Identification of Human T-lymphotropic Virus Type I (HTLV-I) Subtypes Using Restrited Fragment Length Polymorphism in a Cohort of Asymptomatic Carriers and Patients with HTLV-I-associated Myelopathy/tropical Spastic Paraparesis from São Paulo, Brazil

Although human T-lymphotropic virus type I (HTLV-I) exhibits high genetic stability, as compared to other RNA viruses and particularly to human immunodeficiency virus (HIV), genotypic subtypes of this human retrovirus have been characterized in isolates from diverse geographical areas. These are currently believed not to be associated with different pathogenetic outcomes of infection. The present study aimed at characterizing genotypic subtypes of viral isolates from 70 HTLV-I-infected individuals from São Paulo, Brazil, including 42 asymptomatic carriers and 28 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), using restricted fragment length polymorphism (RFLP) analysis of long terminal repeat (LTR) HTLV-I proviral DNA sequences. Peripheral blood mononuclear cell lysates were amplified by nested polymerase chain reaction (PCR) and amplicons submitted to enzymatic digestion using a panel of endonucleases. Among HTLV-I asymptomatic carriers, viral cosmopolitan subtypes A, B, C and E were identified in 73.8%, 7.1%, 7.1% and 12% of tested samples, respectively, whereas among HAM/TSP patients, cosmopolitan A (89.3%), cosmopolitan C (7.1%) and cosmopolitan E (3.6%) subtypes were detected. HTLV-I subtypes were not statistically significant associated with patients' clinical status. We also conclude that RFLP analysis is a suitable tool for descriptive studies on the molecular epidemiology of HTLV-I infections in our environment.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Prevalence of Chlamydia trachomatis in human immunodeficiency virus-infected women in Cuba

To determine the prevalence rates and serovar distribution of Chlamydia trachomatis cervical infections in Cuban women, two different groups were selected. Group I consisted of 60 human immunodeficiency virus (HIV-1) seropositive women from different regions of Cuba and group II of 60 randomly selected women HIV seronegative and apparently healthy. C. trachomatis was detected in cervical scrapes by mean of nested polymerase chain reaction (PCR) specific for major out membrane protein. The overall prevalence rate of C. trachomatis in cervical scrapes determined by nested PCR was 10% in group I and the estimated prevalence was 6.6% for group II; 83.3% of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50%). The control group C. trachomatis-infected women referred a history of cervicitis in 75% of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50%. The present study is the first report of C. trachomatis prevalence in Cuba. It showed that there was not significantly difference in the prevalence rate of C. trachomatis between both groups.

Detection of hilA gene sequences in serovars of Salmonella enterica sufigbspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.

Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR) amplification fragments for the precise tuberculosis (TB) diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture) test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients). Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

Recent advances in the diagnosis of Schistosoma infection: the detection of parasite DNA

As Schistosoma sp. control programs are chiefly based on treatment of infected population, adequate case finding has a crucial role. The available diagnostic methods are far from ideal, since the search for eggs in stools and the detection of circulating antigens lack sensitivity in low prevalence and post-treatment situations and antibody detection lacks specificity. In most endemic foci, repeated treatment of infected people leaves a number of non-diagnosed and consequently non-treated persons, enough to maintain a persistent residue of 5 to 10% prevalence. In an attempt to surpass these diagnostic limitations we have developed a polymerase chain reaction (PCR) for the detection of Schistosoma sp. in feces that, in a first population study, has shown to be more sensitive than three-repeated stool Kato-Katz examination. The PCR may constitute a valuable tool for the diagnosis of the Schistosoma sp. infection in special situations, when high sensitivity and specificity are required and infrastructure is available.

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

Persistent human parvovirus B19 infection in children under maintenance chemotherapy for acute lymphocytic leukemia.

OBJECTIVE: To report on B19 infection management and chemotherapy schedule consequences in five children treated for acute lymphocytic leukemia (ALL). PATIENTS AND METHODS: Between May 2001 and February 2002, five patients between 4 and 12 years of age, receiving maintenance chemotherapy for ALL, presented with symptoms suggesting B19 infection (pallor, fatigue, petechiae and pancytopenia in four patients; generalized rash in two patients; acute hepatitis in one patient). Qualitative polymerase chain reaction (PCR) on peripheral blood was used for diagnosis and follow-up of infection; quantitative PCR was used for viral load measurement. Intravenous nonspecific high-dose immunoglobulin therapy was administered until PCR was negative. RESULTS: Qualitative B19 DNA was found in the peripheral blood of all patients, confirming the infection. Viral load at diagnosis ranged from 10 to 10 particles/mL blood. B19 DNA was detectable in four patients at 45, 21, 40, and 44 weeks, respectively. Chemotherapy was delayed in all patients. No clear benefit of intravenous immunoglobulin was noted. CONCLUSIONS: Infection with B19 is rarely reported in patients with ALL, but it should be suspected when unexplained pancytopenia occurs during chemotherapy. Persistent B19 infection remains a challenge in the management of patients receiving maintenance chemotherapy for ALL, as no specific therapy such as a specific immunoglobulin or vaccine exists. The role of viral load measurement needs to be established in terms of its use in follow-up and evaluation of the therapeutic response.

Evaluation of adherence, hemagglutination, and presence of genes codifying for virulence factors of Acinetobacter baumannii causing urinary tract infection

Acinetobacter baumannii is a strictly aerobic bacterium which causes severe infections, however its pathogenic characteristics are not well defined. Thirteen A. baumannii strains isolated from urine of hospitalized and nonhospitalized patients with different ages were investigated for the presence of virulence factors. The isolates belonged to biotypes 2, 6, and 9 and were sensitive to imipenem. The majority of them showed resistance to amikacin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, norfloxacin, and trimethoprim-sulfamethoxazole. None of A. baumannii strains presented genes codifying for 17 different virulence factors previously described in uropathogenic Escherichia coli, when tested by polymerase chain reaction (PCR). Nine isolates agglutinated human group AB erythrocytes, in presence of mannose, but none of them agglutinated group O erythrocytes. Adherence to polystyrene was observed in 7 isolates, and this result did not correlate with that obtained in hemagglutination assay. All the isolates were able to grow in iron-limiting conditions, showing that A. baumannii produces some type of siderophore. However, the genes iutA and fyuA, from iron uptake system of E. coli and Yersinia sp., respectively, were not present in the isolates, suggesting the presence of a different type of siderophore. The fimbriae of A. baumannii strains that mediates the adherence are possibly mannose-resistant, eventhough the mechanism of adherence to human epithelial cells still remains to be elucidated.

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

A total of 206 serum samples from children (3-14 years old) living in the Amador County (La Chorrera District, Province of Panama) were screened by indirect immunofluorescence antibody test (IFAT) for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR) followed by dot blot hybridization. The results indicated a prevalence of 9.7% for trypanosome infections, a seroprevalence of 2.9% against T. cruzi and a predominance of T. rangeli infection (6.8%). The immunological and clinical implications of these findings are discussed.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Detection of Rickettsia rickettsii in the tick Amblyomma cajennense in a new Brazilian spotted fever-endemic area in the state of Minas Gerais

The present study evaluated rickettsial infection in Amblyomma spp. ticks collected in a farm in Coronel Pacheco, a Brazilian spotted fever (BSF) endemic area. A total of 78 A. cajennense and 78 A. dubitatum free-living adult ticks were collected and tested by polymerase chain reaction (PCR) targeting a fragment of the rickettsial gene gltA. Only one pool of three A. cajennense ticks showed the expected product by PCR. This pool was further tested by PCR using sets of primers targeting the rickettsial genes gltA, ompA, and ompB. All reactions yielded the expected bands that by sequencing, showed 100% identity to the corresponding sequences of the Rickettsia rickettsii gene fragments gltA (1063-bp), ompA (457-bp), and ompB (720-bp). The minimal infection rate of R. rickettii in the A. cajennense population was 1.28% (at least one infected tick within 78 ticks).The present study showed molecular evidence for the presence of R. rickettsii in A. cajennense from a BSF-endemic area in Coronel Pacheco, state of Minas Gerais. Although R. rickettsii has been previously reported infecting A. cajennense ticks in Brazil and other Latin American countries, the present study performed the first molecular characterization of R. rickettsii from the tick A. cajennense.

Identification of human T-cell lymphotropic virus infection in a semi-isolated Afro-Brazilian quilombo located in the Marajó Island (Pará, Brazil)

Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5´LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5´LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.

A new challenge for malaria control in Brazil: asymptomatic Plasmodium infection - a review

The evolution of malaria in Brazil, its morbidity, the malaria control programs, and the new challenges for these programs in the light of the emergence of asymptomatic infection in the Amazon region of Brazil were reviewed. At least six Brazilian research groups have demonstrated that asymptomatic infection by Plasmodium is an important impediment to malaria control, among mineral prospectors in Mato Grosso and riverside communities in Rondônia and, in our group, in the middle and upper reaches of the Negro river, in the state of Amazonas. Likewise, other researchers have studied the problem among indigenous communities in the Colombian, Peruvian, and Venezuelan parts of the Amazon basin, adjacent to Brazil. The frequency of positive results from the polymerase chain reaction (PCR) among asymptomatic individuals has ranged from 20.4 to 49.5%, and the presence of Plasmodium in the thick blood smears, from 4.2 to 38.5%. Infection with Anopheles darlingi has also been demonstrated by xenodiagnosis among asymptomatic patients with positive PCR results. If a mean of 25% is taken for the asymptomatic infection caused by Plasmodium sp. in the Amazon region of Brazil, malaria control will be difficult to achieve in that region with the measures currently utilized for such control.