RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Técnicas de Biología Molecular en el desarrollo de la investigación. Revisión de la literatura / Molecular Biology Techniques for research development. A literature review

troducción: Epidemiología etimológicamente significa ciencia que estudia enfermedades que afecta a las comunidades; esta ha venido evolucionando a través de los siglos describiendo y explicando la dinámica de la salud poblacional; ha integrado nuevas ramas, como la epidemiología molecular definida como una disciplina en la cual se implementa técnicas moleculares para aportes científicos, de investigación y clínico. Objetivo: Presentar las técnicas con fundamento en biología molecular, que han aportado al desarrollo de la investigación. Material y Métodos: Se realizó revisión de artículos científicos durante los meses de agosto a octubre de 2016 y julio a septiembre de 2017, en inglés, portugués, francés y español en revistas científicas Pubmed, Scielo, Biomed Central, Free Medical Journals, LILACS, Redalyc, Inbiomed, Dialnet, usando términos DeCs descriptores de Ciencias de la Salud y MeSH; se emplearon artículos publicados en el período de 2012 a 2017, usando publicaciones de años anteriores como aporte a la historia del tema. Resultados: se presentan 05 técnicas de Biología molecular que han aportado a la investigación: RCP, Secuenciación, Hibridación de sondas de ADN, RAPD y RFLP. Conclusiones: Hoy en día el uso técnicas moleculares permite el estudio de genoma completo o secuencias específicas de ADN cortas o largas con el fin de detectar y analizar secuencias de interés para la investigación en las ciencias agronómicas, forenses, diagnóstico clínico e investigación básica, traslacional y aplicada; cada una de ellas se caracteriza por la confiabilidad y rapidez en la obtención del resultado, robustez, especificidad, sensibilidad y flexibilidad, comparado con métodos fenotípicos(AU)

Introduction: Epidemiology, from the etymological point of view, means science that studies the diseases that affect the communities. It has been developing through centuries, describing and explaining the dynamics of population health; it has integrated new branches such as molecular epidemiology defined as a discipline in which molecular techniques are implemented for clinical, research, and scientific contributions. Objective: To present techniques with basis in molecular biology, which have contributed to research development. Material and methods: A review of scientific articles was made during the months of August-October of 2016, and July-September, 2017 in English, Portuguese, French, and Spanish languages, in scientific journals such as Pubmed, Scielo, Biomed Central, Free Medical Journals, LILACS, Redalyc, Inbiomed, and Dialnet, using DeCs term descriptors of Health Sciences, and the MeSH descriptor; articles published during the time period from 2012-2017 were used, and publications of previous years were also taken into consideration as a contribution to the history of this topic. Results: 05 techniques of molecular biology which have contributed to research development were presented: PCR, sequencing, hybridization with DNA probes, RAPD, and RFLP. Conclusions: At present, the use of molecular techniques allows the complete genome or short and long sequences of DNA with the aim of detecting and analyzing sequences of interest for research in agronomy and forensic sciences, clinical diagnosis and basic, translational, and applied research, each of them characterized by reliability and quickness in obtaining result, strength, specificity, sensitivity, and flexibility, compared to phenotypic methods(AU)

Tipificación con oligonucleótidos espaciadores de Mycobacterium tuberculosis en Cuba / Spacer oligonucleotide typing of Mycobacterium tuberculosis in Cuba

Introducción: el conocimiento de los linajes de Mycobacterium tuberculosis es importante para entender el origen, evolución y propagación de la bacteria. Objetivo: determinar los patrones genéticos de M. tuberculosis circulantes en Cuba. Métodos: estudio descriptivo de corte transversal con un componente analítico en Cuba, en el período comprendido de enero de 2009 a diciembre de 2010. Se aplicó la tipificación con oligonucleótidos espaciadores (Spoligotyping) a 308 aislamientos de M. tuberculosis del período 2009-2010. La clasificación en genotipos se realizó según la base de datos internacional SpolDB4. Los resultados se analizaron además con la herramienta web en línea MIRU-VNTRplus y se compararon con los patrones genéticos de M. tuberculosis identificados en 1993-1995 en Cuba. Resultados: se definieron 79 patrones genotípicos diferentes, de los cuales 46 (62 por ciento) fueron patrones no reportados anteriormente en SpolDB4. Los 22 agrupamientos definidos incluyeron al 75,4 por ciento de los aislamientos estudiados. Se encontraron cinco familias genéticas fundamentales: Beijing (25,6 por ciento), S (19,2 por ciento), LAM (16,9 por ciento), Haarlem (16,9 por ciento) y T (5,8 por ciento). La familia S prevaleció en la región Occidental (OR=3,4; 95 por ciento IC:1,8-6,3; p<0,05), Beijing en el Centro de Cuba (OR=6,7; 95 por ciento IC:3,7-11,9; p<0,05) y LAM (OR=3,0; 95 por ciento IC:1,6-5,6; p<0.05) y Haarlem en la zona Oriental (OR=1,8; 95 por ciento IC:1,0-3,4; p<0,05). Conclusiones: se observó una gran diversidad genética entre los aislamientos de M. tuberculosis circulante en Cuba en 2009-2010. En el país, la estructura genética de la población de M. tuberculosis ha variado en el tiempo con una disminución de genotipos endémicos como Haarlem y T y un aumento significativo de S y Beijing. Estos datos aportan elementos importantes para la epidemiología y control de la TB en Cuba(AU)

Introduction: knowledge about Mycobacterium tuberculosis lineages is important to understand the origin, evolution and spread of this bacterium. Objective: determine the genetic patterns of M. tuberculosis circulating in Cuba. Methods: adescriptive cross-sectional study was conducted with an analytical component in Cuba in the period extending from January 2009 to December 2010. Spacer oligonucleotide typing (Spoligotyping) was applied to 308 M. tuberculosis isolates from the period 2009-2010. Classification into genotypes was carried out according to the international database SpolDB4. Results were additionally analyzed with the online tool MIRU-VNTRplus and compared with the M. tuberculosis genetic patterns found in Cuba in 1993-1995. Results: 79 different genotypic patterns were defined, of which 46 (62 percent) had not been previously reported in SpolDB4. The 22 clusters defined included 75.4 percent of the isolates studied. Five main genetic families were found: Beijing (25.6 percent), S (19.2 percent), LAM (16.9 percent), Haarlem (16.9 percent) and T (5.8 percent). The S family prevailed in the Western region (OR=3.4; CI 95 percent:1.8-6.3; p<0.05), Beijing in Central Cuba (OR=6.7; CI 95 percent:3.7-11.9; p<0.05), and LAM (OR=3.0; CI 95 percent:1.6-5.6; p<0.05) and Haarlem in the Eastern region (OR=1.8; CI 95 percent:1.0-3.4; p<0.05). Conclusions: great diversity was observed among the M. tuberculosis isolates circulating in Cuba in the period 2009-2010. The genetic structure of M. tuberculosis has changed in the country with the passing of time, with a reduction in endemic genotypes like Haarlem and T, and a significant increase in S and Beijing. These data contribute important information for epidemiology and TB control in Cuba(AU)

Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current enotyping procedures: a population-based study in northeast Mexico

The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman’s rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

Molecular typing of Mycobacterium bovis isolates: a review

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

Epidemiologia molecular de Streptococcus pneumoniae sorotipos 1 e 5 isolados de doenças invasiva em Moçambique / Molecular epidemiology of Streptococcus pneumoniae serotypes 1 and 5 isolated from invasive disease in Mozambique

Streptococcus pneumoniae é a principal causa de morbi-mortalidade no mundo. Em Moçambique, os sorotipos 1 e 5 são os mais prevalentes. Este estudo investigou a relação clonal de isolados de pneumococo obtidos de doença invasiva entre o período de 2002-2007, utilizando três procedimentos laboratoriais: Box-PCR, PFGE e MLST. Um total de 105 isolados (sendo 72 do sorotipo 1 e 33 do sorotipo 5), foram submetidos a técnica de Box-PCR. O sorotipo 5 apresentou cinco padrões, sendo um clonal com 20 isolados e quatro padrões não clonais. O padrão A foi o predominante com 61% dos isolados, enquanto que por PFGE, 100% dos isolados foram agrupados em um único clone (clone A), e pela técnica de MLST foi identificado apenas um único ST (ST 289). Por outro lado, o sorotipo 1 apresentou maior diversidade clonal pelos três métodos; por Box-PCR os isolados foram agrupados em 3 clones, sendo predominante o padrão B com 58 isolados, padrão C e N com dois isolados cada. Um total de 12 isolados foram não clonais. O clone B apresentou 20 subtipos, sendo o mais frequente o sub-tipo B1 com 20 amostras idênticas, seguido por B2, B6, B7 com 5 amostras idênticas cada. Por PFGE, 19 amostras confirmaram ser do mesmo perfil clonal (clone B), enquanto que 12 amostras demonstraram ser não clonais. Quando submetidos ao MLST, foram identificados 6 STs; ST 217 (7 isolados), ST 853 (1 isolado), e quatro novos STs, ST 4125, ST 2909, ST 3779 e ST 4166. O ST 217 pertence ao clone Suécia1-27 (ST217), identificados previamente em surtos de meningite na África, enquanto o ST 289 foi identificado como um representante do clone Colômbia5-19 (ST289) que circulam na América Latina desde 1994. A taxa de não susceptibilidade à penicilina foi de 3%, e à cotrimoxazole foi de 39%. A maior taxa de resistência foi encontrada entre os isolados de sorotipo 1. Este trabalho mostra a persistência de dois sorotipos responsáveis por causar doença pneumocócica invasiva graves, bem como seus respectivos clones em uma região do sul de Moçambique.

Resistencia primaria del VIH a los antirretrovirales: informe preliminar del capítulo de Guatemala del estudio de epidemiología molecular del VIH en la región Mesoamericana / Primary HIV resistance to antiretroviral drugs: preliminary report of Guatemala chapter of molecular epidemiology study of HIV in Mesoamerica

Métodos: se seleccionaron pacientes adultos con diagnóstico reciente de infección por VIH, o con diagnóstico previo y que no habían iniciado terapia antirretroviral, que fueron enrolados como parte del estudio Epidemiología Molecular y vigilancia de farmacorresistencia del VIH-1 en la Región Mesoamericana, durante los meses de octubre de 2010 y agosto de 2011 en el Hospital Roosevelt, ciudad de Guatemala. La participación fue debidamente informada y voluntaria...

Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

Background & objectives: El Tor Vibrio cholerae O1 carrying ctxBC trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpAC, tcpAE, hlyAC and hlyAE were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. Interpretation & conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxBC).

Genotyping of Brazilian Giardia duodenalis human axenic isolates

Giardia duodenalis is a complex species that comprises at least seven distinct genetic groups (A to G), but only genotypes A and B are known to infect humans and a wide variety of other mammals. Regardless of biological, biochemical and antigenic analysis, several isolates maintained in vitro were not genetically typed yet. So, in the present study, five Brazilian axenic isolates obtained from asymptomatic and symptomatic patients were typed in order to determine the major genetic groups to which the isolates belonged. DNA was extracted from axenic trophozoites, fragments of glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes were amplified by PCR and the isolate genotyping was carried out using restriction fragment length polymorphism (RFLP) and DNA sequencing for both genes. The results revealed that all isolates were assigned to genotype A at both analyzed loci. Indeed, DNA sequence analysis classified the four isolates obtained from asymptomatic individuals into subtype AII, while the isolate obtained from the symptomatic patient was typed as subtype AI. Despite of the limited number of isolates assessed, the findings presented herein provide interesting insights on the occurrence of Giardia genotypes in Brazil and hold the perspective for future molecular and epidemiological investigations.

Uso de ferramentas de bioinformática para estudos de epidemiologia molecular, filogegrafia e filodinâmica viral / Use of bioinformatics tools for molecular epidemiology studies, phylogeography and viral filodinâmica

As ferramentas de bioinformática tem sido amplamente utilizada para o melhor entendimento de diversos microorganismos. Neste trabalho foram realizados três estudos utilizando estas ferramentas para avaliar diferentes questões biológicas. No primeiro estudo realizou-se uma caracterização molecular de 57 sequências do gene pol, provenientes de pacientes infectados pelo HIV-1 de Salvador, Bahia, Brasil. Para identificar os subtipos e formas recombinantes do HIV-1 circulante na cidade de Salvador foi realizado análises filogenéticas, e através do algoritmo do banco de dados Stanford HIV resistance as mutações associadas à resistência aos ARVs foram detectadas. Entre as 57 sequências analisadas foram identificados neste estudo 45 (77,2%) pertencem ao subtipo B, 11 (21,0%) recombinantes BF e uma (1,8%) do subtipo F1. Além disto, uma alta frequência de eventos de recombinação entre os subtipos B e F foram detectados com 5 padrões de recombinação, duas intergênicas e três intragênicas, mostrando uma alta diversidade. As mutações encontradas com uma maior prevalência foram: I54V (PI) em 7,0%; M184V (NRTI) em 14,0% e K103N (NNRTI) em 10,5% das sequências analisadas. Estes resultados contribuem para traçar o perfil da epidemiologia molecular e diversidade do HIV-1 em Salvador. O segundo estudo avaliou a filodinâmica do HIV-1 em pares de mãe e filho infectados, e em diferentes fases da infecção, três pares na fase aguda e um na fase crônica, e que apresentavam sequências de diferentes tempos. Para este fim foi realizado inferências filogenéticas bayesianas, onde a hipótese do relógio molecular e de diferentes crescimentos populacional foram testadas. Não foi possível observar uma diferença entre a dinâmica da população viral da mãe e a encontrada no filho. Porém, quando observamos o crescimento populacional e o tamanho da população efetiva, ao longo do tempo, sequências provenientes de pares em fase crônica da infecção tem um crescimento mais constante, enquanto as sequências dos pares na fase aguda da infecção se observa uma dinâmica das populações virais, provavelmente devido à pressão do sistema imune e a não adaptação destes vírus. No terceiro estudo, 104 sequências do genoma completo do WNV, disponíveis no Genebank, foram estudadas para identificar a região genômica que apresenta máximo poder interpretativo para inferir relações temporais e geográficas entre as cepas do vírus. Alinhamentos de cada gene foram submetidos à avaliação do sinal filogenético através do programa TREEPUZZEL. As regiões NS3 e NS4 apresentaram um sinal filogenético acima de 70%, sendo as regiões mais indicadas para construção filogenética. Além disto, árvores bayesianas foram inferidas utilizando as regiões NS3, NS5 e E, onde os clados das árvores NS3 e NS5 apresentaram um maior suporte e estrutural temporal geográfica, diferente da região E. Estes achados mostram que os genes NS3 e NS5 são os mais indicados para análises filogenéticas. Neste trabalho foi demonstrando o uso de ferramentas de bioinformática para a melhor caracterização da diversidade, epidemiologia molecular, dinâmica populacional e determinação das relações temporal e geográfica dos vírus.

Identification and Application of Biomarkers in Molecular and Genomic Epidemiologic Research / 예방의학회지

Biomarkers are characteristic biological properties that can be detected and measured in a variety of biological matrices in the human body, including the blood and tissue, to give an indication of whether there is a threat of disease, if a disease already exists, or how such a disease may develop in an individual case. Along the continuum from exposure to clinical disease and progression, exposure, internal dose, biologically effective dose, early biological effect, altered structure and/or function, clinical disease, and disease progression can potentially be observed and quantified using biomarkers. While the traditional discovery of biomarkers has been a slow process, the advent of molecular and genomic medicine has resulted in explosive growth in the discovery of new biomarkers. In this review, issues in evaluating biomarkers will be discussed and the biomarkers of environmental exposure, early biologic effect, and susceptibility identified and validated in epidemiological studies will be summarized. The spectrum of genomic approaches currently used to identify and apply biomarkers and strategies to validate genomic biomarkers will also be discussed.

Gene amplification using DNA from human spot urine samples.

The purpose of this study was to test the amplification of DNA from human urinary sediment for molecular epidemiological studies. Twenty-six urine samples were obtained from healthy volunteers. Polymerase chain reactions (PCR) for methylenetetrahydrofolate reductase (MTHFR), beta-globin, and N-acetyltransferase 2 (NAT2) was conducted using genomic DNA isolated from the urine. The MTHFR and beta-globin genes were amplified successfully from all the urine DNA samples while the NAT2 gene was amplified in 88.5% of cases. The median yield of DNA was 0.28 microg from the 10 ml urine samples, sufficient amounts of DNA being contained in urinary sediments for amplification of all three genes. This result indicates that urine can be used as a DNA source for PCR-based molecular epidemiological studies.

Aplicação da técnica de REP – PCR no rastreamento de Staphylococcus aureus em sala de ordenha, para o monitoramento da qualidade do leite / Using the REP-PCR technic in the tracking of Staphylococcus aureus in milking room, for milk quality production

A identificação e classificação bacteriana são de suma importância no ambiente, na indústria, na medicina veterinária, na microbiologia e na ecologia microbiana. Um número de diferentes métodos genotípicos e fenotípicos estão sendo empregados para identificação e classificação microbiana. A técnica de REP-PCR é baseada no uso de primers sintetizados a partir de sequências repetidas de DNA, chamadas depalindrômicas extragênicas repetidas (REP), e têm sido descrita como um método o qual gera impressões digitais (fingerprints) de DNA que podem diferenciar bactérias entre gêneros e espécies. Neste estudo, o método de fingerprint foi usado para Staphylococcus aureus com o objetivo de avaliar a higiene de ordenha em duas fazendas leiteiras.Foram obtidos vários fingerprints de todos os isolados coletados das diferentes fontes estudadas (mãos de ordenhadores, tetos das vacas,leite e ordenhadeira), e foram obtidos comportamentos muito similares das bandas indicando que os isolados podem ser relatados como clones epidemiológicos. Em nosso estudo, a técnica mostrou ser eficiente para a análise da similaridade entre indivíduos da mesma espécie, no caso, o Staphylococcus aureus, mostrando ser uma ferramenta útil para investigação de falhas no manejo e, em um controle mais eficiente para evitar e/ou diminuir a disseminação de microrganismos causadores de sérias enfermidades em humanos e em animais, que podem ser transmitidas através de produtos como o leite e seus derivados.

The identification and classification of bacteria are of crucial importance in environmental, industrial, veterinary, microbiology and microbial ecology. A number of different phenotypic and genotypic methods are presently being employed for microbial identification and classification. Repetitive-element PCR (Rep-PCR) with primers basedon repetitive extragenic palindromic (REP) repeated DNA sequencesis a recently described method which generates DNA fingerprints that descriminate between bacterial species and strains. In this study, this method was used for genomic fingerprinting of Staphylococcus aureus in control of hygiene in milk line production on two farms. Complex fingerprinting patterns were obtained for all isolates from different sources (milking handlers, cows teats, milk and milk machine) were very similar, and the data indicated that the isolated were closely related.In our study, this technic shows to be usefull for investigation in fails on milk line production and for more efficient control for patogenic microrganisms that cause serious illness in humans and animals.

Molecular epidemiology of tuberculosis.

Molecular epidemiology (ME), a blend of molecular biology and epidemiology, is very useful to study the spread of tubercle bacilli in mini epidemics, outbreaks, to analyse the transmission dynamics of tuberculosis (TB) and to determine the risk factors for TB transmission in a community. ME has a great role in distinguishing between exogenous reinfection and endogenous reactivation. In the laboratory, molecular epidemiology can be used to identify cross contamination. Many new DNA typing methods have been introduced after the initial introduction of restriction fragment length polymorphism (RFLP) in 1993. An internationally accepted, standardized protocol for RFLP typing of the Mycobacterium tuberculosis complex using IS6110 was published in 1993 and is still used today. Most of the newer DNA typing methods are PCR based and microarray based methods are also available. This will enable individual strains of M. tuberculosis or clonal groups to be identified by specific phenotypic traits. ME will continue to be a useful tool in future to measure the impact of any public health intervention strategy for control of tuberculosis in the community.

Molecular evidence of mixed P. vivax and P. falciparum infections in northern Islamic Republic of Iran

This study compared basic microscopy with molecular detection of Plasmodium species. According to thick-film microscopy, 100% of 142 malaria cases in Pars-Abad, Ardebil province, were infected with a single species, P vivax. However, nested polymerase chain reaction [PCR] detected mixed species infections of both P. vivax and P. falciparum in 7.0%. In Maz and eran province, 2/20 blood films were diagnosed with only P. falciparum and 18/20 with only P. vivax. However, nested PCR detected 17/20, 2/20 and 1/20 with P. vivax only, P. falciparum only and mixed species respectively. The unexpected presence of P. falciparum urges prompt investigation and immediate treatment of malaria cases in this region