Performance of OptiMAL® in the diagnosis of Plasmodium vivax and Plasmodium falciparum infections in a malaria referral center in Colombia

Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL®, is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL® in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL® for both sets of patients was 96.9%. Efficiency was higher for P. vivax (98.1%) than for P. falciparum (94.9%). These results corroborate the diagnostic utility of OptiMAL® in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility.

Morphological and polymerase chain reaction-restriction fragment lenght polymorphism characterization of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia

In Colombia, five Biomphalaria planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B. canonica and B. oligoza(var. B. philippiana). Among them, B. straminea is intermediate host of Schistosoma mansoni and B. peregrina has been found to be experimentally susceptible to this parasite. B. straminea is commonly confused with B. kuhniana and they have been clustered together with B. intermedia in the complex named B. straminea. The difficulties involved in the specific identification, based on morphological data, have motivated the use of new techniques as auxiliary tools in cases of inconclusive morphological identification of such planorbid. In the present study, five Biomphalaria populations from the Colombian Amazon region and from Interandian Valleys were morphologically identified and characterized by polymerase chain reaction-restriction fragment lenght polymorphism directed at the internal transcribed spacer region of the rRNA gene, followed by digestion of the generated fragment with restriction enzymes (DdeI, AluI, RsaI, MvaI and HaeIII). Known profiles of the Brazilian species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B. amazonica, besides B. kuhniana from Colombia, were used for comparison. The five populations under study were morphologically and molecularly identified as B. kuhniana and B. amazonica.

IS6110 fingerprinting of sensitive and resistant strains (1991-1992) of Mycobacterium tuberculosis in Colombia

The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 ± 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.

Morphometric discrimination of females of five species of Anopheles of the subgenus Nyssorhynchus from Southern and Northwest Colombia

The most important vectors of human Plasmodium in the neotropics belong to the subgenus Nyssorhynchus. These species are generally sympatric in terms of their geographical distributions. Some are difficult to identify based solely on examination of adult females using the available morphological keys, in these cases examination of immature stages and male genitalia is required to make correct determinations. However, in epidemiological studies it is necessary to identify the species of adult females which are found near humans, i.e. in studies of malaria transmission or evaluation of control measures. The purpose of the present study was to evaluate the discrimination of adult females of different species of Nyssorhynchus isolated mainly from Southern Colombia (department of Putumayo), using morphometric analysis. Adult females were obtained after rearing larvae collected in natural breeding places and from the progeny of females collected on humans. The morphological characteristics of the immature stages allowed the identification of four species of the subgroup Oswaldoi from Southern Colombia: Anopheles rangeli Gabaldon, Cova Garcia & Lopez, An. oswaldoi (Peryassu), An. benarrochi Gabaldon, Cova Garcia & Lopez and An. triannulatus (Neiva & Pinto). The species An. nuneztovari (Gabaldon) from the Northwest of Colombia was included for comparison. Morphometric analysis allowed differentiation of the females of all species to a confidence level approaching 90% using principal components analysis of 10 wing and leg variables, followed by canonical variate analysis of the first four principal components. We conclude that morphometrics may represent a useful taxonomic tool for this group and that its use should be further studied.

Effects of chloroquine and sulfadoxine/pyrimethamine on gametocytes in patients with uncomplicated Plasmodium falciparum malaria in Colombia

The effect of antimalarials on gametocytes can influence transmission and the spread of drug resistance. In order to further understand this relationship, we determined the proportion of gametocyte carriers over time post-treatment in patients with uncomplicated Plasmodium falciparum malaria who were treated with either chloroquine (CQ) or sulfadoxine/pyrimethamine (SP). The overall proportion of gametocyte carriers was high (85%) and not statistically significantly different between the CQ and SP treatment groups. However, an increased risk of carrying gametocytes on day 14 of follow up (1.26 95% CI 1.10-1.45) was found among patients having therapeutic failure to CQ compared with patients having an adequate therapeutic response. This finding confirms and extends reports of increased risk of gametocytaemia among CQ resistant P. falciparum.

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and Northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001). The variation among individuals within populations (phiST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Sequence and phylogenetic analysis of human T cell lymphotropic virus type 1 from Tumaco, Colombia

Human T cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes leukemia and the neurological disorder HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP). Infection with this virus - although it is distributed worldwide - is limited to certain endemic areas of the world. Despite its specific distribution and slow mutation rate, molecular epidemiology on this virus has been useful to follow the movements of human populations and routes of virus spread to different continents. In the present study, we analyzed the genetic variability of a region of the env gene of isolates obtained from individuals of African origin that live on the Pacific coast of Colombia. Sequencing and comparison of the fragment with the same fragment from different HTLV-1 isolates showed a variability ranging from 0.8% to 1.2%. Phylogenetic studies permit us to include these isolates in the transcontinental subgroup A in which samples isolated from Brazil and Chile are also found. Further analyses will be necessary to determine if these isolates were recently introduced into the American continent or if they rather correspond to isolates introduced during the Paleolithic period.

Isozyme analysis of Taenia solium isolates from Mexico and Colombia

Mexican and Colombian Taenia solium cysticerci and some species of Taenia adults were assayed using cellulose acetate electrophoresis to distinguish between isolates. Isozyme patterns for ARK, GOT, G3PD, GPI, and MPI were identical in all cysticerci suggesting homozygotic profiles. G6PD and MDH showed different patterns between Mexican and Colombian cysticerci, suggesting regional differences. ME activity was mainly detected in the adult stage suggesting that this enzyme is active in anaerobic environment, while MDH, detected in cysticerci, could be related to an environment that contains oxygen. Finally, the species of taeniid adults analyzed showed different patterns among them.

Feeding sources and natural infection of Belminus herreri (Hemiptera, Reduviidae, Triatominae) from dwellings in Cesar, Colombia

Belminus herreri, originally described from specimens collected in Panama, was considered entirely silvatic until to 2000 when it was found for the first time in a domestic habitat in Colombia. In 2001, during a new search of houses in the Department of Cesar, Colombia, 121 specimens were collected. Study of their feeding sources using an ELISA test revealed that 96% of these specimens had fed on cockroaches (Blattidae). However, a small proportion of these B. herreri specimens also showed the presence of Trypanosoma cruzi in their gut contents, suggesting a possible role for these insects in the epidemiology of Chagas disease.

Prevalence of blood parasites in Tyrannidae (flycatchers) in the Eastern plains of Colombia

Blood samples from 159 birds of the New-world family Tyrannidae (the flycatchers) from the eastern plains of Colombia, were examined for haematozoa parasites, in 1999-2000. Haematozoa were detected in six of 20 species. The overall prevalence was 10.1%. The most common parasites detected were microfilariae, followed by Trypanosoma and Plasmodium. The highest prevalence (9.6%) was found in the Ochre-bellied Flycatcher (Mionectes oleaginea). Mixed infections with more than one genus of blood parasite were rare and most infections encountered were of low intensity. The results of this study suggest an important role of ecologically diverse conditions determining composition, transmission, and prevalence of a blood parasite fauna, presumably through host interaction population density. Some new host parasite relationship records are presented.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Infection by Mycobacterium leprae of household contacts of lepromatous leprosy patients from a post-elimination leprosy region of Colombia

The Leprosy Control Program of Antioquia, (post-elimination leprosy state of Colombia), had registered by 1999, 56 lepromatous leprosy patients and their household contacts (HHC). Our interest was to detect Mycobacterium leprae infection in these HHC. Clinical examination, acid-fast bacillary staining (AFB) in nasal secretions, and slit skin samples, IgM anti-PGL-I in serum and Lepromine A (Mitsuda) reactivity were tested. Two hundred forty eight HHC were studied, 49% were male. After clinical examination, two HHC were diagnosed as multi bacillary patients; 13% showed positive IgM anti-PGL-I titers; Mitsuda reaction (> 4 mm) was positive in 59%; AFB was negative in all samples, except in the two new patients. HHC were classified according to test results.Group 1: two new multi bacillary patients. Group 2: 15 HHC seropositive, Mitsuda-negative. Group 3: 13 HHC seropositive, Mitsuda-positive. Group 4: 130 HHC seronegative, Mitsuda-positive. Group 5: 88 HHC seronegative, Mitsuda-negative. These results are an indication that the transmission of the infection is still happening in a region considered in the post elimination phase. The two new patients represent an infection source for others contacts, and groups 2 and 3 are infected HHC that could develop the disease in future. Follow up of high risk population is necessary to achieve real elimination of leprosy.

Analysis of antenal sensilla patterns of Rhodnius prolixus from Colombia and Venezuela

Antennal sensilla patterns were used to analyze population variation of domestic Rhodnius prolixus from six departments and states representing three biogeographical regions of Colombia and Venezuela. Discriminant analysis of the patterns of mechanoreceptors and of three types of chemoreceptors on the pedicel and flagellar segments showed clear differentiation between R. prolixus populations east and west of the Andean Cordillera. The distribution of thick and thin-walled trichoids on the second flagellar segment also showed correlation with latitude, but this was not seen in the patterns of other sensilla. The results of the sensilla patterns appear to be reflecting biogeographic features or population isolation rather than characters associated with different habitats and lend support to the idea that domestic R. prolixus originated in the eastern region of the Andes.

Incrimination of Anopheles (Nyssorhynchus) rangeli and An. (Nys.) oswaldoi as natural vectors of Plasmodium vivax in Southern Colombia

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.