Cost-effectiveness analysis of PCR for the rapid diagnosis of pulmonary tuberculosis.

BACKGROUND: Tuberculosis is one of the most prominent health problems in the world, causing 1.75 million deaths each year. Rapid clinical diagnosis is important in patients who have co-morbidities such as Human Immunodeficiency Virus (HIV) infection. Direct microscopy has low sensitivity and culture takes 3 to 6 weeks 123. Therefore, new tools for TB diagnosis are necessary, especially in health settings with a high prevalence of HIV/TB co-infection. METHODS: In a public reference TB/HIV hospital in Brazil, we compared the cost-effectiveness of diagnostic strategies for diagnosis of pulmonary TB: Acid fast bacilli smear microscopy by Ziehl-Neelsen staining (AFB smear) plus culture and AFB smear plus colorimetric test (PCR dot-blot).From May 2003 to May 2004, sputum was collected consecutively from PTB suspects attending the Parthenon Reference Hospital. Sputum samples were examined by AFB smear, culture, and PCR dot-blot. The gold standard was a positive culture combined with the definition of clinical PTB. Cost analysis included health services and patient costs. RESULTS: The AFB smear plus PCR dot-blot require the lowest laboratory investment for equipment (US$ 20,000). The total screening costs are 3.8 times for AFB smear plus culture versus for AFB smear plus PCR dot blot costs (US$ 5,635,760 versus US$ 1,498, 660). Costs per correctly diagnosed case were US$ 50,773 and US$ 13,749 for AFB smear plus culture and AFB smear plus PCR dot-blot, respectively. AFB smear plus PCR dot-blot was more cost-effective than AFB smear plus culture, when the cost of treating all correctly diagnosed cases was considered. The cost of returning patients, which are not treated due to a negative result, to the health service, was higher in AFB smear plus culture than for AFB smear plus PCR dot-blot, US$ 374,778,045 and US$ 110,849,055, respectively. CONCLUSION: AFB smear associated with PCR dot-blot associated has the potential to be a cost-effective tool in the fight against PTB for patients attended in the TB/HIV reference hospital.

The relation between seroprevalence of antibodies against phenolic glycolipid-I among school children and leprosy endemicity in Brazil.

Leprosy control programs would benefit expressively from an easy method to estimate disease prevalence and to assess the effect of leprosy control measures on disease prevalence. Determination of the seroprevalence of antibodies to PGL-I through school children surveys might be a useful indicator of leprosy prevalence at the district level. To investigate whether seropositivity rates could be related to leprosy detection rates and whether seropositivity could be used as a proximal indicator to predict the leprosy incidence in other areas, 7,073 school children in three different leprosy-endemic states in Brazil were tested. The results show a widely varying distribution of seropositivity in the communities independent of the number of leprosy cases detected. Seroprevalence was significantly lower at private schools. No differences in the patterns of seropositivity between ELISA and dipstick were observed. No correlation between leprosy detection rate and seropositivity rates could be established.

A relação entre soroprevalência de anticorpos contra o glicolipídeo fenólico-I entre crianças em idade escolar e endemicidade da hanseníase no Brasil / The relation between seroprevalence of antibodies against phenolic glycolipid-I among school children and leprosy endemicity in Brazil

Os programas de controle da hanseníase se beneficiariam de um método fácil para estimar prevalência e avaliar o impacto das ações de controle na prevalência da doença. A determinação da soroprevalência de anticorpos contra PGL-I através de estudos com crianças em idade escolar foi sugerida como indicador útil da taxa de prevalência da hanseníase a nível municipal.Para investigar se a soropositividade estaria associada aos coeficientes de detecção da hanseníase e se poderia ser usada como indicador da prevalência em outras áreas, 7.073 crianças em três estados endêmicos de hanseníase no Brasil foram testadas. Resultados mostram uma considerável variação da distribuição de soropositividade nas comunidades, independente do número de casos de hanseníase detectados. A soroprevalência foi significativamente menor nos colégios. Nenhuma diferença na distribuição da soropositividade determinada por ELISA ou dipstick foi observada. Nenhuma correlação entre o coeficiente de detecção da hanseníase e soropositividade pôde ser estabelecida.

Leprosy control programs would benefit expressively from an easy method to estimate disease prevalence and to assess the effect of leprosy control measures on disease prevalence. Determination of the seroprevalence of antibodies to PGL-I through school children surveys might be a useful indicator of leprosy prevalence at the district level. To investigate whether seropositivity rates could be related to leprosy detection rates and whether seropositivity could be used as a proximal indicator to predict the leprosy incidence in other areas, 7,073 school children in three different leprosy-endemic states in Brazil were tested. The results show a widely varying distribution of seropositivity in the communities independent of the number of leprosy cases detected. Seroprevalence was significantly lower at private schools. No differences in the patterns of seropositivity between ELISA and dipstick were observed. No correlation between leprosy detection rate and seropositivity rates could be established.

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.

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05) on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease

T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients.

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-gamma in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-gamma in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-gamma production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.

Epidemiology of leprosy on five isolated islands in the Flores Sea, Indonesia

We conducted a population-based survey on five small islands in South Sulawesi Province (Indonesia) to collect baseline data previous to a chemoprophylactic intervention study aiming at interrupting the transmission of Mycobacterium leprae. Here we describe the present leprosy epidemiology on these geographically isolated islands. Of the 4774 inhabitants living in the study area 4140 were screened for leprosy (coverage: 87 per cent). We identified 96 leprosy patients (85 new and 11 old patients), representing a new case detection rate (CDR) of 205/10 000 and a prevalence rate of 195/10 000. CDRs were similar for males and females. Male patients were more often classified as multibacillary (MB) than women. Of the new patients, 33 (39 per cent) were classified as MB, 16 (19 per cent) as paucibacillary (PB) 2-5 lesions and 36 (42 per cent) as PB single lesion. In this area of high leprosy endemicity leprosy patients were extensively clustered, i.e. not equally distributed among the islands and within the islands among the houses.

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens.

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05) on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.

T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50 per cent of the leprosy patients and 60 per cent of the non-TB controls. Nevertheless, the levels of IFN-gamma in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59 per cent of the vaccinated TB patients responded to ESAT in vitro, whereas 100 per cent of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-gamma in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-gamma production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.

Use of ML dipstick as a tool to classify leprosy patients

Leprosy control services face the problem of leprosy patients being misclassified by the lack of or the poor quality of skinsmear examination services. Misclassification increases the risk of relapse due to insufficient treatment if a multibacillary (MB) patient is classified as paucibacillary (PB), thereby also prolonging the time that the patient is infectious. The World Health Organization (WHO) recommends at present an alternative classification based on the number of skin lesions. Its reliability, however, has been questioned. Our investigation sought to determine the usefulness of the ML Dipstick, a simple field assay to detect IgM antibodies to phenolic glycolipid-I of Mycobacterium leprae, for the classification of leprosy patients in addition to lesion count. In this study, 264 leprosy patients were investigated. Of 130 patients with a positive bacterial index (BI), 19 (14.6%) had less than 6 lesions and would have been classified as PB. Out of 134 patients with a negative BI, 26 (19.4%) had 6 or more lesions and would have been classified as MB patients if the lesion counting system would apply. Thus, the classification based on the number of lesions only was found to be 85% sensitive and 81% specific (using the BI as the gold standard) at detecting MB cases among the studied population. Sensitivity would have increased if patients would have been classified according to a combination of the number of lesions and the dipstick result. In that case patients are classified as MB when they are either dipstick positive (N = 16), have more than 6 lesions (N = 43), or both (N = 94). Patients negative for both dipstick and number of lesions would have been classified as PB (N = 111). The classification based on the number of lesions alone left 19 BI-positive cases classified as PB, while the combination method of the ML Dipstick and number of lesions left only 8 BI-positive cases classified as PB (5 borderline, 2 borderline lepromatous and 1 tuberculoid), thus preventing undertreatment. The combination method of the ML Dipstick and lesion counting was found to be 94% sensitive and 77% specific, which is an improvement of 9% (chi-squared test, p = 0.025) in sensitivity compared to lesion counting only. The results of this study indicate that testing all patients initially classified by lesion counting as PB (48% in our study population) with the dipstick can significantly contribute to improved classification of leprosy patients for treatment purposes.

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.

Patient contact is the major determinant in incident leprosy: implications for future control

Notwithstanding the elimination efforts, leprosy control programs face the problem of many leprosy patients remaining undetected. Leprosy control focuses on early diagnosis through screening of household contacts, although this high-risk group generates only a small proportion of all incident cases. For the remaining incident cases, leprosy control programs have to rely on self-reporting of patients. We explored the extent to which other contact groups contribute to incident leprosy. We examined retrospectively incident leprosy over 25 years in a high-endemic village of 2283 inhabitants in Sulawesi, Indonesia, by systematically reviewing data obtained from the local program and actively gathering data through interviews and a house-to-house survey. We investigated the contact status in the past of every incident case. In addition to household contact, we distinguished neighbor and social contacts. Of the 101 incident cases over a 25-year period, 79 (78%) could be associated to contact with another leprosy patient. Twenty-eight (28%) of these 101 cases were identified as household contacts, 36 (36%) as neighbors, and the remaining 15 (15%) as social contacts. Three patients had not had a traceable previous contact with another leprosy patient, and no information could be gathered from 19 patients. The median span of time from the registration of the primary case to that of the secondary case was 3 years; 95% of the secondary cases were detected within 6 years after the primary case. The estimated risk for leprosy was about nine times higher in households of patients and four times higher in direct neighboring houses of patients compared to households that had had no such contact with patients. The highest risk of leprosy was associated with households of multibacillary patients. The risk of leprosy for households of paucibacillary patients was similar to the risk of leprosy for direct neighboring houses of multibacillary patients, indicating that both the type of leprosy of the primary case and the distance to the primary case are important contributing factors for the risk of leprosy. Contact with a leprosy patient is the major determinant in incident leprosy; the type of contact is not limited to household relationships but also includes neighbor and social relationships. This finding can be translated into a valuable and sustainable tool for leprosy control programs and elimination campaigns by focusing case detection and health promotion activities not only on household contacts but also on at least the neighbors of leprosy cases.

DNA amplification for detection of leprosy and assessment of efficacy of leprosy chemotherapy

Polymerase chain reaction (PCR) for the detection of Mycobacterium leprae was applied to fresh skin biopsies and slit-skin smears from 122 untreated leprosy patients. The PCR positivity rates in biopsies were 95.6% in multibacillary (MB) cases and 44.2% in paucibacillary (PB) cases. Following 1 month of treatment, MB cases declined by 54.3% and PB cases by 61.8% of initial values. Six-month values also declined from initial positivity rates to 50.3% and 53.8% of initial values in MB and PB, respectively. Larger declines in the rate of positivity were seen for skin-smear samples at 1 and 6 months in both MB and PB, but overall PCR positivity rates were lower than biopsy rates for M. leprae.

Humoral and cellular immune reactivity to recombinant M. leprae antigens in HLA-Typed leprosy patients and healthy controls

In our search for Mycobacterium leprae antigens that might specifically induce immunity or immunopathology, we have tested both humoral and cellular immune reactivity against purified recombinant M. leprae antigens in 29 paucibacillary (PB), 26 multibacillary (MB) leprosy patients, and 47 matched healthy contacts. The following M. leprae antigens were tested: 2L-1 (65L-1, GroEl-1), 2L-2 (65L-2, GroEl-2), 4L (SoDA), 43L, 10L (B) and 25L (Sra). The individuals were also typed for HLAD-RB1 and DQB1 in order to see whether leprosy status and/or immune reactivity to these antigens might be associated with certain HLA types. We also tested sera from another 48 patients before, during and after multidrug therapy (MDT) to study the relationship between antibody reactivity to recombinant M. leprae antigens and MDT. Antibody titers to the four recombinant M. leprae antigens tested and to D-BSA were higher in MB patients compared to PB patients and healthy controls, and declined with treatment. From a diagnostic or monitoring point of view none of the recombinant antigens seemed to be an improvement over D-BSA, mainly due to the lower sensitivity. IgG subclasses were measured in positive sera of untreated patients. These were mainly of the IgG1 and IgG3 subclasses, but subclass diversity was also observed and antigen dependent: all four subclasses could be detected against 10L (B), only IgG1 and IgG3 against 43L and only IgG1 against 25L and 2L-1. Cellular immune reactivity against the purified recombinant M. leprae antigens was measured in a lymphocyte stimulation test (LST). As for M. leprae, there was an inverse correlation between antibody and T-cell reactivity. However, the number of LST responders to recombinant antigens was much lower than to M. leprae. The 43L antigen was recognized most often (19%-24% of individuals tested) and more often than the 10L (B) antigen (10%-12%). No clear correlation was observed with leprosy type or protection and, in general, M. leprae nonresponders were also negative with recombinant antigens. Finally, we confirmed that HLA-DRB1*02 is associated with leprosy in this population, and we observed an association between DQB1*0601 and lepromatous leprosy. The number of positive individuals was too small to allow a meaningful analysis of the relationship between HLA type and immune reactivity. Although these data do not allow a conclusion as to one of these purified recombinant antigens being either protection or disease related, the antigen-dependent IgG subclass diversity warrants further study on antigen-specific qualitative differences in immune reactivity that may be relevant for the outcome of an infection with M. leprae.