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.     

Female and male partner age and menotrophin requirements influence pregnancy rates with human menopausal gonadotrophin therapy in combination with intrauterine insemination

This study analyses the influence of female and male patient age and human menopausal gonadotrophin (HMG) requirements on clinical pregnancy rates and live birth rates with ovulation stimulation using HMG in combination with intrauterine insemination (IUI). In this study, 363 consecutive HMG/IUI treatment cycles in 184 patients carried out at a university fertility centre were analysed in a retrospective fashion. The main outcomes measured were clinical pregnancy rates and live birth rates. Increased female partner age (> or = 35) and male partner age (> or = 40) were found to negatively influence pregnancy rates with HMG/ IUI therapy. In addition, this study demonstrated a critical threshold of HMG requirements beyond which pregnancy did not occur. No pregnancies occurred in treatment cycles requiring > 25 ampoules (1875 IU) of menotrophins to achieve follicular maturity, irrespective of patient age. In conclusion, female partner age, male partner age, and HMG requirements all significantly influence pregnancy rates with HMG/IUI therapy.