A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.

HTLV-1/2 seroprevalence and coinfection rate in Brazilian first-time blood donors: an 11-year follow-up

The seroprevalence and geographic distribution of HTLV-1/2 among blood donors are extremely important to transfusion services. We evaluated the seroprevalence of HTLV-1/2 infection among first-time blood donor candidates in Ribeirão Preto city and region. From January 2000 to December 2010, 1,038,489 blood donations were obtained and 301,470 were first-time blood donations. All samples were screened with serological tests for HTLV-1/2 using enzyme immunoassay (EIA). In addition, the frequency of coinfection with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Chagas disease (CD) and syphilis was also determined. In-house PCR was used as confirmatory test for HTLV-1/2. A total of 296 (0.1%) first-time donors were serologically reactive for HTLV-1/2. Confirmatory PCR of 63 samples showed that 28 were HTLV-1 positive, 13 HTLV-2 positive, 19 negative and three indeterminate. Regarding HTLV coinfection rates, the most prevalent was with HBV (51.3%) and HCV (35.9%), but coinfection with HIV, CD and syphilis was also detected. The real number of HTLV-infected individual and coinfection rate in the population is underestimated and epidemiological studies like ours are very informative.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

QUANTITATIVE REAL-TIME PCR (Q-PCR) FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

Objective: To assess quantitative real-time polymerase chain reaction (q-PCR) for the sputum smear diagnosis of pulmonary tuberculosis (PTB) in patients living with HIV/AIDS with a clinical suspicion of PTB.Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard.Results: Of the 140 sputum samples, 47 (33.6%) were positive with the gold standard. q-PCR was positive in 42 (30%) of the 140 patients. Only one (0.71%) did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93%) of the 42 q-PCR positive cases, the CT (threshold cycle) was equal to or less than 37.Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

Glomerulonephritis in schistosomiasis mansoni: a time to reappraise

INTRODUCTION: The current prevalence of glomerulonephritis in patients with hepatosplenic schistosomiasis mansoni in Brazil was evaluated. METHODS: Sixty three patients (mean age 45.5±11 years) attending the outpatient infectious disease clinic of a University Hospital in Belo Horizonte, Brazil, from 2007 to 2009, were consecutively examined and enrolled in the present investigation. Diagnosis of hepatosplenic schistosomiasis was based on epidemiological, clinical and parasitological data and imaging techniques. Eight patients, who presented >30mg/day albuminuria, were submitted to percutaneous ultrasound guided renal biopsy. Kidney tissue fragments were examined under light, direct immunofluorescence and electron microscopy. RESULTS: All patients showed mesangial enlargement. In five, mesangial hypercellularity was observed and four presented duplication of the glomerular basement membrane. Areas of glomerular sclerosis were diagnosed in four. Deposits of immunoglobulin M and C3 were present in six samples; deposits of IgG in four, IgA in three and C1q in two samples. In all patients, immunoglobulin A was reported in the lumen of renal tubules. Deposits of kappa and lambda were observed in six samples. Electron microscopy revealed dense deposits in the glomerular tissue of three patients. Arterial hypertension, small esophageal varices, slight increases in serum creatinine and decreases in serum albumin were associated with glomerular disease. CONCLUSIONS: Renal disease associated with hepatosplenic schistosomiasis was verified in 12.7% of patients and type I membranoproliferative glomerulonephritis was observed in 50% of them. Schistosomal glomerulopathy still is an important problem in patients with hepatosplenic schistosomiasis in Brazil.

Chagas disease in the State of Pernambuco, Brazil: analysis of admissions and mortality time series

INTRODUCTION: A time series study of admissions, deaths and acute cases was conducted in order to evaluate the context of Chagas disease in Pernambuco. METHODS: Data reported to the Information Technology Department of the Brazilian National Health Service between 1980 and 2008 was collected for regions and Federal Units of Brazil; and microregions and municipalities of Pernambuco. Rates (per 100,000 inhabitants) of hospitalization, mortality and acute cases were calculated using a national hospital database (SIH), a national mortality database (SIM) and the national Information System for Notifiable Diseases (SINAN), respectively. RESULTS: The national average for Chagas disease admissions was 0.99 from 1995 to 2008. Pernambuco obtained a mean of 0.39 in the same period, with the highest rates being concentrated in the interior of the state. The state obtained a mean mortality rate of 1.56 between 1980 and 2007, which was lower than the national average (3.66). The mortality rate has tended to decline nationally, while it has remained relatively unchanged in Pernambuco. Interpolating national rates of admissions and deaths, mortality rates were higher than hospitalization rates between 1995 and 2007. The same occurred in Pernambuco, except for 2003. Between 2001 and 2006, rates for acute cases were 0.56 and 0.21 for Brazil and Pernambuco, respectively. CONCLUSIONS: Although a decrease in Chagas mortality has occurred in Brazil, the disease remains a serious public health problem, especially in the Northeast region. It is thus essential that medical care, prevention and control regarding Chagas disease be maintained and improved.

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA) assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP) presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62). In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively), whereas ELISA using egg antigens (ELISA-SEA) detected samples after 140 days (p=0.03). CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Pulmonary tuberculosis in São Luis, State of Maranhão, Brazil: space and space-time risk clusters for death (2008-2012)

INTRODUCTION: The objective was to identify space and space-time risk clusters for the occurrence of deaths in a priority city for the control of tuberculosis (TB) in the Brazilian Northeast. METHODS: Ecological research was undertaken in the City of São Luis/Maranhão. Cases were considered that resulted in deaths in the population living in the urban region of the city with pulmonary TB as the basic cause, between 2008 and 2012. To detect space and space-time clusters of deaths due to pulmonary TB in the census sectors, the spatial analysis scan technique was used. RESULTS: In total, 221 deaths by TB occurred, 193 of which were due to pulmonary TB. Approximately 95% of the cases (n=183) were geocoded. Two significant spatial clusters were identified, the first of which showed a mortality rate of 5.8 deaths per 100,000 inhabitants per year and a high relative risk of 3.87. The second spatial cluster showed a mortality rate of 0.4 deaths per 100,000 inhabitants per year and a low relative risk of 0.10. A significant cluster was observed in the space-time analysis between 11/01/2008 and 04/30/2011, with a mortality rate of 8.10 deaths per 100,000 inhabitants per year and a high relative risk (3.0). CONCLUSIONS: The knowledge of priority sites for the occurrence of deaths can support public management to reduce inequities in the access to health services and permit an optimization of the resources and teams in the control of pulmonary TB, providing support for specific strategies focused on the most vulnerable populations.

Two decades of tuberculosis in a city in Northeastern Brazil: advances and challenges in time and space

Abstract: INTRODUCTION This study presents two decades of epidemiological data on tuberculosis (TB), in order to understanding the disease profile and its spatiotemporal dynamics. METHODS This descriptive study was performed in the City of Olinda/Pernambuco, Brazil, from 1991-2010, and it analyzed new patients with TB living in the city. We used the χ²-test with a p-value <0.05 to identify differences in trends. Incidence and cluster distribution were identified using spatial scan statistics. RESULTS In total, 6202 new cases were recorded during the two decades. The highest incidence occurred in 1995 (110 cases/100,000 inhabitants), and the lowest occurred in 2009 (65 cases/100,000 inhabitants) (β=-1.44; R²=0.43; p=0.0018). The highest mortality occurred in 1998 (16 deaths/100,000 inhabitants), and the lowest occurred in 2008 (5 deaths/100,000 inhabitants) (β=-0.19; R²=0.17; p=0.07). There was a male predominance (65%), and ages ranged from 20-49 years (65%). There was a substantial increase in the number of patients that were cured after treatment (60% to 67%; p<0.001) as well as those tested for HIV (1.9% to 58.5%; p<0.001). During the first decade, clusters with p-values <0.05 included 29% of the total notified cases, and in the second decade, that percentage was 12%. CONCLUSIONS We observed a decreasing trend in incidence, which was significant, and mortality rates, which was not significant. The increased number of laboratory tests performed reflects advances in surveillance, and a reduction in the proportion of cases in primary clusters suggests, among other things, that the disease is spreading across the region.

Primate populations in continuous forest and forest fragments in Central Amazonia

Population densities of six primate species (Saguinus midas, Pithecia pithecia, Cebus apella, Chiropotes satanas, Alouatta seniculus and Ateles paniscus) were estimated in continuous forest and in isolated reserves (one of 100 ha and four of 10 ha). Saguinusdensities in the continuous forest were found to be low, probably due to the lack of edge habitat and second growth favoured by them; Pithecia, Cebus and Ateles populations are also low, possibly because of more widely distributed and/or less abundant food sources than is true for other Amazonian regions, although hunting in the past, particularly of Ateles may also be a contributing factor; and Chiropotes and Alouatta densities were found to be similar to those observed in other areas of Amazonas forests. Ateles and Chiropotes, which occupy ranges on the order of three km2 were excluded from the 100-ka reserve at the time of its isolation. Unfortunately populations were not known prior to isolation of this reserve but during isolation there remained four groups of Saguinus, two Pitheciagroups, one Cebus groups and five Alouatta groups. One Saguinus group disappeared two months later, and one year post-isolation the Cebus group also left the reserve. Single Alouatta groups survive in the isolated 10-ha reserves. Saguinus, present in the four 10-ha reserves following isolation, have disappeared from two of them. One 10-ha reserve retains a group of Pithecia.