Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

Study of aged central auditory function using the auditory middle latency response.

OBJECTIVE: Investigate the auditory function of the elderly using the middle latency potentials. METHODOLOGY: Group 1 (G1): 20 healthy individuals of both genders, older than 60 years, without hearing loss. Group 2 (G2): 20 healthy individuals of both sexes, older than 60 years, with hearing loss in frequencies from 4 to 8 kHz. Potential recording was performed with unilateral and bilateral stimulation and the Binaural Interaction Component was calculated. RESULTS: Na latency in C3A1 was greater in the stimulation of the right ear in G2 and the amplitude of Na-Pa was greater in the stimulation of the right ear and recording in C3A1 in G1. The latency of the Pa component was higher in the stimulation of the right ear recorded in C4A2. The Pb component in G2 by bilateral stimulation and recorded in C4A2 had higher latency. The first and second negative and positive peaks presented greater amplitude in G1. In C3A1, the 1st negative peak was more negative in G1 and the 2nd positive peak showed greater amplitude in C4A2 in both groups. CONCLUSION: The transmission of auditory information to the primary auditory cortex is impaired with aging, especially in unilateral stimulation, reinforced by losses in elderly people with peripheral hearing loss, such as in the binaural interaction at the cortical and subcortical levels. Thus, the AMLR has shown to be a sensitive examination to investigate neuroauditory disorders in the elderly, especially related to high-frequency hearing loss and primary auditory cortex dysfunctions caused by the aging process.

Viral Coinfection of Children Hospitalized with Severe Acute Respiratory Infections during COVID-19 Pandemic.

The main pathogens of severe respiratory infection in children are respiratory viruses, and the current molecular technology allows for a rapid and simultaneous detection of a wide spectrum of these viral pathogens, facilitating the diagnosis and evaluation of viral coinfection. METHODS: This study was conducted between March 2020 and December 2021. All children admitted to the ICU with a diagnosis of SARI and who were tested by polymerase chain reaction on nasopharyngeal swabs for SARS-CoV-2 and other common respiratory viral pathogens were included in the study. RESULTS: The result of the viral panel identified 446 children, with one infected with a single virus and 160 co-infected with two or more viruses. This study employed descriptive analyses, where a total of twenty-two coinfections among SARI-causing viruses were identified. Thus, the five most frequent coinfections that were selected for the study are: hRV/SARS-CoV-2 (17.91%), hRV/RSV (14.18%), RSV/SARS-CoV-2 (12.69%), hRV/BoV (10.45%), and hRV/AdV (8.21%). The most significant age group was 38.1%, representing patients aged between 24 and 59 months (61 individuals). Patients older than 59 months represented a total of 27.5%, comprising forty-four patients. The use of oxygen therapy was statistically significant in coinfections with Bocavirus, other CoVs, Metapneumovirus, and RSV. Coinfections with SARS-CoV-2 and the other different coinfections presented a similar time of use of oxygen therapy with a value of (p > 0.05). In the year 2020, hRV/BoV was more frequent in relation to other types of coinfections, representing a total of 35.1%. The year 2021 presented a divergent profile, with hRV/SARS-CoV-2 coinfection being the most frequent (30.8%), followed by hRV/RSV (28.2%). Additionally, 25.6% and 15.4% represented coinfections between RSV/SARS-CoV-2 and hRV/AdV, respectively. We saw that two of the patients coinfected with hRV/SARS-CoV-2 died, representing 9.52% of all deaths in the study. In addition, both hRV/hBoV and hRV/RSV had death records for each case, representing 8.33% and 6.67% of all deaths, respectively. CONCLUSION: Coinfections with respiratory viruses, such as RSV and hBoV, can increase the severity of the disease in children with SARI who are admitted to the ICU, and children infected with SARS-CoV-2 have their clinical condition worsened when they have comorbidities.

Staphylococcus spp. Causatives of Infections and Carrier of blaZ, femA, and mecA Genes Associated with Resistance.

Staphylococcus spp. have been associated with cases of healthcare associated infections due to their high incidence in isolates from the hospital environment and their ability to cause infections in immunocompromised patients; synthesize biofilms on medical instruments, in the case of negative coagulase species; and change in genetic material, thus making it possible to disseminate genes that code for the acquisition of resistance mechanisms against the action of antibiotics. This study evaluated the presence of blaZ, femA, and mecA chromosomal and plasmid genes of Staphylococcus spp. using the qPCR technique. The results were associated with the phenotypic expression of resistance to oxacillin and penicillin G. We found that the chromosomal femA gene was present in a greater proportion in S. intermedius when compared with the other species analyzed, while the plasmid-borne mecA gene was prevalent in the S. aureus samples. The binary logistic regression performed to verify the association among the expression of the genes analyzed and the acquisition of resistance to oxacillin and penicillin G were not significant in any of the analyses, p > 0.05.

Study of aged central auditory function using the auditory middle latency response

Abstract Objective Investigate the auditory function of the elderly using the middle latency potentials. Methodology Group 1 (G1): 20 healthy individuals of both genders, older than 60 years, without hearing loss. Group 2 (G2): 20 healthy individuals of both sexes, older than 60 years, with hearing loss in frequencies from 4 to 8 kHz. Potential recording was performed with unilateral and bilateral stimulation and the Binaural Interaction Component was calculated. Results Na latency in C3A1 was greater in the stimulation of the right ear in G2 and the amplitude of Na-Pa was greater in the stimulation of the right ear and recording in C3A1 in G1. The latency of the Pa component was higher in the stimulation of the right ear recorded in C4A2. The Pb component in G2 by bilateral stimulation and recorded in C4A2 had higher latency. The first and second negative and positive peaks presented greater amplitude in G1. In C3A1, the 1st negative peak was more negative in G1 and the 2nd positive peak showed greater amplitude in C4A2 in both groups. Conclusion The transmission of auditory information to the primary auditory cortex is impaired with aging, especially in unilateral stimulation, reinforced by losses in elderly people with peripheral hearing loss, such as in the binaural interaction at the cortical and subcortical levels. Thus, the AMLR has shown to be a sensitive examination to investigate neuroauditory disorders in the elderly, especially related to high-frequency hearing loss and primary auditory cortex dysfunctions caused by the aging process.

Esforço auditivo e registro do controle parassimpático do coração durante o reconhecimento de sentenças: estudo piloto / Listening effort and recording of parasympathetic heart control during sentence recognition: Pilot study / Esfuerzo auditivo y registro del control parasimpático del corazón durante el reconocimiento de sentencias: Estudio piloto

Introdução: Medidas psicofisiológicas do sistema nervoso autônomo em resposta a tarefas auditivas podem fornecer um meio de quantificar os efeitos do esforço auditivo. Objetivo: Comparar a modulação parassimpática da frequência cardíaca em situações de escuta distintas e determinar se a variabilidade da frequência cardíaca (VFC) é um índice psicofisiológico sensível para mensurar o esforço auditivo. Método: Estudo piloto, observacional, transversal e prospectivo. Participaram 14 adultos normo-ouvintes, de ambos os sexos e com idade entre 18 e 30 anos. Estes responderam a anamnese e realizaram audiometria tonal limiar para verificação de limiares auditivos. Para o registro da VFC, colocou-se a cinta do cardiofrequencímetro na região do processo xifoide e este registro foi coletado por 10 minutos para três situações distintas: "repouso", "recuperação pós-reconhecimento de sentenças no silêncio" e "recuperação pós-reconhecimento de sentenças no ruído". O reconhecimento de sentenças foi realizado utilizando-se duas listas de sentenças, de forma randomizada, binaural, nas situações de escuta silêncio e na relação sinal/ruído de -5 dB e, também foi registrada VFC por um período de dois minutos. Aplicou-se o teste de ANOVA de medidas repetidas seguido pelo pós-teste de Bonferroni para comparar o índice de RMSSD nos diferentes momentos de registro da VFC. Resultados: A análise estatística demonstrou que não houve diferença significante do registro da VFC nos diferentes momentos, inclusive durante a tarefa de reconhecimento de sentenças no ruído, considerada a tarefa mais árdua. Conclusão: Para esta amostra, o registro da VFC não foi considerado um índice sensível para a mensuração do esforço auditivo.

Introduction: Psychophysiological measures of the autonomic nervous system in response to auditory tasks may provide a means of quantifying the effects of listening effort. Objective: To compare parasympathetic modulation of the heart rate in different listening situations and to determine if the heart rate variability is a sensitive psychophysiological index to measure listening effort. Methods: Pilot, observational, transversal and prospective study. 14 normal healthy young adults, both genders and between 18 and 30 years of age, participated in this study. They answered the anamnesis and performed pure tone audiometry to verify hearing thresholds. In order to record HRV, it was placed the cardiofrequency transducer tape in the xiphoid process region and this record was collected for 10 minutes for the "rest", "post-recovery of sentence recognition in quiet" and "post-recovery of sentence recognition in noise". Recognition task was performed using two lists of sentences presented in a randomized way, binaural, in the quiet listening condition and in a signal-to-noise ratio of -5 dB, and HRV was also recorded for a period of two minutes. ANOVA repeated measures test was applied followed by the Bonferroni posttest to compare the RMSSD index in the different HRV recording moments. Results: Statistical analysis showed that there was no significant difference in HRV recording at different moments, including the sentence recognition task in noise, considered the most arduous task. Conclusion: For this sample, the HRV recording was not considered a sensitive index for auditory effort measurement.

Introducción: Las medidas psicofisiológicas del sistema nervioso autónomo en respuesta a las tareas auditivas pueden proporcionar un medio para cuantificar los efectos del esfuerzo auditivo. Objetivo: Comparar la modulación parasimpática de la frecuencia cardíaca en situaciones de escucha distintas y determinar si la variabilidad de la frecuencia cardíaca (VFC) es un índice psicofisiológico sensible para medir el esfuerzo auditivo. Método: Estudio piloto, observacional, transversal y prospectivo. Participaron 14 adultos normo-oyentes, de ambos sexos y con edad entre 18 y 30 años. Estos respondieron la anamnesis y realizaron audiometría tonal umbral para verificación de umbrales auditivos. Para el registro de la VFC, se colocó la cinta del cardiofrecuencímetro en la región del proceso xifoide y este registro fue recolectado por 10 minutos para tres situaciones distintas "reposo", "recuperación post-reconocimiento de sentencias en el silencio" y "recuperación post-reconocimiento de las sentencias en el ruido ". El reconocimiento de sentencias se realizó utilizando dos listas de sentencias, de forma aleatorizada, binaural, en las situaciones de escucha silenciosa y en la relación señal / ruido de -5dB y, también se registró VFC por un período de dos minutos. Se aplicó la prueba de ANOVA de medidas repetidas seguida por el post-test de Bonferroni para comparar el índice de RMSSD en los diferentes momentos de registro de la VFC. Resultados: El análisis estadístico demostró que no hubo diferencia significante del registro de la VFC en los diferentes momentos, incluso durante la tarea de reconocimiento de sentencias en el ruido, considerada la tarea más ardua. Conclusión: Para esta muestra, el registro de la VFC no fue considerado un índice sensible para la medición del esfuerzo auditivo.

Proof of Concept for a Portable Platform for Molecular Diagnosis of Tropical Diseases: On-Chip Ready-to-Use Real-Time Quantitative PCR for Detection of Trypanosoma cruzi or Plasmodium spp.

Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic real-time quantitative PCR (qPCR) reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit use of molecular-based tests in low-resource scenarios. Herein, Trypanosoma cruzi or Plasmodium spp. DNA were detected with qPCR using commercial equipment (ABI7500 instrument) and a prototype platform comprising a portable device and a silicon chip, named Q3-Plus. In addition, a ready-to-use reaction format, where all qPCR reagents are stored on plate or on chip, was compared with the traditional freezer-stored format. No significant differences were observed in detecting T. cruzi or Plasmodium spp. DNA between thermocyclers, as well as between reagents' formats, for storage periods of up to 28 days (at 2°C to 8°C or 21°C to 23°C, respectively). When challenged with patients' samples, the Q3-Plus system performed as efficiently as the standard equipment for Plasmodium spp. DNA detection, showing it to be a valuable solution to malaria point-of-care diagnostics. Detection of T. cruzi DNA in chronic patients' samples using the Q3-Plus system yielded approximately 50% efficiency relative to the ABI7500. These results are essential to support future endeavors to bring molecular diagnostics to the point of care, where most needed.

Interaction Between Cortical Auditory Processing and Vagal Regulation of Heart Rate in Language Tasks: A Randomized, Prospective, Observational, Analytical and Cross-Sectional Study.

Cortical auditory evoked potentials (CAEP) throughout a language task is beneficial during psychophysiological evaluation to advance identification of language disorders. So as to better comprehend human communication and to provide additional elements for neuropsychological examinations we aimed to (1) examine the influence of language tasks on cortical auditory processing and vagal control of heart rate and (2) to verify a possible association between the parasympathetic cardiac regulation and cortical auditory processing in language tasks. This study was completed with 49 women. The subjects were separated into two groups: (1) phonological language tasks (N = 21) and (2) semantic (N = 21) language tasks. Heart rate variability (HRV) and CAEP were evaluated before and after the tests. HRV reduced (small effect size) and P3 wave latency increased after the phonological task. Identical variables were significantly correlated after the phonological task and linear regression indicated significant interaction between pNN50 (percentage of adjacent RR intervals with a difference of duration greater than 50 milliseconds) and P3 latency (16.9%). In conclusion, phonological language tasks slightly reduced parasympathetic control of HR and increased cognitive effort. The association between HRV and CAEP are anticipated to be involved in this mechanism.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Association between heart rhythm and cortical sound processing.

Sound signal processing signifies an important factor for human conscious communication and it may be assessed through cortical auditory evoked potentials (CAEP). Heart rate variability (HRV) provides information about heart rate autonomic regulation. We investigated the association between resting HRV and CAEP. We evaluated resting HRV in the time and frequency domain and the CAEP components. The subjects remained at rest for 10 minutes for HRV recording, then they performed the CAEP examinations through frequency and duration protocols in both ears. Linear regression indicated that the amplitude of the N2 wave of the CAEP in the left ear (not right ear) was significantly influenced by standard deviation of normal-to-normal RR-intervals (17.7%) and percentage of adjacent RR-intervals with a difference of duration greater than 50 milliseconds (25.3%) time domain HRV indices in the frequency protocol. In the duration protocol and in the left ear the latency of the P2 wave was significantly influenced by low (LF) (20.8%) and high frequency (HF) bands in normalized units (21%) and LF/HF ratio (22.4%) indices of HRV spectral analysis. The latency of the N2 wave was significantly influenced by LF (25.8%), HF (25.9%) and LF/HF (28.8%). In conclusion, we promote the supposition that resting heart rhythm is associated with thalamo-cortical, cortical-cortical and auditory cortex pathways involved with auditory processing in the right hemisphere.

Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.

Sera of chagasic patients react with antigens from the tomato parasite Phytomonas serpens.

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

Sera of Chagasic patients react with antigens from the tomato parasite Phytomonas serpens

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These fagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identifed, resulting in 22 different putative proteins. The identifed proteins were classifed into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

Gene expression profiling of macrophages following mice treatment with an immunomodulator medication.

Canova (CA) is a complex homeopathic medication used in diseases where the immune system is depressed. Previous studies demonstrated that it is neither toxic nor mutagenic and activates macrophages. We now evaluate CA effects on cytokine production and gene expression from mice macrophages. The global view of changes in expression of genes with known functions can provide a vivid picture of the way in which cell adapts to a changing environment or a challenge. We found a decrease in IL-2 and IL-4 production and a differential expression in 147 genes from CA group. These genes are mainly involved in transcription/translation, cell structure and dynamics, immune response, cytoprotection, enzymatic process, and receptors/ligands. With gene expression analysis we state that this medication provokes a reaction that involves alterations in gene expression profile mainly in the ones involved with macrophages activation, corroborating the laboratorial research and the clinical data.

Análise genômica funcional do Trypanosoma cruzi submetido a diferentes tipos de estresse / Functional genomic analysis of the Trypanosoma cruzi submitted the different types of stress

A diferenciação dos epimastigotas (formas replicativas e não-infectivas) em tripomastigotas metacíclicos (formas não-replicativa e infectivas) é o processo denominado metaciclogênese. Um evento essencial para a metaciclogênese, que pode ser mimetizado in vitro, é o estresse nutricional que os parasitas enfrentam nas porções posteriores do tubo digestivo do inseto vetor. Importantes mudanças no programa de expressão gênica ocorrem durante o estresse e desempenham um papel fundamental nas mudanças morfológicas e fisiológicas observadas durante a metaciclogênese. No entanto, poucas informações estão disponíveis em relação à capacidade e diferentes condições de estresse em disparar a metaciclogênese e, também, sobre os genes envolvidos na regulação dessas respostas. Para tanto, foi realizada uma análise comparativa das diferentes condições estresse (aumento temperatura, diminuição do pH e o estresse nutricional), em experimentos de metaciclogênese in vitro a fim de avaliar a capacidade de diferenciação do parasita diante de tais condições. Foi observado que todos os parasitas submetidos às condições de estresses são capazes de se diferenciar, entretanto, os parasitas provenientes dos estresses de pH e temperatura sofrem um retardo no disparo da metaciclogênese, porém esta diferença é minimizada até o final do processo. Para comparar as propriedades biológicas dos tripomastigotas metacíclicos originados de cada condição de estresse, foram feitos ensaios de infecção em células Vero e camundongos Swiss. Foi possível constatar que todos os ripomastigotas foram capazes de infectar as células Vero, assim como, infectar os camundongos. Para investigar a expressão dos genes foi utilizada a tecnologia de microarranjos. Foi selecionado um conjunto de 32 genes especificamente expressos nos tipos de estresse estudados. Para os genes selecionados foram realizadas análises quantitativas pela técnica de PCR em tempo real (RT-PCR) que validaram os dados de...epimastigotas.

Trypanosoma cruzi: cloning and characterization of two genes whose expression is up-regulated in metacyclic trypomastigotes.

The differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) involves the transformation of a replicative non-infective form of Trypanosoma cruzi into a non-replicative infective stage. The study of genes with stage-specific expression may provide insight into the mechanisms involved in the regulation of gene expression in this parasite. We cloned and characterized two genes whose expression is up-regulated in metacyclic trypomastigote, those encoding metacyclin-II (Met-II) and metacyclin-III (Met-III). Nucleotide sequence analysis identified no sequence similarity with sequences available from genetic databases. The deduced amino acid sequences of the genes indicated that Met-III encodes a basic polypeptide whereas Met-II encodes an acidic polypeptide. Northern and Western blot analyses showed that Met-II and Met-III were expressed by metacyclic trypomastigotes, but not by epimastigotes. Antisera directed against the recombinant Met-II and Met-III proteins recognized two polypeptides on Western blots: a 16-kDa and a 24-kDa polypeptide. Immunocytochemistry analysis using electron microscopy showed that metacyclin-II is localized mainly at the kinetoplast whereas metacyclin-III is localized at the nucleus of the parasite. Southern blot analysis, using genomic DNA and T. cruzi chromosomes separated by pulsed-field gel electrophoresis, indicated that these genes were present as single copies on different chromosomes of T. cruzi Dm28c.

Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis.

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis.