An algorithm based on molecular protocols to improve the detection of Plasmodium in autochthonous malarial areas in the Atlantic Forest biome

ABSTRACT Malaria is the most important vector-borne disease in the world and a challenge for control programs. In Brazil, 99% of cases occur in the Amazon region. In the extra-Amazonian region, a non-endemic area, epidemiological surveillance focuses on imported malaria and on autochthonous outbreaks, including cases with mild symptoms and low parasitemia acquired in the Atlantic Forest biome. In this scenario, cases are likely to be underreported, since submicroscopic parasitemias are not detected by thick blood smear, considered the reference test. Molecular tests are more sensitive, detecting asymptomatic individuals and mixed infections. The aim of this study was to propose a more efficient alternative to detect asymptomatic individuals living in areas of low malaria endemicity, as they are reservoirs of Plasmodium that maintain transmission locally. In total, 955 blood samples from residents of 16 municipalities with autochthonous malaria outbreaks in the Sao Paulo State were analyzed; 371 samples were collected in EDTA tubes and 584 in filter paper. All samples were initially screened by a genus-specific qPCR targeting ssrRNA genes (limit of detection of 1 parasite/µL). Then, positive samples were subjected to a nested PCR targeting ssrRNA and dihydrofolate reductase-thymidylate synthase genes (limit of detection of 10 parasites/µL) to determine Plasmodium species. The results showed a statistically significant difference (K = 0.049; p < 0.0001) between microscopy positivity (6.9%) and qPCR (22.9%) for EDTA-blood samples. Conversely, for samples collected in filter paper, no statistical difference was observed, with 2.6% positivity by thick blood smear and 3.1% for qPCR (K = 0.036; p = 0.7). Samples positive by qPCR were assayed by a species-specific nested PCR that was in turn positive in 26% of samples (16 P. vivax and 4 P. malariae ). The results showed that molecular protocols applied to blood samples from residents in areas with autochthonous transmission of malaria were useful to detect asymptomatic patients who act as a source of transmission. The results showed that the genus-specific qPCR was useful for screening positives, with the subsequent identification of species by nested PCR. Additional improvements, such as standardization of blood plotting on filter paper and a more sensitive protocol for species determination, are essential. The qPCR-based algorithm for screening positives followed by nested PCR will contribute to more efficient control of malaria transmission, offering faster and more sensitive tools to detect asymptomatic Plasmodium reservoirs.

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates / Técnicas sorológicas e moleculares aplicadas na identificação de Plasmodium spp. em amostras de primatas não humanos

Abstract The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples.

Resumo Neste estudo objetivamos identificar Plasmodium spp. em amostras sangue de primatas não humanos (PNH) do estado do Maranhão, utilizando técnicas clássicas e alternativas para o exame da malária humana. Foram analisadas 161 amostras de sangue de PNH, sendo 141 de CETAS (cativeiro) e 20 de reserva particular (vida livre), utilizando microscopia, teste de diagnóstico rápido (RDT), imunofluorescência indireta (IFI) e técnicas moleculares (semi-nested PCR, PCR em tempo real quantitativo e LAMP). Dois métodos sorológicos (dot-ELISA e ELISA indireto) também foram padronizados com antígenos solúveis de roptrias de P. falciparum e P. berghei. Formas trofozoíticas de Plasmodium sp. foram identificadas em lâminas de cinco animais diferentes. Nenhuma amostra foi positiva em TDR e LAMP. Quatro amostras foram soropositivas para P. malariae na IFI. Os soros de PNH mostraram baixa reatividade pelo ELISA indireto. Plasmodium sp. foi detectado em 34,16% (55/161) das amostras utilizando a qPCR baseada no gene 18S rRNA. No sequenciamento, duas amostras mostraram identidade com P. malariae (100%), uma com Plasmodium sp. ZOOBH (97%) e uma com P. falciparum (99%). A PCR mostrou ser a técnica mais sensível para diagnósticos de Plasmodium em amostras de PNH.

Characteristics of Imported Malaria and Species of Plasmodium Involved in Shandong Province, China (2012-2014)

Malaria remains a serious public health problem in Shandong Province, China; therefore, it is important to explore the characteristics of the current malaria prevalence situation in the province. In this study, data of malaria cases reported in Shandong during 2012-2014 were analyzed, and Plasmodium species were confirmed by smear microscopy and nested-PCR. A total of 374 malaria cases were reported, 80.8% of which were reported from 6 prefectures. Of all cases, P. falciparum was dominant (81.3%), followed by P. vivax (11.8%); P. ovale and P. malariae together accounted for 6.4% of cases. Notably, for the first time since 2012, no indigenous case had been reported in Shandong Province, a situation that continued through 2014. Total 95.2% of cases were imported from Africa. The ratio of male/female was 92.5:1, and 96.8% of cases occurred in people 20-54 years of age. Farmers or laborers represented 77.5% of cases. No significant trends of monthly pattern were found in the reported cases. All patients were in good condition after treatment, except for 3 who died. These results indicate that imported malaria has increased significantly since 2012 in Shandong Province, especially for P. falciparum, and there is an emergence of species diversity.

Size changes of erythrocytes infected with Plasmodium malariae in thin pe-ripheral blood smears / 中国血吸虫病防治杂志

Objective To explore the size changes of erythrocytes infected with Plasmodium malariae,so as to improve the basic?level experimenters’microscopy capabilities for P. malariae identification in thin blood smears. Methods The micro?scopic features of erythrocytes infected with P. malariae in thin peripheral blood smears were observed,and a microscope image processing software was used to measure and analyze the diameter changes of the erythrocytes infected with P. malariae. Re?sults The diameter of erythrocytes infected with P. malariae decreased significantly compared with that of the normal erythro?cytes. The three parameters in this study:the diameter of erythrocytes,the value of diameter variation,and the ratio of diameter variation varied at different developmental stages of P. malariae,and the differences were statistically significant(all P<0.01). The variances of the three parameters grouped by different cases or different developmental stages in different cases were ana?lyzed,all showing statistically significant differences(all P<0.01). Conclusion All the developmental stages of P. malariae will cause the decrease of the diameters of infected erythrocytes in peripheral blood smears,but the influence on the diameter of erythrocytes,value of diameter variation,and ratio of diameter variation varies at different developmental stages in different cases.

A Case of Plasmodium malariae Infection Imported from Guinea

Recently, the number of Korean travelers and workers to malaria-endemic regions has increased, and the number of patients with imported malaria cases has increased as well. In Korea, most cases of imported malaria infections are caused by Plasmodium falciparum and P. vivax. Only one report of imported P. malariae infection has been published thus far. Here, we describe a case of imported P. malariae infection that was confirmed by peripheral blood smear and nested PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene. A 53-yr-old man, who had stayed in the Republic of Guinea in tropical West Africa for about 40 days, experienced fever and headache for 3 days before admission. The results of rapid malaria test using the SD Malaria Antigen/Antibody Kit (Standard Diagnostics, Korea) were negative, but Wright-Giemsa stained peripheral blood smear revealed Plasmodium. To identify the Plasmodium species and to examine if the patient had a mixed infection, we performed nested PCR targeting the SSU rRNA gene. P. malariae single infection was confirmed by nested PCR. Sequence analysis of the SSU rRNA gene of P. malariae showed that the isolated P. malariae was P. malariae type 2. Thus, our findings suggest that when cases of imported malaria infection are suspected, infection with P. malariae as well as P. falciparum and P. vivax should be considered. For the accurate diagnosis and treatment of imported malaria cases, we should confirm infection with Plasmodium species by PCR as well as peripheral blood smear and rapid malaria antigen test.

Anopheles (Kerteszia) cruzii (DIPTERA: CULICIDAE) IN PERIDOMICILIARY AREA DURING ASYMPTOMATIC MALARIA TRANSMISSION IN THE ATLANTIC FOREST: MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES INDICATES HUMANS AS PRIMARY INTERMEDIATE HOSTS / Anopheles (Kerteszia) cruzii (Diptera: Culicidae) em área peridomiciliar durante transmissão de malária assintomática na Mata Atlântica: identificação molecular das fontes de repasto sanguíneo indica humanos como hospedeiros intermediários primários

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

Anopheles (Kerteszia) cruzii é o vetor primário das malárias humana e simiana fora da Amazônia Brasileira e especificamente nas regiões de Mata Atlântica. A presença de casos humanos assintomáticos, macacos silvestres positivos para Plasmodium e a similaridade entre os parasitas que os infectam suportam a discussão se essas infecções podem ser consideradas como zoonoses. Embora muitos aspectos da biologia de An. cruzii já tenham sido abordados, estudos conduzidos durante surtos de transmissão de malária, visando a análise de repasto sanguíneo e infectividade, são ausentes na Mata Atlântica. Este estudo foi conduzido na localidade de Palestina, Juquitiba, Mata Atlântica do Estado de São Paulo, onde anualmente a maioria dos casos humanos autóctones é notificada. Locais em peridomicílio foram selecionados para coleta de mosquitos em um perímetro de até 100 m em torno das residências de casos humanos de malária e da floresta circundante. Os mosquitos foram analisados com o objetivo de identificação molecular das fontes de repasto sanguíneo e para examinar a prevalência de Plasmodium. Um total de 13.441 fêmeas de An. (Ker.) cruzii foi coletado. A taxa de infecção mínima foi calculada a 0,03% e 0,01%, respectivamente, para P. vivax e P. malariae e somente sangue humano foi detectado nos mosquitos analisados que se alimentaram com sangue. Nossos dados reforçam a hipótese de que os portadores humanos assintomáticos são a principal fonte de infecção para os anofelinos na área do peridomicílio, tornando a transmissão zoonótica improvável.

Two cases of African imported Plasmodium malariae malaria / 中国血吸虫病防治杂志

The process of diagnosis,epidemiological survey and treatment of two cases of African imported quartan malaria is reported in this paper.

A Case of Imported Plasmodium malariae Malaria

Malaria, the most common vector-borne parasite infection worldwide, results from infection by Plasmodium species. Approximately 80% of malaria cases are caused by P. vivax, which is broadly distributed from tropical to temperate regions; P. falciparum is the second most common infectious species. P. malariae and P. ovale are responsible for a relatively small proportion of malaria cases. Here, we report the case of a 23-yr-old Korean woman who acquired a P. malariae infection while visiting the Republic of Ghana in West Africa for business. She was diagnosed with P. malariae malaria on the basis of peripheral blood smear (PBS) and species-specific conventional and real-time PCR assays for 18S rRNA. She was treated with hydroxychloroquine, and the resulting PBS examination on day 2 suggested that negative conversion occurred. At her 1-month follow-up, however, both the PBS examination and molecular test for malaria demonstrated recurrent parasitemia. We started rescue therapy with mefloquine, and the patient recovered successfully. This is an important finding suggesting possible late recrudescence of a chloroquine-resistant P. malariae strain identified not only by its morphological features, but also by molecular tests.

Epidemiología de malaria por Plasmodium malariae en Venezuela, 1999 - 2008 / Epidemiology of Plasmodium malariae in Venezuela (1999 – 2008)

El paludismo es un problema mundial grave que afecta de forma inaceptable la salud y el bienestar económico de las comunidades más pobres del mundo. En Venezuela, durante el decenio 1999 - 2008, fueron diagnosticados 341.200 casos de malaria, con una incidencia de 0,1% a Plasmodium malariae. Históricamente se conocía que la incidencia a este Plasmodium era exclusiva del estado Amazonas y así lo demuestran los registros del programa nacional antimalárico. Ahora la mayoría de los diagnósticos (61,4%) son realizados en el estado Bolívar, en sesenta y nueve (69) de sus localidades, pudiendo ser consideradas algunas, como focos de P. malariae durante los últimos tres años, especialmente la localidad de Matupo I, en el municipio Sifontes. Aunque el laboratorio Central o de referencia nacional para el diagnóstico de malaria de la Dirección de Salud Ambiental, ubicado en la ciudad de Maracay, no ha podido corroborar los diagnósticos realizados en el país sobre la especie, es conocido el aumento de interés por el mismo, de un grupo de investigadores y la Dirección Regional de Saneamiento Ambiental del estado Bolívar, lo cual podría ser la causa del aumento de su incidencia en el área

Malaria remains a major global problem, affecting in an unacceptable way the health and economic welfare of the world’s poorest communities. In Venezuela, during the decade 1999 - 2008, 341,200 cases of malaria were diagnosed, with an incidence of 0.1% for Plasmodium malariae. Historically it has been known that the incidence of this Plasmodium was unique to the Amazon state as shown by the national malaria program records. Now most of the diagnoses (61.4%) are made in Bolivar state in sixty-nine (69) localities, some may be considered as foci of P. malariae in the last three years, especially the town of Matupo I in the municipality Sifontes. Although the central or national reference laboratory for malaria diagnosis of the Directorate of Environmental Health, located in the city of Maracay, was unable to corroborate the diagnoses on this species, it has an increased interest in it, including a group of researchers and the Regional Bureau of Environmental Sanitation in Bolívar state, who think this could be the cause of increased incidence in the area

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64 percent of all infections, P. vivax for 28 percent, and P. malariae for 4 percent. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4 percent of total cases. Children under 10 years accounted for 58 percent of the cases; the risk for malaria in this age group was 77 percent higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42 percent), P. malariae (0.25 percent), and P. vivax-247 (0.1 percent). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.