. DESIGN: We cultured M. tuberculosis in different systems and measured their buoyant density. We also calculated the centrifuge times and speeds needed to effectively pellet the mycobacteria. RESULTS: In contrast to earlier reports, we were unable to identify cells with a buoyant density <1 g/cm(3). The measured buoyant density of the cells ranged from 1.13 to 1.02 g/cm(3), and we suspect that the less dense cells are more likely to reflect clinically derived mycobacterial cells. CONCLUSION: Based on our results, this means that for effective sedimentation in a typical universal centrifuge, centrifugation for 22 min at 3200 x g would be required. A limitation of this study is that cultured M. tuberculosis was studied. The data from this study should be confirmed in clinical samples. However, based on our results, centrifugation at lower speed for less time is unlikely to result in effective recovery.

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis.

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.

Light emitting diodes for auramine O fluorescence microscopic screening of Mycobacterium tuberculosis.

We describe the simple adaptation of a standard fluorescent microscope for illumination using a 'Royal Blue' Luxeon light emitting diode (LED) and demonstrate that this form of illumination is suitable for the detection of auramine O stained Mycobacterium spp. The low cost, low power consumption, safety and reliability of LEDs makes them attractive alternatives to mercury vapour lamps.

Sex-specific performance of routine TB diagnostic tests.

SETTING: City Council Chest Clinic, Nairobi, Kenya. OBJECTIVE: To determine to what extent the performance of smear microscopy is responsible for sex differences in notification rates. METHODOLOGY: Three sputum samples from TB suspects were subjected to smear microscopy with Ziehl-Neelsen (ZN) and auramine (FM) staining. Lowenstein-Jensen culture was used as the gold standard. RESULTS: Of 998 suspects, 600 (60%) were men and 398 (40%) women. The odds of detecting culture-positive patients with ZN was lower for women (OR 0.67). By examining the first spot specimen, ZN detected 35% of culture-positive males and 26% of culture-positive females. These proportions increased to respectively 63% and 53% when examining three specimens, and to 79% and 74% when using FM. The sex difference reduced and became non-significant (P = 0.19) when adjusted for HIV; however, the numbers involved for HIV stratification were low. CONCLUSION: The performance of a diagnostic tool contributes to sex differences in notification rates and influences male/female ratios. Women were less likely to be diagnosed (P = 0.08), and when ZN was used they were less likely to be labelled as smear-positive TB (P < 0.01). The application of more sensitive diagnostic tools such as FM is to the advantage of women.

Direct detection of Staphylococcus aureus mRNA using a flow through microarray.

The direct detection of mRNAs from bacterial cultures on a DNA array without amplification and labelling would greatly extend the range of applications suitable for microarray analysis. Here we describe the direct detection of 23S rRNA and seven mRNA species from total Staphylococcus aureus RNA prepared using commercially available RNA purification columns followed by fluorescent detection on a flow through microarray. RNA hybridisation was detected using paired secondary labelled probes directly 5' and 3' to immobilised 60 mers. In this way, we were able to detect the effect of 30-min exposure to antimicrobials on mRNA levels within 3 h after column purification of total RNA without the need for enzymatic manipulation. Specifically the expression of mecA was confirmed in a highly resistant strain and induction of katA and ile-tRNA synthetase genes after exposure to mupirocin could be detected.

The role and performance of chest X-ray for the diagnosis of tuberculosis: a cost-effectiveness analysis in Nairobi, Kenya.

BACKGROUND: The objective of this study was to establish 1) the performance of chest X-ray (CXR) in all suspects of tuberculosis (TB), as well as smear-negative TB suspects and 2) to compare the cost-effectiveness of the routine diagnostic pathway using Ziehl-Neelsen (ZN) sputum microscopy followed by CXR if case of negative sputum result (ZN followed by CXR) with an alternative pathway using CXR as a screening tool (CXR followed by ZN). METHODS: From TB suspects attending a chest clinic in Nairobi, Kenya, three sputum specimens were examined for ZN and culture (Lowenstein Jensen). Culture was used as gold standard. From each suspect a CXR was made using a four point scoring system: i: no pathology, ii: pathology not consistent for TB, iii: pathology consistent for TB and iv: pathology highly consistent for TB. The combined score i + ii was labeled as "no TB" and the combined score iii + iv was labeled as "TB". Films were re-read by a reference radiologist. HIV test was performed on those who consented. Laboratory and CXR costs were used to compare for cost-effectiveness. RESULTS: Of the 1,389 suspects enrolled, for 998 (72%) data on smear, culture and CXR was complete. 714 films were re-read, showing a 89% agreement (kappa value = 0.75 s.e.0.037) for the combined scores "TB" or "no-TB". The sensitivity/specificity of the CXR score "TB" among smear-negative suspects was 80%/67%. Using chest CXR as a screening tool in all suspects, sensitivity/specificity of the score "any pathology" was 92%, respectively 63%. The cost per correctly diagnosed case was for the routine process 8.72 dollars, compared to 9.27 dollars using CXR as screening tool. When costs of treatment were included, CXR followed by ZN became more cost-effective. CONCLUSION: The diagnostic pathway ZN followed by CXR was more cost-effective as compared to CXR followed by ZN. When cost of treatment was also considered CXR followed by ZN became more cost-effective. The low specificity of chest X-ray remains a subject of concern. Depending whether CXR was performed on all suspects or on smear-negative suspects only, 22%-45% of patients labeled as "TB" had a negative culture. The introduction of a well-defined scoring system, clinical conferences and a system of CXR quality control can contribute to improved diagnostic performance.

Simple dipstick assay for semi-quantitative detection of neopterin in sera.

Neopterin, a low-molecular-mass pteridine produced by macrophages, is closely associated with activation of the cellular immune system. Neopterin biosynthesis during inflammatory disease is primarily derived from interferon-activated monocytes/macrophages and neopterin concentrations may be significantly increased in a particular disease state compared to controls. A follow-up of serum neopterin concentrations during the course of an infectious disease could be useful for measuring the activity of the disease and the influence of treatment. We have developed a simple dipstick assay for the semi-quantitative detection of the neopterin concentration in the serum of patients during the course of an infectious disease. Assay performance was comparable to an ELISA, but there is no requirement for specialised equipment.

Effect of secondary structure on single nucleotide polymorphism detection with a porous microarray matrix; implications for probe selection.

Oligonucleotide arrays capable of detecting single nucleotide polymorphisms (SNPs) from amplified nucleic acid have many applications. The expected SNP is usually placed approximately in the center of the probe to ensure the maximum shift in Tm between complementary and SNP sequences. Unfortunately, different short probes (< 30 bases) selected using widely accepted criteria do not perform consistently in this type of assay. Here we present a systematic study on the effect of secondary structure on the ability of oligonucleotide probes to detect an SNP, using real-time array monitoring of a porous microarray substrate that incorporates a novel intra-array mixing system. These results demonstrate that, although positioning of an SNP in the middle of the probe is highly destabilizing, the effect of stable secondary structure on the signal obtained is so dramatic that such probes may be very insensitive. Therefore, if the SNP flanking sequence contains significant secondary structure, then more sensitive probes with good specificity may be obtained by positioning the mutation towards one end of the probe.

A simple dipstick assay for the detection of antibodies to phenolic glycolipid-I of Mycobacterium leprae.

Among the many reported applications of the detection of antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae, in particular, the use of seroprevalence as an indicator of the magnitude of the leprosy problem may turn out to be very useful in leprosy control programs. An operational function of serology within the leprosy control services requires a simple test system. We have developed a simple dipstick assay for the detection of antibodies to PGL-I and compared its performance with that of an ELISA. A high degree of agreement (97.2%) was observed between the ELISA and the dipstick assay when tested on 435 sera; the agreement beyond chance (Kappa value) was 0.92. No significant difference was found between the dipstick assay and the ELISA when seropositivity rates obtained in groups of leprosy patients, household contacts, and controls were compared. The interpretation of the dipstick results as positive or negative was unequivocal, as illustrated by the high agreement between different persons reading the test (Kappa values > 0.88). Storage of the only reagents required, the dipsticks and the stabilized detection reagent, up to three weeks under tropical conditions of high temperatures, high humidity, and exposure to light, did not influence the results of the assay. The dipstick assay described here is an easy-to-perform method for the detection of IgM antibodies to PGL-I of M. leprae; it does not require any special equipment and the highly stable reagents make the test robust and suitable for use in tropical countries. An internal control validates the performance of the assay. This dipstick assay may be the method of choice for epidemiologic mapping of leprosy.

Antibodies to sulfatide in leprosy and leprosy reactions.

Antibodies to sulfatide have been reported in various demyelinating peripheral polyneuropathies. We have investigated the diagnostic value of these antibodies in leprosy. Anti-sulfatide IgM in leprosy patients was not significantly elevated. High anti-sulfatide IgG titers were observed in individuals from endemic areas, irrespective of their leprosy status, while western European controls were negative. No significant correlation was found between IgM or IgG antibody titers and leprosy classification, although multibacillary patients had higher anti-sulfatide IgM titers than paucibacillary patients. In addition, 23 patients developing leprosy reactions were followed longitudinally. Antibody titers in these patients fluctuated slightly during the follow-up period. There was no association with the occurrence of leprosy reactions or treatment. Thus, IgG titers against sulfatides are high in both leprosy patients and healthy controls in endemic areas, whereas such antibodies are not found in western European controls, suggesting that these antibodies are induced by environmental factors, such as microorganisms.

The epidemiology of Mycobacterium leprae: recent insight.

Leprosy is still a health problem in many countries. Because the causative organism, Mycobacterium leprae cannot be cultured in vitro, it is virtually impossible to assess exposure, and the onset of infection and disease. As a consequence, the chain of infection, considered as the relationships between M. leprae, transmission and human host, is poorly understood. Here, we discuss a number of organism-, host- and environmental-related factors which may be incriminated in the dynamic process of the development of leprosy disease. The use of modern molecular and immunological tools has become a valuable addition to epidemiological research. Understanding of the epidemiology of leprosy is a prerequisite for effective control of the disease.

Cost-effectiveness of the polymerase chain reaction versus smear examination for the diagnosis of tuberculosis in Kenya: a theoretical model.

SETTING: A major out-patient tuberculosis clinic in Nairobi, Kenya. OBJECTIVE: To ascertain the cost-effectiveness of the polymerase chain reaction (PCR) for the diagnosis of tuberculosis in an urban setting in a developing country. DESIGN: A cost-effectiveness analysis of PCR and direct smear microscopy examination based on theoretical modelling. The cost-effectiveness was expressed in costs per correctly diagnosed tuberculosis patient for each of the two diagnostic techniques. Data were obtained from the literature, from the staff and the register at the health facility and from structured interviews with patients. Assumptions were made when no data were available. RESULTS: The PCR is expected to be more specific and sensitive than the routine procedure for diagnosis, but it is also more costly. The routine procedure based on direct smear microscopy turned out to be 1.8 times as cost-effective as PCR. CONCLUSION: It is concluded that the PCR method can potentially be a cost-effective screening procedure for tuberculosis, provided that the largest contributing cost component, the costs of the PCR-kit, can be reduced substantially.

A comprehensive study of the efficiency of the routine pulmonary tuberculosis diagnostic process in Nairobi.

Tuberculosis (TB) suspects from Rhodes Chest Clinic, Nairobi, Kenya, were subjected to three sputum smear microscopy (Ziehl-Neelsen) examinations and a chest X-ray (CXR). Results were compared with Löwenstein-Jensen culture as the gold standard to establish the efficiency of the routine diagnostic process. All laboratory tests and the CXR were available for 993 (71%) of the 1,398 enrolled suspects. Of these, 554 (56%) were culture-positive. The routine diagnostic process was very sensitive, able to detect 92% of culture-positive cases but missing 8%. The specificity was low (66%), and 23% of the patients started on treatment were culture-negative, mainly due to the low specificity of the CXR. It may be possible to increase the efficiency of the diagnostic process by specifying better criteria for CXR examination, improving the quality of CXR reading and counselling patients to return when complaints persist.

The use of whole blood in a dipstick assay for detection of antibodies to Mycobacterium leprae: a field evaluation.

We describe a further simplification of a dipstick assay for the detection of antibodies to phenolic glycolipid I of Mycobacterium leprae by using whole blood and evaluated the assay performance in the leprosy endemic area of Amazonas in Brazil. The agreement with the 'gold' standard ELISA was 94.9% (kappa value = 0.87). This simple assay may be useful to identify those at risk of developing leprosy, for example among contacts of leprosy patients at lower levels in the health services.

Serum neopterin as a marker for reactional states in leprosy.

Reactions, a relatively common phenomenon among leprosy patients in treatment, require early detection and proper management to prevent serious sequelae. It is generally accepted that reactional states are immunologically mediated and, as such, usually improve with immunomodulatory treatments such as corticosteroids or thalidomide. Neopterin, a product of gamma-interferon-activated macrophages, is a marker for cell-mediated immune activation and may be useful to detect reactional states in leprosy. Here, we compared neopterin levels in single serum samples from leprosy patients with and without reaction with untreated controls and, when available, serial samples among patients with and without reaction. Levels in the single sample measurements, conducted in 22 patients with a reversal reaction (mean 14.5 nmol l(-1), S.D. 8.7) and 13 with erythema nodosum leprosum (mean 16.9 nmol l(-1), S.D. 13.6), were significantly higher (P=0.02 and P=0.001, respectively) than levels in 26 untreated patients (mean 9.1 nmol l(-1), S.D. 7.3). Values above the upper limit of normal (10 nmol l(-1)) were found in seven of 26 untreated patients, 14 of the 22 reversal reaction patients (P=0.01) and 10 of the 13 ENL patients (P=0.003). Serial serum samples, obtained from six patients that developed reactions and 14 that remained free of reaction, indicated that reversal reaction or erythema nodosum leprosum paralleled a concomitant increase in the serum neopterin level. Neopterin levels generally declined upon corticosteroid therapy. Neopterin may be a useful marker for reactional states in leprosy by providing a laboratory parameter to assess the onset, progression, response to therapy and resolution.

Distribution and persistence of Mycobacterium leprae nasal carriage among a population in which leprosy is endemic in Indonesia.

In order to understand better the relationship among Mycobacterium leprae, its transmission and the human host or the chain of infection which may lead to the development of leprosy, we performed a population survey in which nasal carriage of M. leprae was determined by a specific polymerase chain reaction (PCR), 2 years after an earlier survey in the same population. 1923 persons were registered, 1171 were clinically examined for signs of leprosy, and 418 were tested by PCR. The detection rate of leprosy in the study area had not changed significantly during the 2 years' observation period since the introduction of multi-drug therapy, i.e. 6/1000 compared to 7.7/1000 2 years before. Of 6 newly detected cases, 5 were diagnosed as having paucibacillary leprosy. The presence of M. leprae could be demonstrated by PCR in 2.9% (12/418) of the persons. PCR positivity was not persistent over the 2 years. All the PCR positive persons identified in the first survey were negative in the second, indicating that M. leprae nasal carriage is transient. As in the previous survey, we found evidence for widespread M. leprae nasal carriage as determined by PCR among the general population in an area in which leprosy is endemic. In addition, our data indicated that PCR positivity can occur in certain clusters in the community. This clustering seems to be time-dependent, not necessarily related to the presence of patients.

Follow-up of multibacillary leprosy patients using a phenolic glycolipid-I-based ELISA. Do increasing ELISA-values after discontinuation of treatment indicate relapse?

With the introduction of reproducible serological tests it was hoped that relapses in leprosy patients, after discontinuing treatment, could be detected before damaging reactions occurred and before the patients became infectious. The possible value of an ELISA using a semisynthetic analogue of phenolic glycolipid-I to detect antibodies to this antigen in order to predict a relapse in multibacillary patients was investigated. In contrast to that reported for paucibacillary patients, this test was useful to detect early relapses in multibacillary patients. In 3 out of 4 multibacillary patients who relapsed, the ELISA-values were increased. The decreased ELISA-values in the one relapsed patient could be attributed to the corticosteroid therapy. In the multibacillary patients who did not relapse after RFT, the ELISA-values were consistently low or decreased. In only one patient did the ELISA-values increase following his release from treatment and this patient was clinically suspected of developing a relapse.

Evaluation of Mycobacterium leprae antigens in the monitoring of a dapsone-based chemotherapy of previously untreated lepromatous patients in Cebu, Philippines.

Thirty-five previously untreated lepromatous patients receiving dapsone-based therapy were monitored throughout their 5-year period of treatment by serology and by pathology. Sequentially collected sera were used to evaluate the usefulness of four Mycobacterium leprae antigens as used in ELISA to monitor the progress of their therapy. ELISA results were compared with each other and with bacterial load over the treatment period and with duration of treatment. The ELISAs, based on the measurement of IgM antibody reactivity to the two neoglycoproteins (NDO and NTO) representing the phenolic glycolipid antigen of M. leprae, were found to be the most effective in monitoring treatment. A whole M. leprae based ELISA was less efficient in monitoring treatment because it failed to measure antibodies in 8 out of 35 patients and because it provided consistently lower values than either NTO or NDO. The ELISA-inhibition test based on the detection of antibodies to a species-specific epitope on the 36 K antigen of M. leprae was less suitable because of persistent reactivity during therapy, consequently resulting in no significant correlation with ELISA reactivities to NTO or NDO.

Transmission and protection in leprosy: indications of the role of mucosal immunity.

Recent advances in treatment have achieved a large drop in the prevalence of active leprosy cases, but the incidence is at best decreasing slowly. Most people within leprosy-endemic populations have been exposed to Mycobacterium leprae, but few develop disease and it seems likely that the majority of the population develops protective immunity. If the site of initial infection is in the nose, dissemination of bacilli around the body to skin and nerve implies that the initial infection is bacilliferous and it has been shown that nasal M. leprae are detectable by polymerase chain reaction (PCR) of nasal swabs. Since salivary anti-M. leprae IgA (sMLIgA) levels are correlated with protection, we have surveyed groups of leprosy patients, contacts and the general population for both their sMLIgA and nasal PCR positivity. A total of 304 subjects were enrolled in the study: PCR and mucosal challenge tests were performed in 204 of these individuals. sMLIgA was present in 66% of treated patients, 76% of leprosy workers and 72% of healthy contacts. However, only 33% of indigenous subjects were sMLIgA+, in contrast to the earlier studies showing 74% positivity. PCR for M. leprae was present in both household contacts (2%) and indigenous controls (5%). In a subsequent follow-up study, nasal swabs were taken from 97 of those studied in the first series: three PCR+ individuals followed up after one year became negative, while of the remaining 94 PCR- individuals retested, 2 became positive. Of 112 subjects retested with the mucosal challenge test for sMLIgA: 22 converted from positive to negative and 12 from negative to positive. These results suggest that there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response, despite the fact that few individuals will develop clinical disease. This may explain the current lack of effect of multidrug therapy (MDT) control programmes on incidence, although the reduction in general population immunity is consistent with some effect of MDT on transmission.