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.     

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.     

Comparison of PCR with the routine procedure for diagnosis of tuberculosis in a population with high prevalences of tuberculosis and human immunodeficiency virus

Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis (TB) as employed in most low-income countries is cheap and easy to use, but its low sensitivity is a major drawback. The low specificity of chest X-rays, used for the diagnosis of smear-negative TB, risks high levels of overdiagnosis. Major advances in molecular techniques, which rapidly identify mycobacterial DNA in sputa, may overcome these obstacles. In this study, the AMPLICOR PCR system was used to diagnose pulmonary TB in a developing country with high prevalences of both TB and human immunodeficiency virus (HIV). The sensitivity and specificity of this technique were compared to those of the usual diagnostic techniques. Sputum specimens were collected from 1,396 TB suspects attending the Rhodes Chest Clinic, Nairobi, Kenya. The specimens were analyzed for the presence of Mycobacterium tuberculosis by PCR; culture on Löwenstein-Jensen medium was used as the “gold standard.” All culture-positive samples were genotyped to identify the mycobacterial species. The sensitivity and specificity of PCR were 93 and 84%, respectively. HIV status did not affect the sensitivity of PCR. A total of 99.7% of the true smear-positive and 82.1% of the true smear-negative TB patients were correctly identified by PCR. PCR detected M. tuberculosis in 11.7% of the culture-negative suspects, 60% of which had one or two PCR-positive sputum specimens. Of the 490 positive cultures, 486 were identified as M. tuberculosis. The high sensitivity of Amplicor PCR merits usage in a clinical setting with high TB and HIV burdens. Thus, PCR can be considered as an alternative to ZN staining in combination with chest X-ray for diagnosis of TB; however, cost-effectiveness studies and operational studies are required to support an evidence-based decision of introducing PCR for TB control in high-burden environments.Tuberculosis (TB) is one of the most serious infectious diseases and a considerable public health problem due to its high risk of person-to-person transmission, morbidity, and mortality. Both the human immunodeficiency virus (HIV) epidemic and social deterioration have contributed to the overall increase in the Mycobacterium tuberculosis infection rate, especially in developing countries, where resources are scarce (13). In Nairobi the case detection rate increased from 78 per 100,000 in 1991 to 581 per 100,000 in 2001, with a total number of 12,963 cases.Early diagnosis followed by adequate treatment is essential to prevent both morbidity and mortality. Although the conventional technique of direct smear examination with Ziehl-Neelsen staining (ZN) is cheap and easy to perform, its low sensitivity is a major drawback. Depending on the number of specimens examined, ZN detects 30 to 60% of the culture-positive “TB suspects” (7). Furthermore, it requires sputum samples collected on consecutive days, making the procedure slow and making patient compliance with the diagnostic process difficult.New techniques are very much needed (7), and molecular amplification assays such as PCR have been shown to be promising alternatives even for developing countries (2). PCR has the potential to be a cost-effective alternative, provided the diagnosis can be determined with one sputum examination (8). If diagnosis can be established faster, and the diagnostic process becomes less cumbersome for the patient, PCR may reduce delay both in diagnosis and in the start of treatment.Depending on the “gold standard” and other methodological factors, studies show PCR sensitivities ranging from 77% to more than 95% and PCR specificities of >95% for smear-positive specimens (4, 9, 10, 12). However, sensitivities for smear-negative TB patients have been reported to be below 90% (9). Most PCR studies have been performed in industrial countries (4, 9, 10, 12) where the TB and HIV burdens are low.To investigate the performance and feasibility of PCR in an environment of TB endemicity and high prevalences of HIV and AIDS, a study was conducted in Nairobi, Kenya, comparing PCR to conventional routine diagnostic methods within a program setup. In this study, the Roche Amplicor Mycobacteria PCR test for the direct detection of M. tuberculosis was used on sputum specimens from TB suspects attending a chest clinic in Nairobi. Its performance was compared with those of the basic routine diagnostic procedures according to the national guidelines (6), including clinical findings, ZN, and chest X-rays (CXR), on smear-negative suspects. Löwenstein-Jensen (LJ) culture results were used as the gold standard.     

Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.     

Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples.

We developed a colorimetric microwell plate hybridization assay (CoMPHA) for the specific detection of 5'-biotinylated amplified Mycobacterium tuberculosis DNA. The optical densities of the CoMPHA corresponded to the initial amounts of purified template DNA. Here, we show that the CoMPHA is useful in distinguishing the PCR-positive and PCR-negative samples.     

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.     

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.     

Seroprevalence rates of antibodies to phenolic glycolipid-I among school children as an indicator of leprosy endemicity

In order to study whether the seroprevalence of antibodies to phenolic glycolipid-I (PGL-I) among school children is a useful indicator of the leprosy problem in certain areas, school surveys were carried out. These surveys have the advantage of targeting an easily accessible, stable and standardized population. Antibodies to the species-specific PGL-I of Mycobacterium leprae were detected in a simple gelatin particle agglutination test. We have determined the seroprevalence rates in 2835 school children from five different areas in three provinces of Sulawesi, Indonesia. Three areas with a case-detection rate of over 3.4/10,000 were designated as high-endemic areas. The other two were designated as low-endemic areas, having a case-detection rate of less than 1/10,000. The seroprevalence rates in the three high-endemic areas ranged from 26% to 28% (95% CI 21%-31%). In both low-endemic areas the seroprevalence rate was 7% (95% CI 5%-10%). In a second survey conducted in one high-endemic area 3 years after the first survey, the seroprevalence rate was the same as in the first survey. These results indicate that seropositivity rates among school children may reflect the leprosy incidence. They illustrate the potential applicability of seroprevalence as an indicator of the magnitude of the leprosy problem in a selected area.     

Diagnosis of tuberculous lymphadenitis in Ethiopia: correlation with culture, histology and HIV status

SETTING: Butajira, Southern Ethiopia. OBJECTIVE: To compare the diagnostic capacity of the clinical criteria for tuberculous lymphadenitis (TBLN) with histological and/or culture results and to assess the association of human immunodeficiency virus (HIV) with tuberculosis (TB) lymphadenitis. DESIGN: Patients (n=171) were included in the study from October 2005 until July 2006 at Butajira Hospital. Laboratory tests were performed to confirm TBLN. HIV status was identified in TBLN patients and retrospectively in 1608 healthy individuals. RESULT: A total of 136/161 (84.5%) patients were diagnosed with TBLN by histology. TBLN was culture-confirmed in 107/156 (68.6%) patients. The sensitivity, specificity, positive and negative predictive values of histology were respectively 92.5%, 49%, 79.8% and 75% when compared to culture as gold standard. Patients positive for TBLN by cytology and Ziehl-Neelsen (ZN) were also positive by histology and culture. Among the 143 confirmed TBLN patients, nine (6.3%) were HIV-positive. Of the 1608 healthy individuals, 77 (4.8%) were HIV-positive. Younger age (P=0.0001), female sex (P=0.016), not being married (P=0.0001) and illiteracy (P=0.016) showed a strong association with HIV in healthy individuals. CONCLUSION: Clinical criteria alone over-diagnosed TBLN by 15.4% compared to histological and/or bacteriological results. The HIV prevalence in TBLN patients and healthy individuals was the same.