Molecular surveillance for anti-malarial drug resistance and genetic diversity of Plasmodium falciparum after chloroquine and sulfadoxine-pyrimethamine withdrawal in Quibdo, Colombia, 2018.

BACKGROUND: Resistance to anti-malarial drugs is associated with polymorphisms in target genes and surveillance for these molecular markers is important to detect the emergence of mutations associated with drug resistance and signal recovering sensitivity to anti-malarials previously used. METHODS: The presence of polymorphisms in genes associated with Plasmodium falciparum resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated by Sanger sequencing, in 85 P. falciparum day of enrollment samples from a therapeutic efficacy study of artemether-lumefantrine conducted in 2018-2019 in Quibdo, Colombia. Samples were genotyped to assess mutations in pfcrt (codons 72-76), pfdhfr (codons 51, 59, 108, and 164), and pfdhps genes (codons 436, 437, 540, and 581). Further, the genetic diversity of infections using seven neutral microsatellites (NMSs) (C2M34, C3M69, Poly α, TA1, TA109, 2490, and PfPK2) was assessed. RESULTS: All isolates carried mutant alleles for pfcrt (K76T and N75E), and for pfdhfr (N51I and S108N), while for pfdhps, mutations were observed only for codon A437G (32/73, 43.8%). Fifty samples (58.8%) showed a complete neutral microsatellites (NMS) profile. The low mean number of alleles (2 ± 0.57) per locus and mean expected heterozygosity (0.17 ± 0.03) showed a reduced genetic diversity. NMS multilocus genotypes (MMG) were built and nine MMG were identified. CONCLUSIONS: Overall, these findings confirm the fixation of chloroquine and pyrimethamine-resistant alleles already described in the literature, implying that these drugs are not currently appropriate for use in Colombia. In contrast, mutations in the pfdhps gene were only observed at codon 437, an indication that full resistance to sulfadoxine has not been achieved in Choco. MMGs found matched the clonal lineage E variant 1 previously reported in northwestern Colombia.

Concordance analysis of three diagnostic tests for malaria in the symptomatic population of Colombian endemic municipalities / [Análisis de concordancia de tres pruebas para el diagnóstico de malaria en la población sintomática de los municipios endémicos de Colombia].

Introduction: Taking into account the difficulty of performing malaria microscopic diagnosis in rural areas, rapid diagnostic tests (RDT) are a good alternative, but it is important to verify their diagnostic performance. Objective: To evaluate the diagnostic performance of the RDTs used in five Colombian departments by comparing them with the microscopic diagnosis and using PCR as the reference standard. Materials and methods: Thick blood film and RDTs were used to diagnose symptomatic individuals; additionally, the filter paper was impregnated with blood for the molecular test. Results: We included 314 samples whose percentage of positivity for malaria was 49% by PCR, 48% by microscopy and 46% by RDT; parasitemia ranged between 180 and 23,800 p/µL of blood. The concordance of the results from the microscopy units and those of the PCR (National Laboratory of Reference) was as follows: Cohen's kappa coefficient, 0.975 (95% CI: 0.950-0.999); sensitivity, 97% (95% CI 95-100); specificity 100% (95% CI: 100-100), and kappa index of species, 0.958 (IC95%: 0.912-1.00). The concordance between the Pf/Pv RDT (at the microscopy units) and the PCR (National Laboratory of Reference) was as follows: kappa coefficient, 0.878 (95% CI: 0.784-0.973); sensitivity, 94% (95% CI: 87-100); specificity, 95% (95% CI: 90-100), and kappa index of species, 1.0 (95% CI: 1.00-1.00). The concordance between the Pf/Pan RDT versus PCR was: Cohen's kappa coefficient, 0.920 (95 % CI: 0.865- 0.974); sensitivity, 94% (95% CI: 90-98); specificity, 99% (95% CI 95-100), and kappa index of species, 0.750 (IC95% 0,637-0,863). Conclusion: The results of this study support the use of RDTs in Colombia; however, more training of the personnel is required to accurately differentiate Plasmodium species.

Introducción. Dadas las dificultades del diagnóstico microscópico de la malaria o paludismo en las áreas rurales, las pruebas de diagnóstico rápido constituyen una buena alternativa, por lo que es importante conocer su desempeño. Objetivo. Evaluar el desempeño de las pruebas de diagnóstico rápido utilizadas en cinco departamentos para al diagnóstico microscópico de la malaria usando la reacción en cadena de la polimerasa (PCR) como estándar de referencia. Materiales y métodos. Se usaron la prueba de gota gruesa y las pruebas de diagnóstico rápido y, además, se impregnó papel de filtro con sangre para la prueba molecular (PCR), en individuos sintomáticos. Resultados. Se incluyeron 314 muestras cuyo porcentaje de positividad para malaria fue de 49 % con la PCR, de 48 % con microscopía y de 46 % con las pruebas de diagnóstico rápido; la parasitemia fluctuó entre 180 y 23.800 parásitos/µl de sangre. La concordancia de los resultados de los puestos de microscopía comparados con la PCR (Laboratorio Nacional de Referencia) fueron los siguientes: coeficiente kappa de Cohen de 0,975 (IC95% 0,950-0,999), sensibilidad de 97 % (IC95% 95-100) y especificidad de 100 % (IC95% 100-100), e índice kappa de especie de 0,958 (IC95% 0,912-1,00). La concordancia de los resultados de la prueba de diagnóstico rápido Pf/Pv en los puestos de microscopía y los de la PCR (Laboratorio Nacional de Referencia), fue la siguiente: coeficiente kappa de 0,878 (IC95% 0,784-0,973), sensibilidad de 94 % (IC95% 87-100), especificidad de 95 % (IC95% 90-100), e índice kappa de especie de 1,0 (IC95% 1,00-1,00). La concordancia entre la prueba de diagnóstico rápido Pf/Pan y la PCR fue la siguiente: coeficiente kappa de Cohen de 0,920 (IC95% 0,865-0,974), sensibilidad de 94 % (IC95% 90-98), especificidad de 99 % (IC95% 95-100), e índice kappa de especie de 0,750 (IC95% 0,637-0,863). Conclusión. Los resultados de este estudio respaldan el uso de las pruebas de diagnóstico rápido en Colombia, aunque se requiere un mejor entrenamiento del personal para diferenciar eficientemente las especies de Plasmodium.

Artemether-Lumefantrine Efficacy for the Treatment of Uncomplicated Plasmodium falciparum Malaria in Choco, Colombia after 8 Years as First-Line Treatment.

Artemether-lumefantrine (AL) is the first-line treatment for uncomplicated Plasmodium falciparum infection in Colombia. To assess AL efficacy for uncomplicated falciparum malaria in Quibdo, Choco, Colombia, we conducted a 28-day therapeutic efficacy study (TES) following the WHO guidelines. From July 2018 to February 2019, febrile patients aged 5-65 years with microscopy-confirmed P. falciparum mono-infection and asexual parasite density of 250-100,000 parasites/µL were enrolled and treated with a supervised 3-day course of AL. The primary endpoint was adequate clinical and parasitological response (ACPR) on day 28. We attempted to use polymerase chain reaction (PCR) genotyping to differentiate reinfection and recrudescence, and conducted genetic testing for antimalarial resistance-associated genes. Eighty-eight patients consented and were enrolled; four were lost to follow-up or missed treatment doses. Therefore, 84 (95.5%) participants reached a valid endpoint: treatment failure or ACPR. No patient remained microscopy positive for malaria on day 3, evidence of delayed parasite clearance and artemisinin resistance. One patient had recurrent infection (12 parasites/µL) on day 28. Uncorrected ACPR rate was 98.8% (83/84) (95% CI: 93.5-100%). The recurrent infection sample did not amplify during molecular testing, giving a PCR-corrected ACPR of 100% (83/83) (95% CI: 95.7-100%). No P. falciparum kelch 13 polymorphisms associated with artemisinin resistance were identified. Our results support high AL efficacy for falciparum malaria in Choco. Because of the time required to conduct TESs in low-endemic settings, it is important to consider complementary alternatives to monitor antimalarial efficacy and resistance.

Polymorphism in the Plasmodium vivax msp 3: gene in field samples from Tierralta, Colombia.

We evaluated the Plasmodium vivax polymorphism by studying the Pvmsp-3alpha gene's polymorphic region by PCR-RFLP in 55 samples from patients living in Tierralta, Colombia. Three different sizes of the Pvmsp-3 alpha gene were found, type A (1,900 bp), type B (1,500 bp) and type C (1,100 bp); most of the samples were type A (96.4 %). The Pvmsp-3alpha gene exhibited high polymorphism. Seven restriction patterns were found when using Alu I, and nine were found with Hha I; 12 different alleles were obtained when these patterns were combined. The findings suggest that this gene could be used in Colombia as a molecular epidemiologic marker for genotyping P. vivax.

Asymptomatic Plasmodium spp. infection in Tierralta, Colombia.

With the aim of determining the prevalence of asymptomatic Plasmodium spp. infection by thick smear and PCR and its association with demographic and epidemiological characteristics in the village of Nuevo Tay, Tierralta, Córdoba, Colombia, a cross-sectional population study was carried out, using random probabilistic sampling. Venous blood samples were taken from 212 people on day 0 for thick smear and PCR. Clinical follow-up and thick smears were carried out on days 14 and 28. The prevalence of Plasmodium spp. infection was 17.9% (38/212; 95% CI: 12.5-23.3%) and the prevalence of asymptomatic Plasmodiumspp. infection was 14.6% (31/212; 95% CI: 9.6-19.6%). Plasmodium vivax was found more frequently (20/31; 64.5%) than Plasmodium falciparum (9/31; 29%) and mixed infections (2/31; 6.5%). A significantly higher prevalence of asymptomatic infection was found in men (19.30%) than in women (9.18%) (prevalence ratio: 2.10; 95% CI: 1.01-4.34%; p = 0.02). People who developed symptoms had a significantly higher parasitemia on day 0 than those who remained asymptomatic, of 1,881.5 +/- 3,759 versus 79 +/- 106.9 (p = 0.008). PCR detected 50% more infections than the thick smears. The presence of asymptomatic Plasmodium spp. infection highlights the importance of carrying out active searches amongst asymptomatic populations residing in endemic areas.

[Efficacy of the amodiaquine+sulfadoxine-pyrimethamine combination and of chloroquine for the treatment of malaria in Córdoba, Colombia, 2006]. / Eficacia de la combinación amodiaquina más sulfadoxina-pirimetamina y de la cloroquina para el tratamiento del paludismo en Córdoba, Colombia, 2006.

INTRODUCTION: The decrease in the efficacy of antimalarial drugs in the world and in Colombia hampers its control. OBJECTIVE: The in vivo therapeutic efficacy of the amodiaquine+sulfadoxine-pyrimethamine combination was evaluated in the treatment of uncomplicated Plasmodium falciparum malaria and of chloroquine for P. vivax malaria. MATERIALS AND METHODS: From May to November 2006, in vivo efficacy studies of malaria treatments were undertaken in Tierralta, Córdoba, northeastern Colombia. Standard protocols were followed as recommended by the World Health Organization/Panamerican Health Organization, with some modifications. Patients older than two years with single P. falciparum or P. vivax infection, with asexual parasitemia between 500 and 50,000 parasites/microl, were selected according to established inclusion and exclusion criteria. Supervised treatment was administered, and clinical and parasitological follow-up was carried out on days 0 (inclusion), 1, 2, 3, 7, 14, 21 and 28. The outcome was defined as adequate clinical and parasitological response, early therapeutic failure, or late therapeutic failure. RESULTS: Of 53 subjects selected, 50 (94.3%; CI 70%-100%) presented adequate clinical and parasitological response to the amodiaquine+sulfadoxine-pyrimethamine treatment for uncomplicated falciparum malaria. One patient presented early therapeutic failure, and two developed late therapeutic failure. All of the 50 patients (95%CI: 74%-100%) in the invivo efficacy study of chloroquine for vivax malaria presented adequate clinical and parasitological response. CONCLUSION: In Cordoba, the amodiaquine+sulfadoxine-pyrimethamine combination and chloroquine show a high efficacy for the treatment of uncomplicated falciparum and vivax malaria, respectively.

[Clinical profiles of patients with uncomplicated Plasmodum falciparum malaria in northwestern Colombia]. / Perfiles clínicos del paludismo no complicado por Plasmodium falciparum en Córdoba, Colombia.

INTRODUCTION: Few studies describe the clinical presentations of uncomplicated Plasmodium falciparum malaria in the province of Córdoba in an endemic area of northwestern Colombia. OBJECTIVE: Profiles of patients with uncomplicated Plasmodium falciparum malaria were described from two twons of Córdoba, Tierrata and Puerto Libertador, based on clinical, epidemiological and laboratory variables. MATERIALS AND METHODS: Patients were examined according to standard WHO/PAHO protocols for assessment of antimalarial drug efficacy. Clinical data and parasitological information was collected as well. A multiple correspondence multivariate analysis was used to compare the profiles of 127 patients with uncomplicated Plasmodium falciparum malaria. RESULTS: Of the 127 patients,105 completed the 14-day follow-up and 7 had adequate clinical response. Between 80% and 98% of patients exhibited at least one of the most frequent symptoms of uncomplicated malaria, and 80.3% had asthenia as the most frequent symptom. The multivariate analysis grouped the variables into five distinguishable clusters of clinical profiles. These groups showed similarities with the classical clinical descriptions of uncomplicated malaria encountered in the literature. The low frequency of patients with adequate clinical response hampered the association analysis. CONCLUSIONS: In Córdoba, therapeutic failure to chloroquine treatment is high in treating uncomplicated Plasmodium falciparum malaria. Multivariate analysis summarized variables related to epidemiological and clinical aspects and permitted a more objective approach to the interpretation of the findings.

Baseline in vivo, ex vivo and molecular responses of Plasmodium falciparum to artemether and lumefantrine in three endemic zones for malaria in Colombia.

Background: Colombia began using artemisinin-based combination therapies for the treatment of uncomplicated Plasmodium falciparum malaria in 2006. It is necessary to implement resistance surveillance to antimalarial drugs in order to promptly detect changes in parasite susceptibility. The aim of this study was to establish a susceptibility baseline of P. falciparum to artemether-lumefantrine using three monitoring tools. Methods: Patients with uncomplicated malaria treated with artemether-lumefantrine underwent clinical and parasitological follow-up over 28 days. Ex vivo test was performed using the microtest technique for chloroquine, arthemeter, dihydroartemisinin and lumefantrine. Pfmdr1 copy number and polymorphisms in Pfk13, Pfatp6, Pfcrt and Pfmdr1 genes were analyzed. Results: From a total of 150 screened patients, 49 completed follow-up for 28 days. All treated patients had adequate clinical and parasitological responses. Parasitic clearance showed a drastic reduction of parasite biomass at 24 hours and complete elimination at 48 hours. One hundred eleven isolates were processed, all exhibited high susceptibility to artemisinins and a slight decrease in susceptibility to lumefantrine. No genetic polymorphisms associated with resistance to artemisinin were found. Conclusion: This study generated a susceptibility baseline in response to therapy with Coartem (artemether-lumefantrine) with numerical reference values, which will allow data comparison with future studies to systematically monitor changes in the parasite and to provide an early alert to the health authorities.

[Genotyping of the Plasmodium falciparum msp1 (block 2) and dhfr (codon 108) genes in field samples collected in four endemic Colombian localities]. / Genotipificación de los genes msp1 (bloque 2) y dhfr (codón108) de Plasmodium falciparum en muestras de campo recolectadas en cuatro localidades endémicas de Colombia.

INTRODUCTION: Plasmodium falciparum is a highly polymorphic parasite, which allows it to evade the host's immune response, spread drug resistance and favours transmission. OBJECTIVES: To analyse the genetic diversity of P. falciparum populations in samples from four endemic localities in Colombia. MATERIALS AND METHODS: 123 blood samples were collected on filter paper from patients with non-complicated P. falciparum malaria during 2002 to 2004. The samples were genotyped using polymerase chain reaction with specific primers for the polymorphic region of block 2 of the msp1 gene and the 108 codon of the dhfr gene. RESULTS: In msp1 block 2, 95.9% (118/123; 95% CI: 90.8-98.7) of the samples harboured MAD20; 6.5% K1 (8/123; 95% CI: 2.8-12.4) and 2.4% RO33 (3/123; 95% CI: 0.5-6.9). For the dhfrgene the mutant allele N 108 was found in all the samples amplified, T 108 in 3.2% and the wild type S108 in 34.1%. Taking together all the results from both genes, 61.8% (76/123; 95% CI: 52.6-70.4) of the samples were simple infections and 38.2% (47/123; 95% CI: 29.6-47.4) were mixed infections. MAD20/N108-S108 (30.1%) was the most frequent combination among the latter. CONCLUSIONS: Simple infections, i.e, a single allelic type in each one of the genes studied, prevailed among the circulating parasite populations. In this study the genetic composition of P. falciparum parasite populations was very homogeneous.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

INTRODUCTION: The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  OBJECTIVE: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  MATERIALS AND METHODS: The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  RESULTS: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  CONCLUSION: The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Análisis de concordancia de tres pruebas para el diagnóstico de malaria en la población sintomática de los municipios endémicos de Colombia / Concordance analysis of three diagnostic tests for malaria in the symptomatic population of Colombian endemic municipalities

Introducción. Dadas las dificultades del diagnóstico microscópico de la malaria o paludismo en las áreas rurales, las pruebas de diagnóstico rápido constituyen una buena alternativa, por lo que es importante conocer su desempeño. Objetivo. Evaluar el desempeño de las pruebas de diagnóstico rápido utilizadas en cinco departamentos para al diagnóstico microscópico de la malaria usando la reacción en cadena de la polimerasa (PCR) como estándar de referencia. Materiales y métodos. Se usaron la prueba de gota gruesa y las pruebas de diagnóstico rápido y, además, se impregnó papel de filtro con sangre para la prueba molecular (PCR), en individuos sintomáticos. Resultados. Se incluyeron 314 muestras cuyo porcentaje de positividad para malaria fue de 49 % con la PCR, de 48 % con microscopía y de 46 % con las pruebas de diagnóstico rápido; la parasitemia fluctuó entre 180 y 23.800 parásitos/pl de sangre. La concordancia de los resultados de los puestos de microscopía comparados con la PCR (Laboratorio Nacional de Referencia) fueron los siguientes: coeficiente kappa de Cohen de 0,975 (IC95% 0,950-0,999), sensibilidad de 97 % (IC95% 95-100) y especificidad de 100 % (IC95% 100-100), e índice kappa de especie de 0,958 (IC95% 0,912-1,00). La concordancia de los resultados de la prueba de diagnóstico rápido Pf/Pv en los puestos de microscopía y los de la PCR (Laboratorio Nacional de Referencia), fue la siguiente: coeficiente kappa de 0,878 (IC95% 0,784-0,973), sensibilidad de 94 % (IC95% 87-100), especificidad de 95 % (IC95% 90-100), e índice kappa de especie de 1,0 (IC95% 1,00-1,00). La concordancia entre la prueba de diagnóstico rápido Pf/Pan y la PCR fue la siguiente: coeficiente kappa de Cohen de 0,920 (IC95% 0,865-0,974), sensibilidad de 94 % (IC95% 90-98), especificidad de 99 % (IC95% 95-100), e índice kappa de especie de 0,750 (IC95% 0,637-0,863). Conclusión. Los resultados de este estudio respaldan el uso de las pruebas de diagnóstico rápido en Colombia, aunque se requiere un mejor entrenamiento del personal para diferenciar eficientemente las especies de Plasmodium.

Introduction: Taking into account the difficulty of performing malaria microscopic diagnosis in rural areas, rapid diagnostic tests (RDT) are a good alternative, but it is important to verify their diagnostic performance. Objective: To evaluate the diagnostic performance of the RDTs used in five Colombian departments by comparing them with the microscopic diagnosis and using PCR as the reference standard. Materials and methods: Thick blood film and RDTs were used to diagnose symptomatic individuals; additionally, the filter paper was impregnated with blood for the molecular test. Results: We included 314 samples whose percentage of positivity for malaria was 49% by PCR, 48% by microscopy and 46% by RDT; parasitemia ranged between 180 and 23,800 p/µl of blood. The concordance of the results from the microscopy units and those of the PCR (National Laboratory of Reference) was as follows: Cohen's kappa coefficient, 0.975 (95% CI: 0.9500.999); sensitivity, 97% (95% CI 95-100); specificity 100% (95% CI: 100-100), and kappa index of species, 0.958 (IC95%: 0.912-1.00). The concordance between the Pf/Pv RDT (at the microscopy units) and the PCR (National Laboratory of Reference) was as follows: kappa coefficient, 0.878 (95% CI: 0.784-0.973); sensitivity, 94% (95% CI: 87-100); specificity, 95% (95% CI: 90-100), and kappa index of species, 1.0 (95% CI: 1.00-1.00). The concordance between the Pf/Pan RDT versus PCR was: Cohen's kappa coefficient, 0.920 (95 % CI: 0.865- 0.974); sensitivity, 94% (95% CI: 90-98); specificity, 99% (95% CI 95-100), and kappa index of species, 0.750 (IC95% 0,637-0,863). Conclusion: The results of this study support the use of RDTs in Colombia; however, more training of the personnel is required to accurately differentiate Plasmodium species.

Comparison of asymptomatic Plasmodium spp. infection in two malaria-endemic Colombian locations.

BACKGROUND: Asymptomatic infection by Plasmodium spp. could help explain the dynamics of malaria transmission in low-transmission regions. We therefore compared the prevalence of asymptomatic infection by Plasmodium spp. in two Colombian locations, Tierralta and Tumaco, with different transmission patterns, different ecological and epidemiological characteristics and potentially different associated factors. METHOD: Two cross-sectional studies were carried out between September 2006 and November 2007. Infection by Plasmodium spp. was determined using polymerase chain reaction (PCR) and microscopy. RESULTS: Prevalence of asymptomatic infection at day 0 in Tierralta was 11.3% (95% CI 7.2-16.8) by microscopy and 16.5% (95% CI 11.5-22.9) by PCR, while in Tumaco, values were 2.4% (95% CI 0.7-5.5) and 5.8% (95% CI 2.3-9.2) respectively. General prevalence of asymptomatic plasmodium infection after 28 days of follow-up was 5% (95% CI 3.2-7.7), 6.6% (95% CI 3.8-10.6%) in Tierralta and 3.4% (95% CI 1.5-6.6%) in Tumaco. Plasmodium vivax was predominant in Tierralta, P. falciparum in Tumaco. The main associated factors were male sex (aOR 2.5; 95% CI 1.0-6.7) and two to five previous malaria episodes (aOR 3.0; 95% CI 1.0-9.4). PCR detected 61% more infections than microscopy. CONCLUSION: Measurement of the prevalence of asymptomatic Plasmodium spp. infection is suggested as part of the epidemiologic evaluation of malaria in low-transmission areas such as Colombia.

Haplotypes associated with resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum in two malaria endemic locations in Colombia.

Colombia has four main malaria transmission zones. In vivo efficacy studies carried out in these areas showed big differences in the response of Plasmodium falciparum to treatment with sulphadoxine-pyrimethamine. In addition, there is still insufficient information about the genetics of P. falciparum populations. The objective of this study was to determine the haplotypes in dhfr and dhps genes of P. falciparum circulating in two distinct endemic zones. Samples from patients with non-complicated P. falciparum malaria were collected: 135 from Tumaco and 206 from Tierralta. Alleles 108 and 51 of the dhfr gene, and 437 and 540 of the dhps gene were analyzed by PCR/enzymatic restriction, while alleles 59 and 164 (dhfr), and 581(dhps) by PCR/dot blot/hybridization. Five different haplotypes were found, of which the triple mutant 51I/C59/108N/I164/437G/K540/A581 was the most frequent (54.6%). In Tumaco, the parasites with wild haplotype predominated, while mutant parasites predominated in Tierralta. Another interesting finding is the presence of the C59R mutation in the dhfr gene in two samples, a mutation rarely found in South America. These data provide information about parasite population genetics and highlight the importance of starting a long term molecular surveillance program.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli / Producción de proteínas recombinantes de Plasmodium falciparum en Escherichia coli

Introduction: The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum . However, the proteins of P . falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies. Objective: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8). Materials and methods: The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice. Results: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli , compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies. Conclusion: The use of genetically modified strains of E . coli was essential to achieve high expression levels of the four evaluated P . falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P . falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Introducción. La producción de proteínas recombinantes es fundamental para el estudio funcional de las proteínas de Plasmodium falciparum . Sin embargo, las proteínas recombinantes de P . falciparum están entre las más difíciles de expresar y, cuando lo hacen, usualmente se agregan dentro de cuerpos de inclusión insolubles. Objetivo. Evaluar la producción de cuatro proteínas de P. falciparum usando como sistema de expresión dos cepas de Escherichia coli genéticamente modificadas para favorecer la producción de proteínas heterólogas y establecer una reserva de proteínas recombinantes puras y solubles, y producir anticuerpos policlonales a partir de ellas. Materiales y métodos. Las proteínas recombinantes, las cuales correspondían a secuencias parciales de PfMyoA (Miosina-A) y PfGAP50 (proteína-asociada a glideosoma de 50 kDa) y a las secuencias completas de PfMTIP (proteína de interacción con miosina-A) y PfGAP45 (proteína asociada a glideosoma de 45 kDa), fueron expresadas como proteínas de fusión con la glutatión S-transferasa y luego purificadas y usadas para producir anticuerpos policlonales en ratón. Resultados. La expresión de las proteínas recombinantes fue mucho más eficiente en la cepa BL21-CodonPlus (la cual expresa tRNAs escasos en las bacterias silvestres), que en la cepa BL21-pG-KJE8. Por el contrario, aunque la cepa BL21-pG-KJE sobreexpresa chaperonas, no redujo la formación de cuerpos de inclusión. Conclusión. El uso de cepas de E . coli genéticamente modificadas fue fundamental para alcanzar altos niveles de expresión de las cuatro proteínas recombinantes evaluadas y permitió obtener dos de ellas en forma soluble. La estrategia utilizada permitió expresar cuatro proteínas recombinantes de P . falciparum en cantidad suficiente para inmunizar ratones y producir anticuerpos policlonales y, además, conservar proteína pura y soluble de dos de ellas para ensayos futuros.

[Genotypic survey of Plasmodium falciparum based on the msp1, msp2 and glurp genes by multiplex PCR]. / Genotipificación de Plasmodium falciparum por PCR múltiple por medio de los genes msp1, msp2 y glurp, en cuatro localidades de Colombia.

INTRODUCTION: The genetic diversity of Plasmodium falciparum has been one of the major obstacles for the success of anti-malaria drug therapy. It provides the parasite an ability to evade the host's immune response by generating changes in its antigenic composition and resistance to antimalarial drugs. OBJECTIVE: The genetic diversity of P.falciparum was characterized in 4 Colombian localities through the analysis of polymorphic genes. MATERIALS AND METHODS: Eighty-one samples were obtained from patients with uncomplicated P. falciparum malaria and screened for polymorphic variants of msp1, msp2 (merozoite surface proteins) and glurp (glutamate-rich protein) with a multiplex PCR assay. The geographic regions sampled were Tierralta (Córdoba), in northwestern Colombia and in the Orinoco river watershed of eastern Colombia--Inírida (Guainía), La Carpa (Guaviare), and Casuarito (Vichada). RESULTS: The MAD20 variant was detected in all samples analyzed for the msp1 gene. For the msp2 gene, the IC allelic family was found in 96.3% of the samples as compared to 4.9% of the samples with the FC family. Both families showed size polymorphism with bands between 467 and 513 basepairs (bp) for IC and 286 and 300 bp for FC. PCR products of differing sizes were detected for the glurp gene and grouped into 5 size classes: I (600-699 bp) 2.5%, II (700-799 bp) 19.8%, III (800-899 bp) 72.8%, IV (900-999 bp) 1.2% and V (1000-1099 bp) 3.7%. CONCLUSIONS: The msp1 molecular marker did not provide information for differentiating P. falciparum parasite populations. The msp2 gene was more suitable for studying the genetic diversity, however, further studies are required to identify polymorphisms within the two allelic families. The glurp gene showed a great genetic diversity of circulating P. falciparum populations, and suggested that this gene may be useful for distinguishing between recrudescence and reinfection.

[Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum from three endemic malaria regions in Colombia]. / Mutaciones puntuales en los genes dhfr y dhps de Plasmodium falciparum de tres regiones endémicas para malaria en Colombia.

INTRODUCTION: Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure. OBJECTIVES: To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba. MATERIALS AND METHODS: Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism. RESULTS: The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito. CONCLUSIONS: The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.

Genotipificación de plasmodium falciparum por PCR múltiple por medio de los genes msp1, msp2 y glurp, en cuatro localidades de Colombia / Genotypic survery of plasmodium falciparum based on the msp1, msp2 and glurp genes by multiplex PCR

Introducción. La diversidad genética de Plasmodium falciparum constituye un obstáculo para el éxito de la terapia antipalúdica, pues le permite al parásito evadir la respuesta inmunitaria del huésped, lo cual genera cambios en su composición antigénica y resistencia a los medicamentos antipalúdicos.Objetivo. Estudiar la diversidad genética de P. falciparum procedente de cuatro localidades colombianas mediante el análisis de los genes polimórficos msp1, msp2 y glurp. Materiales y métodos. Se genotipificaron 81 muestras mediante PCR múltiple de pacientes infectados con P. falciparum, procedentes de Tierralta (Córdoba), Inírida (Guainía), La Carpa (Guaviare) y Casuarito (Vichada).Resultados. Para el gen msp1 se detectó MAD20 en todas las muestras analizadas. Para el gen msp2 se halló con mayor frecuencia la familia alélica IC (96,3%) comparada con FC (4,9%). En ambas familias se evidenció polimorfismo de tamaño, y se encontraron bandas en un rango entre 467 y 513 pares de bases (pb) para IC y entre 286 y 300 pb para FC. Para el gen glurp se detectaron diferentes tamaños de productos de PCR, los cuales se agruparon en cinco genotipos: I (600-699 pb) 2,5%, II (700-799 pb) 19,8%, III (800-899 pb) 72,8%, IV (900-999 pb) 1,2% y V (1.000-1.099 pb) 3,7%.Conclusiones. Nuestros resultados demuestran que el marcador molecular msp1 no proporciona información útil para diferenciar las poblaciones parasitarias de P. falciparum. El gen msp2 es apropiado para evaluar la diversidad genética, pero requiere ensayos más finos que permitan diferenciar claramente el polimorfismo de tamaño en las dos familias. Los resultados obtenidos con el gen glurp evidenciaron una gran diversidad genética en las poblaciones de P. falciparum que circulan en el país y sugieren que este gen puede ser útil para diferenciar nuevas infecciones de infecciones recurrentes o recrudecimientos.

Introduction. The genetic diversity of Plasmodium falciparum has been one of the major obstacles for the success of anti-malaria drug therapy. It provides the parasite an ability to evade the host’s immune response by generating changes in its antigenic composition and resistance to antimalarial drugs.Objective. The genetic diversity of P.falciparum was characterized in 4 Colombian localities through the analysis of polymorphic genes. Materials and methods. Eighty-one samples were obtained from patients with uncomplicated P. falciparum malaria and screened for polymorphic variants of msp1, msp2 (merozoite surface proteins) and glurp (glutamate-rich protein) with a multiplex PCR assay. The geographic regions sampled were Tierralta (Córdoba), in northwestern Colombia and in the Orinoco river watershed of eastern Colombia-- Inírida (Guainía), La Carpa (Guaviare), and Casuarito (Vichada). Results. The MAD20 variant was detected in all samples analyzed for the msp1 gene. For the msp 2 gene, the IC allelic family was found in 96.3% of the samples as compared to 4.9% of the samples with the FC family. Both families showed size polymorphism with bands between 467 and 513 basepairs (bp) for IC and 286 and 300 bp for FC. PCR products of differing sizes were detected for the glurp gene and grouped into 5 size classes: I (600-699 bp) 2.5%, II (700-799 bp) 19.8%, III (800-899 bp) 72.8%, IV (900-999 bp) 1.2% and V (1000-1099 bp) 3.7%.Conclusions. The msp1 molecular marker did not provide information for differentiating P. falciparum parasite populations. The msp 2 gene was more suitable for studying the genetic diversity, however, further studies are required to identify polymorphisms within the two allelic families. The glurp gene showed a great genetic diversity of circulating P. falciparum populations, and suggested that this gene may be useful for distinguishing between recrudescence and reinfection.

Mutaciones puntuales en los genes dhfr y dhps de Plasmodium falciparum de tres regiones endémicas para malaria en Colombia / Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum from three endemic malaria regions in Colombia

Introducción. La acumulación progresiva de mutaciones en los genes dhfr y dhps lleva al parásito Plasmodium falciparum a evadir la acción de la sulfadoxina-pirimetamina, situación que aumenta el nivel de resistencia del parásito a estos medicamentos y conlleva a la aparición de fallas del tratamiento. Objetivos. Determinar la frecuencia de mutaciones en los genes dhfr y dhps de P. falciparum asociadas con resistencia a sulfadoxina-pirimetamina, en muestras de pacientes de tres zonas endémicas de Colombia: La Carpa, Guaviare; Casuarito, Vichada; Tierralta y Puerto Libertador, Córdoba. Materiales y métodos. Se incluyeron 40 muestras de pacientes con malaria no complicada por P. falciparum. Los alelos 108, 59 y 164 del gen dhfr se analizaron mediante PCR específica de alelo y los alelos 51 del gen dhfr y 436, 437 y 540 del gen dhps por PCR y restricción enzimática. Resultados. En el gen dhfr encontramos en todas las muestras las mutaciones asparagina 108 e isoleucina 51. No se detectaron alelos mutantes en los codones 59 y 164 del gen dhfr, ni en el codón 436 del gen dhps. La mutación glicina 437 estuvo presente en 36 muestras y el alelo silvestre alanina en tres de Tierralta y una de La Carpa. La mutación ácido glutámico 540 sólo se halló en Casuarito. Conclusiones. En las poblaciones de P. falciparum analizadas prevalecen los alelos asparagina 108, isoleucina 51 y glicina 437, lo que indica un efecto acumulativo de mutaciones y la necesidad de vigilar la aparición de nuevos alelos mutantes que puedan conducir a la pérdida total de la eficacia de la sulfadoxina-pirimetamina.

Introduction. Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure. Objectives. To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba. Materials and methods. Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism. Results. The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito. Conclusions. The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.

Polymorphism in the Plasmodium vivax msp 3: gene in field samples from Tierralta, Colombia

We evaluated the Plasmodium vivax polymorphism by studying the Pvmsp-3 gene's polymorphic region by PCR-RFLP in 55 samples from patients living in Tierralta, Colombia. Three different sizes of the Pvmsp-3 gene were found, type A (1,900 bp), type B (1,500 bp) and type C (1,100 bp); most of the samples were type A (96.4 percent). The Pvmsp-3 gene exhibited high polymorphism. Seven restriction patterns were found when using Alu I, and nine were found with Hha I; 12 different alleles were obtained when these patterns were combined. The findings suggest that this gene could be used in Colombia as a molecular epidemiologic marker for genotyping P. vivax.

Eficacia de la combinación amodiaquina más sulfadoxina-pirimetamina y de la cloroquina para el tratamiento del paludismo en Córdoba, Colombia, 2006 / Efficacy of the amodiaquine+sulfadoxine-pyrimethamine combination and of chloroquine for the treatment of malaria in Córdoba, Colombia, 2006

Introducción. La disminución de la eficacia de los medicamentos antipalúdicos en el mundo y en Colombia, dificulta el control de la enfermedad. Objetivo. Evaluar la eficacia terapéutica in vivo de la combinación amodiaquina más sulfadoxina-pirimetamina para el tratamiento del paludismo no complicado por Plasmodium falciparum y de la cloroquina para el tratamiento del paludismo por P. vivax en Tierralta, Córdoba. Materiales y métodos. Durante el período de mayo a noviembre de 2006, se realizaron estudios de eficacia in vivo siguiendo los protocolos estandarizados por la Organización Mundial de la Salud y la Organización Panamericana de la Salud, con algunas modificaciones. Se estudiaron pacientes mayores de dos años, con parasitemia entre 500 y 50.000 formas asexuales/µl, seleccionados conforme a los criterios de inclusión y exclusión previamente definidos. Se administró tratamiento supervisado y se realizó seguimiento clínico y parasitológico en los días 0 (inclusión), 1, 2, 3, 7, 14, 21 y 28. El desenlace se definió como respuesta clínica y parasitológica adecuada, fracaso terapéutico precoz o fracaso tardío al tratamiento. Resultados. De los pacientes evaluados, 50/53 (94,3 por ciento) (IC95 por ciento: 70 por ciento-100 por ciento) presentaron respuesta clínica y parasitológica adecuada al tratamiento con amodiaquina más sulfadoxina-pirimetamina para paludismo no complicado por P. falciparum, un paciente presentó fracaso terapéutico precoz y dos presentaron fracaso terapéutico tardío. Los 50 pacientes evaluados (100 por ciento) (IC95 por ciento: 74 por ciento-100 por ciento) presentaron respuesta clínica y parasitológica adecuada al tratamiento con cloroquina para el paludismo por P. vivax. Conclusiones. En Córdoba, la combinación amodiaquina más sulfadoxina-pirimetamina y la cloroquina son eficaces para el tratamiento del paludismo no complicado por P. falciparum y por P. vivax, respectivamente.