Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Introduction Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. Methods We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

Frequency of the toxic shock syndrome toxin-1 gene in methicillin-susceptible and -resistant Staphylococcus aureus isolates from teaching hospitals in Shiraz, Iran

INTRODUCTION: Staphylococcus aureus produces a range of virulence factors such as toxic shock syndrome toxin-1. METHODS: In this cross-sectional study of 345 clinical S. aureus isolates, the presence of the tst gene was assessed by polymerase chain reaction (PCR). RESULTS: The study revealed 53/345 (15.4%) isolates were positive for the tst gene. The tst gene was present in 18.1% of methicillin-susceptible S. aureus (MSSA) isolates and 11.6% of methicillin-resistant S. aureus (MRSA) isolates (p = 0.136). CONCLUSIONS: These results reveal the remarkable risk of S. aureus infections in hospitals, regardless of methicillin-resistance status.

HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil.

Specific Identification of Biomphalaria tenagophila and Biomphalaria occidentalis Populations by the Low Stringency Polymerase Chain Reaction

Although Biomphalaria occidentalis and B. tenagophila are indistinguishable on the basis of shell morphology and the majority of their genital organs, only the latter is susceptible to infection with Schistosoma mansoni. Thus, the identification of these species is fundamental to epidemiological studies of schistosomiasis. Here we describe a simple and rapid method for differentiating B. tenagophila from B. occidentalis based on low stringency polymerase chain reaction and using a pair of primers specific for the amplification of the 18S rRNA gene. Analysis of the low stringency product profiles of populations of these snails from different geographical regions confirmed this approach as being applicable to the identification of B. tenagophila and B. occidentalis in cases where classical morphology is inconclusive

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Diagnosis of Dengue by Using Reverse Transcriptase-Polymerase Chain Reaction

A rapid identification of dengue viruses from clinical samples by using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was carried out for diagnostic and epidemiological purposes. RT-PCR identified DEN-1 and DEN-2 viruses in 41% (41/100) of previously confirmed cases and provided an accurate confirmation of DHF in four fatal cases. RT-PCR was also useful for detecting and typing dengue viruses in suspected cases, allowing a rapid identification of new serotypes in endemic areas

Allelic Diversity at the Merozoite Surface Protein-1 (MSP-1) Locus in Natural Plasmodium falciparum Populations: a Brief Overview

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n = 54), the mesoendemic southern Vietnam (n = 238) and the holoendemic northern Tanzania (n = 79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with parasites carrying more than one MSP-1 version) ranged from 39% in Brazil to 44% in Vietnam and 60% in Tanzania. The vast majority (90%) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven gene types corresponded to only 61% of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution of MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mnl I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.