RESUMO
The human malaria-Aotus monkey model has served the malaria research community since its inception in 1966 at the Gorgas Memorial Laboratory (GML) in Panama. Spanning over five decades, this model has been instrumental in evaluating the in vivo efficacy and pharmacokinetics of a wide array of candidate antimalarial drugs, whether used singly or in combination. The animal model could be infected with drug-resistant and susceptible Plasmodium falciparum and Plasmodium vivax strains that follow a characteristic and reproducible course of infection, remarkably like human untreated and treated infections. Over the years, the model has enabled the evaluation of several synthetic and semisynthetic endoperoxides, for instance, artelinic acid, artesunate, artemether, arteether, and artemisone. These compounds have been evaluated alone and in combination with long-acting partner drugs, commonly referred to as artemisinin-based combination therapies, which are recommended as first-line treatment against uncomplicated malaria. Further, the model has also supported the evaluation of the primaquine analog tafenoquine against blood stages of P. vivax, contributing to its progression to clinical trials and eventual approval. Besides, the P. falciparum/Aotus model at GML has also played a pivotal role in exploring the biology, immunology, and pathogenesis of malaria and in the characterization of drug-resistant P. falciparum and P. vivax strains. This minireview offers a historical overview of the most significant contributions made by the Panamanian owl monkey (Aotus lemurinus lemurinus) to malaria chemotherapy research.
Assuntos
Antimaláricos , Artemisininas , Modelos Animais de Doenças , Animais , Antimaláricos/uso terapêutico , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Artemisininas/uso terapêutico , Artemisininas/farmacologia , Humanos , Panamá , Aotidae , Plasmodium falciparum/efeitos dos fármacos , Malária/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Artesunato/uso terapêutico , Artesunato/farmacologia , Artesunato/farmacocinética , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , História do Século XX , AminoquinolinasRESUMO
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 , TemperaturaRESUMO
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/químicaRESUMO
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/imunologiaRESUMO
Chloroquine-resistant (CQR) vivax malaria has emerged as a threat to the malaria elimination agenda. The objective of this study was to assess if a combination of chloroquine (CQ) and prochlorperazine was able to reverse CQ resistance of the Plasmodium vivax AMRU-1 strain from Papua New Guinea in infected Aotus monkeys. For this purpose, in two independent experimental drug efficacy trials, a total of 18 Aotus monkeys infected with blood obtained from donor animals were randomly assigned to treatment and control groups and orally administered CQ at 10 mg/kg or prochlorperazine at 20 mg/kg, alone or in combination, for five consecutive days. Reversal of CQR was achieved in animals that received the drug combination, whereas neither drug alone produced cures. This same drug combination reverses CQR in P. falciparum and could be an alternative for treatment in humans with chloroquine-resistant P. vivax infections.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Haplorrinos/microbiologia , Malária Vivax/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Animais , Resistência a Medicamentos/efeitos dos fármacos , Feminino , Malária Falciparum/tratamento farmacológico , Malária Vivax/microbiologia , Masculino , Papua Nova Guiné , Plasmodium falciparum/efeitos dos fármacosRESUMO
BACKGROUND: Identification of risk factors is important for the establishment of malaria elimination programmes tailored to specific regions. Type of house construction had been associated with increasing risk of acquiring malaria. This study aimed at establishing the association between determinants of low socio-economic status (SES) and type of house construction with the likelihood of living in a Plasmodium vivax malarious corregimiento (smallest political division) in Panama during 2009-2012. METHODS: To determine the association between type-2 houses (build with deciduous materials) and other determinants of low SES, with living in a malarious corregimiento, this study analyzed demographic and housing census data (2010), and malaria incidence aggregated at the corregimiento level (2009-2012), using a Spearman's non-parametric correlation test to explore for associations, followed by a case-control study and a reduced multivariate logistic regression approach for confirmation. RESULTS: A descriptive temporal and spatial analysis indicated that P. vivax in Panama was associated with Amerindian reservations. Moreover, this study demonstrated that a strong correlation (deleterious effect) existed between living in a malarious corregimiento and being exposed to a type-2 house (OR = > 1.0) (p < 0.001), while, it showed an inverse correlation for exposure to type-1 houses (protective effect) (build with permanent materials) (OR = < 1.0) (p < 0.001). In the same way, a significant association between exposure to type-2 houses and the outcome of living in a malarious corregimiento was found using a case-control study approach (Chi(2) test = p < 0.001), that was confirmed applying a reduced multivariate logistic regression fitted model. CONCLUSIONS: This study demonstrated that living in a P. vivax malarious corregimiento in Panama during 2009-2012 was strongly correlated with those corregimientos having a high proportion of type-2 houses. A multivariate logistic regression approach at the house and corregimiento level indicated a strong association of type-2 houses, dirt floors and illiteracy with the likelihood of living in a malarious corregimiento. It is expected that these findings will help implement a multi-sectorial approach for the elimination of malaria in poor areas of Panama where malaria is endemic, which emphasizes house improvements such as mosquito-proofing and socio-economic development.
Assuntos
Malária Vivax/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Habitação , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Panamá/epidemiologia , Fatores de Risco , Fatores Socioeconômicos , Adulto JovemRESUMO
Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination. Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas where malaria has been eliminated.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Surtos de Doenças , Resistência a Medicamentos/genética , Malária Falciparum/epidemiologia , Plasmodium falciparum/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , Colômbia , Código de Barras de DNA Taxonômico , Feminino , Loci Gênicos/genética , Haplótipos , Humanos , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Panamá/epidemiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Adulto JovemRESUMO
BACKGROUND: The long-term effect of a PVC pipe nest-box on the reproductive efficiency and other life traits of an Aotus monkey-breeding colony have not been characterized. METHODS AND RESULTS: We analyzed laboratory records of the Gorgas Memorial Institute (GMI) Aotus monkey colony in Panama for the period 1999-2010 and found a 273% increase in the annual mean life births in the following 7 years after the introduction of a PVC pipe nest-box in 2002, as well as increases in the mean body mass and survival of laboratory-bred monkeys. Other life traits such as inter-birth interval, parity, birth sex distribution, mortality, and longevity were also determined. CONCLUSIONS: The use of a PVC pipe nest-box significantly improved the reproductive efficiency and other life traits of the GMI Aotus breeding colony.
Assuntos
Criação de Animais Domésticos/métodos , Aotidae/fisiologia , Reprodução , Animais , Aotidae/crescimento & desenvolvimento , Causas de Morte , Modelos Animais de Doenças , Feminino , Malária , Masculino , Panamá , Dinâmica PopulacionalRESUMO
A multidrug-resistant (MDR) clone of Plasmodium falciparum (C2A) from Thailand was adapted through serial passage to Aotus monkeys. During adaptation, the parasite showed resistance to a single 20 or 40 mg/kg oral dose of mefloquine (MQ). Infection was only cured when MQ was administered orally at 40 mg/kg once in combination with intravenous artesunic acid at 20 mg/kg for 3 days. Similarly, the parasite clone was found to be resistant to quinine, failing at 20 mg/kg orally for 5 days in combination with an experimental dihydrofolate reductase (DHFR) inhibitor (WR297608) at 10, 20, or 40 mg/kg orally for 3 days, and with atovaquone/proguanil at 25 mg/kg for 3 days. This new model will allow in vivo testing of new antimalarial compounds or their combinations against a currently circulating MDR P. falciparum strain.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Adaptação Fisiológica , Animais , Aotidae , Resistência a Múltiplos Medicamentos , Feminino , Malária Falciparum/parasitologia , Masculino , Parasitemia , Tailândia , Fatores de TempoRESUMO
Artemisone (single oral dose, 10 mg/kg of body weight) cured nonimmune Aotus monkeys of their Plasmodium falciparum infections when combined with mefloquine (single oral dose, 5 and 10 mg/kg but not 2.5 mg/kg). In combination with amodiaquine (20 mg/kg/day), artemisone (10 mg/kg/day) given orally for 3 days cured all infected monkeys. Three days of treatment with artemisone (30 mg/kg/day) and clindamycin (100 mg/kg/day) was also curative.
Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/fisiologia , Amodiaquina/uso terapêutico , Animais , Aotus trivirgatus , Quimioterapia Combinada , Mefloquina/uso terapêutico , Testes de Sensibilidade Parasitária , Resultado do TratamentoRESUMO
Panama is the first country in the Central American region that has officially discarded chloroquine as a first-line drug to treat Plasmodium falciparum cases. Here we describe the clinical and molecular findings from autochthonous P. falciparum fatal cases, and the epidemiological situation that led to a change in the national antimalarial drug policy. Our results illustrate the potential pathogenicity of the strain of P. falciparum circulating in the country and provide molecular evidence of parasite resistance to chloroquine and antifolate drugs. The public health threats of these findings for the Central American region are discussed.
Assuntos
Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Adulto , Animais , Pré-Escolar , DNA de Protozoário/análise , Evolução Fatal , Feminino , Genótipo , Política de Saúde/legislação & jurisprudência , Humanos , Masculino , Mutação/genética , Panamá , Plasmodium falciparum/genética , GravidezRESUMO
During routine antimalarial drug efficacy trials, we observed, for the first time, severe thrombocytopenia developing in Aotus monkeys infected with Plasmodium vivax. Data obtained from 26 Aotus infected with the AMRU-1 strain showed that 77% developed severe thrombocytopenia, whereas only 15% had severe anemia, with hemorrhagic diathesis ensuing in 31%. In general, thrombocytopenic monkeys either failed primary treatment with experimental antimalarial drugs or were found to have higher-density parasitemias, longer patency duration, and lower hematocrits. In these monkeys, severe thrombocytopenia inversely correlated to parasitemia (R = -1.0), and animals that received a blood transfusion had significantly higher platelet counts (P < 0.05) by day 38 after inoculation. In conclusion, the AMRU-1 strain of P. vivax, was considered to be highly pathogenic to Aotus monkeys, and thrombocytopenia rather than anemia should be regarded an early indicator of drug treatment failure with this strain.
Assuntos
Antimaláricos/administração & dosagem , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Plasmodium vivax/patogenicidade , Anemia/sangue , Animais , Aotus trivirgatus , Modelos Animais de Doenças , Resistência a Medicamentos , Feminino , Malária Vivax/sangue , Masculino , Parasitemia/sangue , Índice de Gravidade de Doença , Trombocitopenia/sangueRESUMO
A molecular epidemiology study was conducted to determine the distribution of antimalarial drug resistance alleles among field isolates of Plasmodium falciparum. Samples were obtained during an epidemic affecting Kuna Amerindians in Panama. A high prevalence of mutations associated with chloroquine, pyrimethamine, and sulfadoxine was observed. Genotype analysis of msp2 revealed a low genetic diversity of P. falciparum parasites circulating in the studied area. The public health implications of these findings for the Central American region are discussed.