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1.
Life Sci Alliance ; 7(3)2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38158220

RESUMO

The malaria parasite Plasmodium vivax remains a major global public health challenge, and no vaccine is approved for use in humans. Here, we assessed whether P. vivax strain-transcendent immunity can be achieved by repeated infection in Aotus monkeys. Sterile immunity was achieved after two homologous infections, whereas subsequent heterologous challenge provided only partial protection. IgG levels based on P. vivax lysate ELISA and protein microarray increased with repeated infections and correlated with the level of homologous protection. Parasite transcriptional profiles provided no evidence of major antigenic switching upon homologous or heterologous challenge. However, we observed significant sequence diversity and transcriptional differences in the P. vivax core gene repertoire between the two strains used in the study, suggesting that partial protection upon heterologous challenge is due to molecular differences between strains rather than immune evasion by antigenic switching. Our study demonstrates that sterile immunity against P. vivax can be achieved by repeated homologous blood stage infection in Aotus monkeys, thus providing a benchmark to test the efficacy of candidate blood stage P. vivax malaria vaccines.


Assuntos
Vacinas Antimaláricas , Malária Vivax , Malária , Animais , Humanos , Malária Vivax/prevenção & controle , Malária Vivax/parasitologia , Aotidae , Haplorrinos
2.
Front Mol Biosci ; 9: 977113, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072435

RESUMO

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for low pH levels (pH < 6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Förster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the intermediate states visited during i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor to establish the time scale for the pH drop in a commonly used oxygen scavenging system.

3.
PLoS Negl Trop Dis ; 16(4): e0010327, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35394999

RESUMO

BACKGROUND: As the elimination of malaria in Mesoamerica progresses, detection of Plasmodium vivax using light microscopy (LM) becomes more difficult. Highly sensitive molecular tools have been developed to help determine the hidden reservoir of malaria transmission in low transmission settings. In this study we compare the performance of PvLAP5 and Pvs25 qRT-PCR assays to LM for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from malaria endemic regions of Panama. METHODS: For this purpose, we collected a total of 83 malaria field samples during 2017-2020 preserved in RNAprotect (RNAp) of which 63 (76%) were confirmed P. vivax by LM and selected for further analysis. Additionally, 16 blood samples from local healthy malaria smear negative volunteers, as well as, from 15 malaria naïve lab-bred Aotus monkeys were used as controls. To optimize the assays, we first determined the minimum blood volume sufficient for detection of PvLAP5 and Pv18SrRNA using P. vivax infected Aotus blood that was preserved in RNAp and kept either at ambient temperature for up to 8 days before freezing or was snap-frozen at -80° Celsius at the time of bleeding. We then compared the mean differences in gametocyte detection rates of both qRT-PCR assays to LM and performed a multivariate correlation analysis of study variables. Finally, we determined the sensitivity (Se) and specificity (Sp) of the assays at detecting gametocytes compared to LM. RESULTS: Blood volume optimization indicated that a blood volume of at least 60 µL was sufficient for detection of PvLAP5 and Pv18SrRNA and no significant differences were found between RNA storage conditions. Both PvLAP5 and Pvs25 qRT-PCR assays showed a 37-39% increase in gametocyte detection rate compared to LM respectively. Strong positive correlations were found between gametocytemia and parasitemia and both PvLAP5 and Pvs25 gametocyte markers. However, no significant differences were detected in the Se and Sp of the Pvs25 and PvLAP5 qRT-PCR assays, even though data from control samples suggested Pvs25 to be more abundant than PvLAP5. CONCLUSIONS: This study shows that the PvLAP5 qRT-PCR assay is as Se and Sp as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes in field samples preserved in RNAp at ambient temperature from malaria endemic regions of Panama. AUTHOR SUMMARY: Plasmodium vivax is one of the five species of malaria (P. falciparum, P. malariae, P. ovale and P. knowlesi) that are transmitted to man by the bite of female anopheles mosquitoes. It causes ~14.3 million cases mainly in Southeast Asia, India, the Western Pacific and the Americas annually. In the Americas, malaria remains a major problem in underdeveloped areas and indigenous communities in the Amazon region and eastern Panama, where it is endemic and difficult to eliminate. As malaria elimination progresses, detection of P. vivax by light microscopy (LM) becomes more difficult. Therefore, highly sensitive molecular tools have been developed that use genetic markers for the parasite to help determine the hidden reservoir of malaria transmission. This study compares the performance of two molecular assays based on the genetic markers of mature gametocytes PvLAP5 and Pvs25 with LM. The study shows that the PvLAP5 qRT-PCR assay is as sensitive and specific as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes. These data suggest that the PvLAP5 qRT-PCR assay can be a useful tool to help determine the hidden reservoir of transmission in endemic foci approaching elimination.


Assuntos
Malária Falciparum , Malária Vivax , Malária , Animais , Feminino , Marcadores Genéticos , Humanos , Malária Falciparum/epidemiologia , Malária Vivax/diagnóstico , Malária Vivax/epidemiologia , Malária Vivax/parasitologia , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura
4.
Malar J ; 19(1): 278, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32746814

RESUMO

BACKGROUND: Filtration of leukocytes (WBCs) is a standard practice of malaria ex vivo cultures. To date, few studies have considered the effect of filtration or the lack thereof on the survival of Plasmodium vivax ex vivo cultures through one cycle of maturation. This study investigates the effect of WBC filtration and culture media supplementation on the survival of 48-72 h ex vivo cultures. METHODS: Using parasitaemia density, the study compares the survival of Plasmodipur® filtered, filter-retained or washed ex vivo cultures, maintained with McCoy's5A medium supplemented with 25% serum alone or 20% in combination with 5% chemically defined lipid concentrate (CDLC), and in washed ex vivo cultures plus GlutaMAX™, benchmarked against IMDM™ or AIM-V™ media; also, assessed the survival of ex vivo cultures co-cultivated with human red blood cells (hRBCs). RESULTS: After 48 h of incubation a statistically significant difference was detected in the survival proportions of filtered and the filter-retained ex vivo cultures supplemented with serum plus CDLC (p = 0.0255), but not with serum alone (p = 0.1646). To corroborate these finding, parasitaemias of washed ex vivo cultures maintained with McCoy's5A complete medium were benchmarked against IMDM™ or AIM-V™ media; again, a statistically significant difference was detected in the cultures supplemented with CDLC and GlutaMAX™ (p = 0.03), but not when supplemented with either alone; revealing a pattern of McCoy's5A medium supplementation for Aotus-derived P. vivax cultures as follows: serum < serum + GlutaMAX™ < serum + CDLC < serum + CDLC + GlutaMAX™; confirming a key role of CDLC in combination with GlutaMAX™ in the enhanced survival observed. Lastly, results showed that co-cultivation with malaria-naïve hRBCs improved the survival of ex vivo cultures. CONCLUSIONS: This study demonstrates that WBC filtration is not essential for the survival of P. vivax ex vivo cultures. It also demonstrates that McCoy's5A complete medium improves the survival of Aotus-derived P. vivax ex vivo cultures, with no significant difference in survival compared to IMDM and AIM-V media. Finally, the study demonstrates that co-cultivation with hRBCs enhances the survival of ex vivo cultures. These findings are expected to help optimize seeding material for long-term P. vivax in vitro culture.


Assuntos
Aotidae , Técnicas de Cultura de Células/métodos , Leucócitos/fisiologia , Plasmodium vivax/fisiologia , Animais , Filtração , Lipídeos/química
5.
Artigo em Inglês | MEDLINE | ID: mdl-30245044

RESUMO

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a devastating and terminal disease in non-human primates (NHPs). Regular TB screenings using the intradermal tuberculin test (TST) have been the mainstay of TB surveillance and control in NHPs. Historically, Aotus monkeys have been considered less susceptible to TB than other NHPs. Here we present the diagnosis and epidemiology of a TB outbreak at The Gorgas Memorial Institute Aotus colony in Panama, and the results of two cross-sectional randomized TB screening studies, using antibody (Ab) and IFN-gamma release assay testing. RESULTS: Epidemiological and spatial analysis confirmed that the outbreak was the result of a continuing intermittent exposure, with human to monkey transmission as the most likely source. During the outbreak that lasted five months (January-June 2015), Mycobacterium kansassi and MTB were isolated from lung caseous granulomas in 1/7 and 3/7 TB suspicious animals respectively. Furthermore, MTB was detected by qRT-PCR in formalin fixed lung and liver granulomas in 2/7 and 1/6 monkeys respectively, suggesting an aerosol route of infection. Likewise, a random sample that included 63 / 313 adult (>2 year-old) monkeys, screened for latent TB with the Primagam® IFN-gamma release assay, between March-May, 2016, were all non-reactors; indicating that the outbreak was self-limiting and the colony was likely free or latent TB infection. Control measures included, quarantine, disinfection and TST screening of all personnel. In conclusion, this study demonstrates that Aotus are highly susceptible to TB, therefore, TB prevention measures should be strictly enforced in Aotus monkey colonies.


Assuntos
Aotidae , Surtos de Doenças , Doenças dos Macacos/epidemiologia , Mycobacterium tuberculosis/imunologia , Tuberculose/veterinária , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Estudos Transversais , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Surtos de Doenças/veterinária , Suscetibilidade a Doenças/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Humanos , Interferon gama/sangue , Interferon gama/imunologia , Interferon gama/metabolismo , Testes de Liberação de Interferon-gama/métodos , Testes de Liberação de Interferon-gama/veterinária , Masculino , Programas de Rastreamento/veterinária , Doenças dos Macacos/diagnóstico , Doenças dos Macacos/microbiologia , Mycobacterium tuberculosis/genética , Panamá/epidemiologia , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Teste Tuberculínico/veterinária , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/imunologia
6.
PLoS Negl Trop Dis ; 10(7): e0004870, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27463518

RESUMO

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.


Assuntos
Malária Vivax/parasitologia , Plasmodium vivax , Animais , Aotidae , Técnicas de Cultura de Células/métodos , Meios de Cultura , Feminino , Plasmodium vivax/classificação , Reticulócitos
7.
Space Weather ; Volume 14(Iss 8): 544-562, 2016 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31708717

RESUMO

This paper presents the Shock ARrival Model (SARM) for predicting shock arrival times for distances from 0.72 AU to 8.7 AU by using coronal mass ejections (CME) and flare data. SARM is an aerodynamic drag model described by a differential equation that has been calibrated with a dataset of 120 shocks observed from 1997 to 2010 by minimizing the mean absolute error (MAE), normalized to 1 AU. SARM should be used with CME data (radial, earthward or plane-of-sky speeds), and flare data (peak flux, duration, and location). In the case of 1 AU, the MAE and the median of absolute errors were 7.0 h and 5.0 h respectively, using the available CME/flare data. The best results for 1 AU (an MAE of 5.8 h) were obtained using both CME data, either radial or cone-model-estimated speeds, and flare data. For the prediction of shock arrivals at distances from 0.72 AU to 8.7 AU, the normalized MAE and the median were 7.1 h and 5.1 h respectively, using the available CME/flare data. SARM was also calibrated to be used with CME data alone or flare data alone, obtaining normalized MAE errors of 8.9 h and 8.6 h respectively for all shock events. The model verification was carried out with an additional dataset of 20 shocks observed from 2010 to 2012 with radial CME speeds to compare SARM with the empirical ESA model [Gopalswamy et al., 2005a] and the numerical MHD-based ENLIL model [Odstrcil et al., 2004]. The results show that the ENLIL's MAE was lower than the SARM's MAE, which was lower than the ESA's MAE. The SARM's best results were obtained when both flare and true CME speeds were used.

8.
IEEE Trans Pattern Anal Mach Intell ; 33(8): 1659-72, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21263164

RESUMO

A new algorithm for incremental construction of binary regression trees is presented. This algorithm, called SAIRT, adapts the induced model when facing data streams involving unknown dynamics, like gradual and abrupt function drift, changes in certain regions of the function, noise, and virtual drift. It also handles both symbolic and numeric attributes. The proposed algorithm can automatically adapt its internal parameters and model structure to obtain new patterns, depending on the current dynamics of the data stream. SAIRT can monitor the usefulness of nodes and can forget examples from selected regions, storing the remaining ones in local windows associated to the leaves of the tree. On these conditions, current regression methods need a careful configuration depending on the dynamics of the problem. Experimentation suggests that the proposed algorithm obtains better results than current algorithms when dealing with data streams that involve changes with different speeds, noise levels, sampling distribution of examples, and partial or complete changes of the underlying function.


Assuntos
Algoritmos , Análise de Regressão , Mineração de Dados , Bases de Dados Factuais , Aprendizado de Máquina
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