EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

FATAL OUTCOME OF INFECTION BY DENGUE 4 IN A PATIENT WITH THROMBOCYTOPENIC PURPURA AS A COMORBID CONDITION IN BRAZIL

Dengue is currently a major public-health problem. Dengue virus (DENV) is classified into four distinct serotypes, DENV 1-4. After 28 years of absence, DENV-4 was again detected in Brazil in 2010 in Roraima State, and one year later, the virus was identified in the northern Brazilian states of Amazonas and Pará, followed by Rio de Janeiro and São Paulo. In Minas Gerais, the first confirmed case of DENV-4 occurred in the municipality of Frutal in 2011 and has now been isolated from a growing number of patients. Although DENV-2 is associated with the highest risk of severe forms of the disease and death due to the infection, DENV-4 has also been associated with severe forms of the disease and an increasing risk of hemorrhagic manifestations. Herein, the first fatal case of confirmed DENV-4 in Brazil is reported. The patient was an 11-year-old girl from the municipality of Montes Claros in northern Minas Gerais State, Brazil. She had idiopathic thrombocytopenic purpura as a comorbid condition and presented with a fulminant course of infection, leading to death due to hemorrhagic complications. Diagnosis was confirmed by detection of Dengue-specific antibodies using IgM capture enzyme-linked immunosorbent assay and semi-nested RT-PCR. Primary care physicians and other health-care providers should bear in mind that DENV-4 can also result in severe forms of the disease and lead to hemorrhagic complications and death, mainly when dengue infection is associated with coexisting conditions.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

FALSE-NEGATIVE DENGUE CASES IN RORAIMA, BRAZIL: AN APPROACH REGARDING THE HIGH NUMBER OF NEGATIVE RESULTS BY NS1 AG KITS

Serum samples from 150 NS1-negative (Platelia ELISA) patients presumptively diagnosed with dengue were analyzed by the TaqMan probed real-time reverse transcription PCR (TaqMan qRT-PCR) method. The qRT-PCR positive samples were tested for serotype by semi-nested RT-PCR and a qualitative immunochromatographic assay for IgG and IgM. Molecular detection methods showed 33 (22%) positive samples out of 150 NS1-antigen negative samples. Of these, 72% were collected up to day 2 after the onset of symptoms, when diagnostic sensitivity of NS1-antigen test assays is significantly enhanced. Most of the cases were not characterized as secondary infection. Twenty-eight samples were successfully serotyped, 75% of which for DENV-4, 14% for DENV-2, 7% for DENV-3 and 4% for DENV-1. These findings reaffirm the hyperendemic situation of the state of Roraima and suggest a lower sensitivity of the NS1 test, mainly when DENV-4 is the predominant serotype. Health care providers should therefore be aware of samples tested negative by NS1 antigen assays, especially when clinical symptoms and other laboratory data results show evidence of dengue infection.

SAINT LOUIS ENCEPHALITIS VIRUS IN MATO GROSSO, CENTRAL-WESTERN BRAZIL

The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012. Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple co-infection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state of Pará. This is the first report of SLEV molecular identification in MT.

QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES

SUMMARY Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

DENGUE OUTBREAK IN MATO GROSSO STATE, MIDWESTERN BRAZIL

Dengue virus (DENV) is the most frequent arbovirus worldwide. In this study, we report a large outbreak in Mato Grosso State (MT). Serum samples from 604 patients with acute febrile illness for less than five days were inoculated in C6/36 cells, then infected cells were subjected to an indirect immunofluorescence test for DENV serotypes and yellow fever virus. Serum samples were submitted to a multiplex-semi-nested-RT-PCR for 11 flaviviruses. DENV-4 was isolated in 150/604 (24.8%) and DENV-1 in 19/604 (3.1%) specimens. By RT-PCR, 331 (54.8%) samples tested positive for DENV; 321 had single infections (DENV-4 n = 305; DENV-1 n = 15; DENV-3 n = 1), nine had co-infections of DENV-1/DENV-4, and one of DENV-2/DENV-4. DENV-4 was detected in 315/331 (95.2%) positive patients from 17 municipalities, and DENV-1 in 24/331 (7.2%) patients from five cities in north-central MT and the city of Cuiaba. The incidence of infection was higher in patients aged 20-39 (142/331; 42.9%). The NS5 partial nucleotide sequence of DENV-1 was most similar to that of genotype V, DENV-2 to Southeast Asian/American, DENV-3 to genotype III, and DENV-4 to genotype II strains, considered the most frequent strains in Brazil. This outbreak coincided with the introduction of DENV-4 in the state. Cuiaba was hyperendemic for the four DENV serotypes, highlighting the necessity for arbovirus surveillance in MT.

How prevalent is Plasmodium malariae in Rondônia, Western Brazilian Amazon?

We have compared results of Plasmodium species identification obtained with conventional on-site microscopy of Giemsa-stained thick smears (GTS) and a semi-nested polymerase chain reaction (PCR) in 96 malaria patients from Rondônia, Western Brazilian Amazon. Mixed-species infections were detected by PCR in 30% patients, but no such case had been found on GTS. Moreover, P. malariae infections were detected in 9 of 96 patients (10%) by PCR, but were not identified by local microscopists. The potential impact of species misidentification on malaria treatment and control is discussed.

Monitoramento de pacientes com AIDS para o desenvolvimento de doença por citomegalovirus (CMV) usando-se PCR multiplex

O citomegalovirus é um patógeno importante em indivíduos infectados pelo vírus da imunodeficiência humana (HIV). A carga viral do CMV parece ser preditora da sintomatologia das infecções citomegálicas em pacientes com a síndrome da imunodeficiência adquirida (AIDS). Um protocolo de PCR multiplex que fornece informações de quantificação de DNA amplificando somente um ou dois genes alvo do CMV foi utilizado neste trabalho. Amostras mensais de sangue foram coletadas de 270 pacientes com AIDS. Vinte pacientes tiveram positividade para dois alvos por 3 ou mais consecutivas e apresentaram doença por CMV no decorrer do estudo. Os pacientes que não apresentaram positividade ou alternância de amostras positivas somente para um gene viral não desenvolveram doença ligada ao CMV. Os resultados sugerem que a PCR multiplex é um protocolo útil para a identificação dos pacientes com alta carga viral e maior risco de desenvolvimento de doença por CMV.

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Differentiation of Candida species obtained from nosocomial candidemia using RAPD-PCR technique

Thirteen strains of the genus Candida were isolated from catheter, urine and surgical wounds from individual patients of the Santa Casa de Misericórdia, Belo Horizonte, MG, Brazil. Ten strains were characterized as Candida albicans, two as Candida glabrata, and one as Candida parapsilosis. Isolates were evaluated for molecular relatedness by random amplified polymorphic DNA technique using 15 primers. The analysis of the genomic DNA obtained revealed a low intraspecific polymorphism and did not allow for the differentiation between strains of the same species obtained from distinct clinical sources (catheter, urine and surgical wounds). The RAPD profiles generated were able to differentiate among the species of Candida albicans, Candida parapsilosis and Candida glabrata strains isolated in this study.

Assessment of chemiluminescence and PCR effectiveness in relation to conventional serological tests for the diagnosis of Chagas' disease

While testing 414 sera for the diagnosis of Chagas' disease, the conventional reactions of indirect hemagglutination, indirect immunofluorescence and the immunosorbent assay showed a sensitivity of 95.7%, 100% and 98.2% and a specificity of 98%, 98% and 96.4%, respectively, and an excellent association using Fisher's exact test. Chemiluminescence presented 100% sensitivity and 89.6% specificity, while PCR showed 100% specificity and 1.2% sensitivity. It is believed that the three conventional serological reactions are still adequate for diagnosing Chagas' disease.