Species-specific Nested Polymerase Chain Reaction Research Articles

Molecular investigation of malaria-infected patients in Djibouti city (2018–2021)

BackgroundThe Republic of Djibouti is a malaria endemic country that was in pre-elimination phase in 2006–2012. From 2013, however, malaria has re-emerged in the country, and its prevalence has been increasing every year. Given the co-circulation of several infectious agents in the country, the assessment of malaria infection based on microscopy or histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDT) has shown its limitations. This study, therefore, aimed to assess the prevalence of malaria among febrile patients in Djibouti city using more robust molecular tools.MethodsAll suspected malaria cases reported to be microscopy-positive were randomly sampled (n = 1113) and included in four health structures in Djibouti city over a 4-year period (2018–2021), mainly during the malaria transmission season (January–May). Socio-demographic information was collected, and RDT was performed in most of the included patients. The diagnosis was confirmed by species-specific nested polymerase chain reaction (PCR). Data were analysed using Fisher’s exact test and kappa statistics.ResultsIn total, 1113 patients with suspected malaria and available blood samples were included. PCR confirmed that 788/1113 (70.8%) were positive for malaria. Among PCR-positive samples, 656 (83.2%) were due to Plasmodium falciparum, 88 (11.2%) Plasmodium vivax, and 44 (5.6%) P. falciparum/P. vivax mixed infections. In 2020, P. falciparum infections were confirmed by PCR in 50% (144/288) of negative RDTs. After the change of RDT in 2021, this percentage decreased to 17%. False negative RDT results were found more frequently (P < 0.05) in four districts of Djibouti city (Balbala, Quartier 7, Quartier 6, and Arhiba). Malaria occurred less frequently in regular bed net users than in non-users (odds ratio [OR]: 0.62, 95% confidence interval [CI]: 0.42–0.92).ConclusionsThe present study confirmed the high prevalence of falciparum malaria and, to a lesser extent, vivax malaria. Nevertheless, 29% of suspected malaria cases were misdiagnosed by microscopy and/or RDT. There is a need to strengthen the capacity for diagnosis by microscopy and to evaluate the possible role of P. falciparum hrp2 gene deletion, which leads to false negative cases of P. falciparum.

Significance of nested PCR testing for the detection of low-density malaria infection amongst febrile patients from the Malaria Elimination Demonstration Project in Mandla, Madhya Pradesh, India

BackgroundLow-density malaria infections (LDMI) are defined as infections that are missed by the rapid diagnostic test (RDT) and/or microscopy which can lead to continued transmission and poses a challenge in malaria elimination efforts. This study was conducted to investigate the prevalence of LDMI in febrile cases using species-specific nested Polymerase Chain Reaction (PCR) tests in the Malaria Elimination Demonstration Project, where routine diagnosis was conducted using RDT.MethodsEvery 10th fever case from a cross-sectional community based fever surveillance was tested with RDT, microscopy and nested PCR. Parasite DNA was isolated from the filter paper using Chelex based method. Molecular diagnosis by nested PCR was performed targeting 18SrRNA gene for Plasmodium species.ResultsThe prevalence of malaria was 2.50% (436/17405) diagnosed by PCR, 1.13% (196/17405) by RDT, and 0.68% (118/ 17,405) by microscopy. Amongst 17,405 febrile samples, the prevalence of LDMI was 1.51% (263/17405) (95% CI 1.33–1.70), which were missed by conventional methods. Logistic regression analysis revealed that illness during summer season [OR = 1.90 (p < 0.05)] and cases screened within three days of febrile illness [OR = 5.27 (p < 0.001)] were the statistically significant predictors of LDMI.ConclusionThe prevalence of malaria among febrile cases using PCR was 2.50% (436/17405) as compared to 1.13% (196/17405) by RDT. Higher number of the LDMI cases were found in subjects with ≤ 3 days mean duration of reported fever, which was statistically significant (p < 0.001). This observation suggests that an early detection of malaria with a more sensitive diagnostic method or repeat testing of the all negative cases may be useful for curtailing malaria transmission. Therefore, malaria elimination programme would benefit from using more sensitive and specific diagnostic methods, such as PCR.

Is Plasmodium vivax becoming more prevalent in parts of Nigeria? A molecular evaluation of the parasite from persons living in Ishielu, South-East Nigeria

Plasmodium vivax, the leading cause of malaria in the vast majority of countries outside of Africa and the second most dominant cause of malaria globally, is fast becoming widespread and domiciled in Nigeria and other parts of sub-Saharan Africa. The objective of this cross-sectional survey conducted in Ishielu, a suburb of Abakaliki, southeastern Nigeria was to identify circulating Plasmodium species (with focus on P. falciparum and P. vivax species) in the population. A total of 120 venous blood samples (2-5 ml) of individuals in the community was examined microscopically; while thirty randomlyselected blood samples were used for polymerase-chain reaction (PCR) assays. Genomic DNA was isolated from the blood samples and species-specific nested PCRs targeting the 18S ribosomal ribonucleic acid (rRNA) gene were applied in detecting the presence of the target parasite species. Microscopy showed that 90% (108/120) of the blood samples were positive for parasitaemia and P. falciparum was the only Plasmodium species identified. Of the 30 selected blood samples assayed by PCR, the amplicon of 18S rRNA gene of the malaria parasite species was amplifiable in 80 % (24/30), combined vivax and falciparum positive cases were identified in three blood specimens and from microscopically negative slides. Mixed infections constituted 25% (6/24) while single infections with vivax and falciparum amounted to 42% (10/24) and 33% (8/24) respectively. These call for large-scale surveys of the target parasite species at community level by molecular PCR method for significant reduction of morbidity due to malaria.

Entamoeba species infection in patients seeking treatment for diarrhea and abdominal discomfort in Mukuru informal settlement in Nairobi, Kenya

Entamoeba histolytica is the only pathogenic species of the Entamoeba genus and is morphologically identical to E. dispar/E. moshkovskii (Entamoeba complex) hence cannot be microscopically differentiated. The other Entamoeba spp. found in humans (E. hartmanni, E. polecki, and E. coli) can be differentiated morphologically from this Entamoeba complex. However, some of their morphologic features overlap making differential diagnosis difficult. This study aimed at determining the occurrence of Entamoeba spp. in patients seeking treatment for diarrhea and/or abdominal discomfort at two clinics in Mukuru informal settlement in Nairobi, Kenya. Faecal samples were collected from 895 patients, examined microscopically following direct wet smear and formal-ether concentration methods. Entamoeba spp. positive faecal samples were subjected to DNA extraction and species-specific nested polymerase chain reaction of the 18S ribosomal RNA (rRNA). By microscopy, Entamoeba spp. cysts or trophozoites were detected in 114/895 (12.7%, 95% Confidence Interval (CI) 10.6–15.1) faecal samples. By nested PCR, the prevalence was: E. histolytica (7.5%, 95% CI 5.9–9.4, 67/895) and E. dispar (8.2%, 95% CI 6.5–10.2, 73/895). Among the Entamoeba spp. complex positive samples, nested PCR detected E. coli and E. hartmanni DNA in 63/114 (55.3%) and 37/114 (32.5%), samples respectively. Among the E. histolytica/E. dispar PCR negative samples (32.5%), 21 (18.4%) contained cysts of either E. coli (19) or E. hartmanni (2) by nested PCR. Entamoeba spp. infections were most common among participants aged 21–30 years; however it was not significant (P = 0.7). Entamoeba spp. infections showed an inverse relationship with diarrhea being most common among participants without diarrhea (P = 0.0). The difference was significant for E. histolytica (P = 0.0) but not significant for E. dispar (P = 0.1). Only E. dispar infections were significantly associated with sex (P = 0.0). This study highlights the need for differentiation of E. histolytica from other Entamoeba spp. by molecular tools for better management of amoebiasis.

An updated geographic distribution of Myxobolus cerebralis (Hofer, 1903) (Bivalvulida: Myxobolidae) and the first diagnosed case of whirling disease in wild-caught trout in the south-eastern United States.

Myxobolus cerebralis (Bivalvulida: Myxobolidae), the aetiological agent of salmonid whirling disease, was detected in 2 river basins of North Carolina during 2015, which initiated the largest spatial-temporal monitoring project for the disease ever conducted within the south-eastern United States (focused mainly in eastern Tennessee and western North Carolina). A total of 2072 rainbow trout Oncorhynchus mykiss, 1,004 brown trout Salmo trutta and 468 brook trout Salvelinus fontinalis were screened from 113 localities within 7 river basins during June 2017 through October 2019. Infections were detected by pepsin-trypsin digest, microscopy and the species-specific nested polymerase chain reaction (PCR) in 19 localities across 6 river basins. Myxospore morphology was indistinguishable from the published literature. In 2019, five rainbow trout that symptomatic for whirling disease (sloping neurocranium and lordosis) were captured and processed for histopathology. Myxospores were detected in the calvarial cartilage of two deformed trout with associated erosion of the cartilage consistent with reported whirling disease lesions. This is the first report of M.cerebralis in Tennessee and the first histologically confirmed cases of whirling disease in southern Appalachian (south-eastern United States) rivers and streams and expands the distribution of M.cerebralis throughout western North Carolina and eastern Tennessee.

Prevalence of asymptomatic Plasmodium falciparuminfection, anaemia and use of ITNs among pregnant women yet to receive IPTp in parts of southern Ghana

Background: Asymptomatic malaria in pregnancy evades most fever-based surveillance systems yet causes significant morbidities such as anaemia in the pregnant woman and low birth weight in the neonate. Objective:This study determined the prevalence of asymptomatic malaria and its association with anaemia among pregnant women who are yet to receive their first dose of Sulphadoxine-Pyrimethamine (SP) as intermittent preventive treatment of malaria in pregnancy (IPTp). Information on the use of insecticide treated nets(ITNs)as vector control by the pregnant women was also sought. Methods: This is a cross-sectional hospital-based study conducted in the Western Region of Ghana. Pregnant women at gestational ages, 16-26 wkwere included. A structured questionnaire was used to collect vital information from the participants. Plasmodiumparasitaemia was determined by rapid diagnostic test (MRDT), microscopy and species-specific nested polymerase chain reaction (PCR). Anaemia was classified using the level of haemoglobin. Results: A total of 413 antenatal clinic attendants were recruited. Prevalence of asymptomatic Plasmodium falciparuminfection was 13.1% by MRDT, 10.1% by microscopy and 13.8% by PCR. The mean haemoglobin was 10.73g/dL. Prevalence of anaemia was 40.49% and the mean parasite density was 149.6 parasite/μL. Pregnant women with asymptomatic malaria were 4 times more at risk of being anaemic (adjusted odds ratio with 95% confidence interval: 4.42, 1.82 -10.70)than those who did not have malaria. There was statistically significant negative correlation between parasite density and anaemia (r=0.0028, p=0.02). Conclusion: Asymptomatic P. falciparuminfection was found among some of the pregnant women and the presence of the parasites make them 4 times at increased risk of developing anaemia. Anaemia when occurring amongst such women was significantly worsened by increasing parasitaemia.

LABORATORY DIAGNOSIS OF MALARIA INFECTION IN CLINICALLY SUSPECTED CASES USING MICROSCOPIC EXAMINATION, RAPID ANTIGEN TESTS & PCR

Accurate diagnosis of malaria is important for providing malaria burden and preventing misdiagnosis. To analyses the diagnostic performance of conventional blood film microscopic examination, OptiMAL Rapid Antigen Test, Hexagon Malaria Combi, Diaquick Malaria Pf/Pan Cassette in comparison with species-specific nested polymerase chain reaction (nest-PCR) in Makkah and Taif in Kingdom of Saudi Arabia. A total of 100 blood samples collected from febrile cases in Makkah and Taif Cities were screened for malaria by microscopic examination, rapid diagnostic tests (RDTs) using nest-PCR as standard method. The results showed differences in detection rates of nested-PCR, conventional blood film microscopic examination, OptiMAL test, Hexagon Malaria Combi, Diaquick Malaria Pf/Pan Cassette (72%, 57%, 59%, 44% & 52% respectively) in 100 clinically suspected cases. Microscopic examination, OptiMAL test, Hexagon Malaria Combi and Diaquick Malaria Pf/Pan Cassette showed sensitivity (72.2%,72.2%, 62.9%, 65.3%), specificity (82.1%, 75%, 82.1%,& 82.1%), accuracy index (75%, 73%, 62%, 70%), positive predictive value (91.2%, 88.1%, 88.6%,& 90.4%) and negative predictive value (53.5%, 51.2%, 41.1%, 47.9) using nested-PCR as the reference technique. The detection rates of nested-PCR for Pasmodium infection was superior than conventional blood film microscopic examination, OptiMAL test, Hexagon Malaria Combi microscopy and Diaquick Malaria Pf/Pan Cassette. The nest-PCR method was more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections.

Malaria Prevalence in a Low Transmission Area, Jazan District of Southwestern Saudi Arabia.

Detailed description of malaria in low transmission areas is crucial for elimination. The current study aimed to provide a comprehensive description for malaria transmission in Jazan, a low transmission district, southwestern Saudi Arabia. Patients at a tertiary care hospital were recruited in our study between August 2016 and September 2018. Malaria diagnosis was performed through a species-specific nested polymerase chain reaction (nested PCR), microscopy and Paramax-3TM rapid detection test (RDT). Malaria was detected in 30 patients by the PCR, with point prevalence of 10.9%. Of these malaria infections, 80% was imported, 26.6% was asymptomatic and 23.3% was sub-microscopic. Malaria was reported throughout the year, with February/March and September/October peaks. Infection was significantly more in males than in females (P=0.01). Likewise, infections were detected more in febrile than in non-febrile patients (P=0.01). Adult aged 15–24 years, fever and travel were identified as high-risk factors. Malaria was primarily attributed to Plasmodium falciparum mono-infections, followed by P. vivax mono-infections and lastly to falciparum/vivax mixed infections accounting 76.6%, 16.6%, and 6.6% of PCR-confirmed malaria cases, respectively. The nested PCR was superior to the smear microscopy (sensitivity 76.6%; specificity 100%) and the RDT (sensitivity 83.3%, specificity 94.2%). The overall percent agreement between microscopy and the RDT was 92.7% (kappa=0.63). High proportion of imported malaria including sub-microscopic and sub-patent cases were described. We suggest that incorporation of molecular tool into the conventional malaria diagnosis is beneficial in Jazan district.

Asymptomatic infections in blood donors harbouring Plasmodium: an invisible risk detected by molecular and serological tools.

Transfusion-transmitted malaria due to asymptomatic Plasmodium infections is a challenge for blood banks. There is a lack of data on the prevalence of asymptomatic infected blood donors and the incidence of transfusion-transmitted malaria in low endemicity areas worldwide. We estimated the frequency of blood donors harbouring Plasmodium in an area in which asymptomatic infections have been reported. To estimate the frequency of blood donors harbouring Plasmodium we used microscopy and molecular tools. Serological tests were applied to measure the exposure of candidates to Plasmodium antigens. Venous blood was collected from 91 candidates attending the "Pró-Sangue" Blood Centre Foundation in São Paulo, who lived in the municipality of Juquitiba, São Paulo, Brazil, where sporadic autochthonous cases of malaria have been described. Blood samples were used for parasitological, molecular and serological studies. Among the 91 samples examined, rare Plasmodium forms were observed in two donors. Genus real-time polymerase chain reaction analysis demonstrated Plasmodium amplification in three candidates and species-specific nested polymerase chain reaction identified P. malariae in two. ELISA-IgG was reactive in 42.9% of samples for P. vivax (Pv-MSP119) and in 6.6% for P. falciparum (Pf-Zw). ELISA-IgM was reactive in 2.2% of samples for P. vivax and in 4.4% for P. falciparum. An indirect immunofluorescence assay was reactive for P. malariae in 15.4% of cases. Reservoirs of Plasmodium represent a challenge for blood banks, since studies have shown that high levels of submicroscopic infections can occur in low transmission areas. The risk of transfusion-transmitted malaria presented here points to the need to conduct molecular investigations of candidate donors with any positive malarial antibody test.

Comparison of the diagnostic performance of microscopic examination with nested polymerase chain reaction for optimum malaria diagnosis in Upper Myanmar

BackgroundAccurate diagnosis of Plasmodium infection is crucial for prompt malaria treatment and surveillance. Microscopic examination has been widely applied as the gold standard for malaria diagnosis in most part of malaria endemic areas, but its diagnostic value has been questioned, particularly in submicroscopic malaria. In this study, the diagnostic performance of microscopic examination and nested polymerase chain reaction (PCR) was evaluated to establish optimal malaria diagnosis method in Myanmar.MethodsA total of 1125 blood samples collected from residents in the villages and towns located in Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships and Mandalay of Upper Myanmar were screened by microscopic examination and species-specific nested PCR method.ResultsAmong the 1125 blood samples, 261 samples were confirmed to be infected with malaria by microscopic examination. Evaluation of the 1125 samples by species-specific nested PCR analysis revealed that the agreement between microscopic examination and nested PCR was 87.3% (261/299). Nested PCR successfully detected 38 Plasmodium falciparum or Plasmodium vivax infections, which were missed in microscopic examination. Microscopic examinations also either misdiagnosed the infected Plasmodium species, or did not detect mixed infections with different Plasmodium species in 31 cases.ConclusionsThe nested PCR method is more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections. Given the observed higher sensitivity and specificity of nested PCR, the molecular method holds enormous promise in malaria diagnosis and species differentiation, and can be applied as an effective monitoring tool for malaria surveillance, control and elimination in Myanmar.

Molecular Identification of an Invasive Wood-Boring Insect Lyctus brunneus (Coleoptera: Bostrichidae: Lyctinae) Using Frass by Loop-Mediated Isothermal Amplification and Nested PCR Assays.

Lyctus brunneus (Stephens) is one of the most destructive and worldwide invasive pests of seasoned woods for wooden products. This and other pest Lyctus species have had their distribution expanded by international and domestic human transportation of infested wood and wood products. Rapid detection and accurate identification of Lyctus species are effective tools for helping to eradicate them in new introduction sites. The accurate species-level identification of adults requires expert knowledge about their morphology. However, it takes much time and effort to recover suitable adult specimens because they are borers inside wood. Frass of Lyctus species can easily be detected and recovered in and around infested wood. Thus, frass was tested to see if it was a suitable sample to allow development of a rapid and technically easy molecular detection and identification method for L.brunneus. Species-specific primers were designed from the cytochrome c oxidase subunit I region of L.brunneus and used in development and testing of methods for successfully identifying them from their frass using the loop-mediated isothermal amplification (LAMP) or species-specific nested polymerase chain reaction (PCR) assays. The LAMP assay was faster and more sensitive for detecting the presence of DNA derived from L.brunneus in their frass than the nested PCR assay. These methodologies will be applicable for the rapid detection and identification of other wood-boring invasive pests in regulatory applications.

Plasmodium genome in blood donors at risk for malaria after several years of residence in Italy

At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy. Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 10(3) -8.5 × 10(4) copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.

High prevalence and genetic diversity of Plasmodium malariae and no evidence of Plasmodium knowlesi in Bangladesh

Although the prevalence of malaria remains high in parts of Bangladesh, there continues to be a substantial shortage of information regarding the less common malaria parasites such as Plasmodium malariae or Plasmodium knowlesi. Recent studies indicate that P. malariae may be extremely rare, and so far, there are no data on the presence (or absence) of P. knowlesi in southeastern Bangladesh. Genus- and species-specific nested polymerase chain reaction (PCR) analysis of the small subunit ribosomal RNA gene was performed to assess the presence and prevalence of P. malariae and P. knowlesi in 2,246 samples originating from asymptomatic and febrile participants of a cross-sectional and a febrile illnesses study in the Chittagong Hill Tracts in southeastern Bangladesh. P. malariae was detected in 60 samples (2.7%) corresponding to 8% of the 746 samples giving positive PCR results for Plasmodium sp., mainly because of the high prevalence (9.5%) among asymptomatic study participants testing positive for malaria. Symptomatic cases were more common (4.3% of all symptomatic malaria cases) during the dry season. Parasitemias were low (1,120-2,560/μl in symptomatic and 120-520/μl in asymptomatic carriers). Symptomatic patients presented mild to moderate symptoms like fever, chills, headache, dizziness, fatigue and myalgia.Although both the intermediate as well as the definite host are known to be endemic in southeastern Bangladesh, no evidence for the presence of P. knowlesi was found. We conclude that the role of P. malariae is highly underestimated in rural Bangladesh with major implications for malaria control and elimination strategies.

Investigating the prevalence of transfusion transmission of Plasmodium within a hyperendemic blood donation system

Prevention of transfusion-transmitted malaria in at-risk children and pregnant women in endemic areas with inexpensive chloroquine is no longer effective due to widespread drug resistance. There is an urgent need for devising new strategies for transfusion malarial safety. We investigated the frequency of transfusion transmission of malaria within the Ghanaian blood donation system using blood donations from 106 asymptomatic adult Ghanaian blood donors. Paired samples from 106 blood donations and recipients (before and after transfusion) were tested for anti-merozoite surface protein-1/2 using the commercial Lab21 malaria enzyme immunoassay (EIA), four antigen-specific in-house EIAs, and Plasmodium lactate dehydrogenase (pLDH) EIA. Additionally, Plasmodium DNA was screened for using a species-specific nested polymerase chain reaction (PCR) and a Pan-Plasmodium quantitative PCR. Donor-recipient parasite identity was defined by two concordant genotyping strategies. Plasmodium antibody prevalence was 100% in both donors and recipients, with at least one antigen. Parasitemia prevalence was 54.7% in both donors and recipients with median levels of 20 and 5.3 copies/μL, respectively, the difference being correlated to age (p = 0.0001). Multiple species infections were frequent (8.5%). Twenty-four units of parasitemic blood were transfused to nonparasitemic recipients, of which 10 (41.7%) became infected after transfusion. Molecular genotyping with 13 distinct markers (antigenic genes and microsatellite loci) identified three to nine parasitemic recipients after transfusion with level of allelic identity suggesting 14% to 28% definite or possible transfusion-related parasitemia. None of the currently available screening assays appear suitable to minimize transfusion malaria without compromising the blood supply in endemic areas.

Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat, Central India: implications for the diagnosis of malaria and its control

During an epidemiological study (January-July 2012) on malaria in forest villages of Central India, Plasmodium malariae-like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species-specific nested polymerase chain reaction (PCR) and DNA sequencing. All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae. Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3-day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum. Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re-affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria.

Molecular Survey ofAnaplasmaandEhrlichiaInfections of Feral Raccoons (Procyon lotor) in Hokkaido, Japan

Infection by Anaplasma and Ehrlichia in feral raccoons (Procyon lotor) in Hokkaido, Japan, was examined by molecular methods. A polymerase chain reaction (PCR) screen for Anaplasmataceae, based on 16S rRNA, showed that 38 (5.4%) of 699 raccoons examined were positive. These 38 positive samples were examined for Anaplasma phagocytophilum, Anaplasma bovis, Ehrlichia chaffeensis, and Ehrlichia canis infection by species-specific nested PCR. Nested PCR results indicated that 36 of the 38 samples were positive for A. bovis. All 38 samples were PCR negative for A. phagocytophilum, E. chaffeensis, and E. canis. This is the first report of the detection of A. bovis in the peripheral blood of raccoons. A total of 124 raccoons were infested with ticks, including Ixodes ovatus, Ixodes persulcatus, and Haemaphysalis spp. The rate of A. bovis infection in raccoons infested with Haemaphysalis spp. (46.7%, 7/15) was significantly higher than that in raccoons without Haemaphysalis spp. infestation (3.7%, 4/109, p < 0.001). No significant differences were observed in A. bovis infection rates between raccoons infested with I. ovatus or I. persulcatus and those not so infested. A total of four ticks (two males and two nymphs) and one larval pools from four raccoons showed positive for A. bovis-specific nested PCR. This results support the correlation between the A. bovis infection of raccoons and Haemaphysalis infestation. In conclusion, raccoons could be possible reservoir animals for A. bovis, and A. bovis infection in raccoons may be related to infestation with Haemaphysalis spp.

Indigenous Plasmodium ovale Malaria in Bangladesh

In spite of the high prevalence of malaria in Southeastern Bangladesh, there remains a significant shortage of information regarding the presence of three of five human malaria parasites: Plasmodium ovale, P. malariae, and P. knowlesi. The presence of P. ovale and P. knowlesi has previously never been reported from Bangladesh. We used a genus- and species-specific nested polymerase chain reaction, targeting highly conserved regions of the small subunit ribosomal RNA (SSU rRNA) gene, to investigate the presence of malaria parasites in a total number of 379 patient samples in a survey of patients with febrile illnesses in the Chittagong Hill Tracts in Southeastern Bangladesh. We identified the first cases of P. ovale in Bangladesh. They were confirmed by sequence analysis; 189 of 379 samples (49.9%; 95% confidence interval = 44.9-54.9%) were positive for Plasmodium sp. by PCR. P. falciparum monoinfections accounted for 68.3% (61.3-74.5%), followed by P. vivax (15.3%; 10.9-21.2%), P. malariae (1.6%; 0.5-4.6%), P. ovale (1.6%; 0.5-4.6%), and mixed infections (13.2%; 9.1-18.8%). We found no evidence of P. knowlesi in this region.

Molecular Detection of Bartonella alsatica in European Wild Rabbits (Oryctolagus cuniculus) in Andalusia (Spain)

A sample of 279 European wild rabbits, Oryctolagus cuniculus (141 males, 138 females), captured alive in Andalusia (Spain) and belonging to the two haplotype classes previously described for this species (230 and 49 corresponding with haplotypes A and B, respectively), were tested for the presence of Bartonella alsatica DNA. Two species-specific nested polymerase chain reaction assays targeting for 16S-23S rRNA intergenic spacer region and RNA polymerase β subunit genes have been developed. Forty-eight (17.20%) rabbits were infected with B. alsatica. Two-way contingency table analyses and the calculation of Cramer's V statistic showed no differences in infection rate, considering haplotype lineage or sex. The risk of infection of human population, especially for hunters in close contact with this demonstrated human pathogen, should be considered.

Molecular Survey for &lt;i&gt;Anaplasma bovis&lt;/i&gt; and &lt;i&gt;Anaplasma phagocytophilum&lt;/i&gt; Infection in Cattle in a Pastureland Where Sika Deer Appear in Hokkaido, Japan

Anaplasma bovis and Anaplasma phagocytophilum infection were examined by species-specific nested polymerase chain reaction in cattle in a pastureland where sika deer appear in Hokkaido, Japan. Of the 78 cattle examined, 12 (15%) and 1 (1%) tested positive for infection by A. bovis and A. phagocytophilum, respectively. One cattle tested positive for both. A. phagocytophilum infection rates were significantly lower in cattle than in sika deer (46%), but the infection rate by A. bovis was not significantly different between cattle (15%) and sika deer (23%). The strain of A. phagocytophilum detected in this study may possess significantly lower virulence or infectivity in cattle hosts. No clinical symptoms were recorded in the positive cattle, and morulae were not detected in the blood smears.

Differences in prevalence of selected bacterial species in primary endodontic infections from two distinct geographic locations

This study intended to compare the prevalence of 7 putative endodontic pathogens in samples of primary endodontic infections taken from patients of 2 distant geographic locations. Samples from infected root canals associated with asymptomatic periradicular lesions or from pus aspirated from acute periradicular abscesses were collected from patients in Rio de Janeiro, Brazil, and Seoul, South Korea. South Korean samples were frozen and delivered to Brazil, where all steps in the molecular analysis were performed. DNA was extracted and a species-specific nested polymerase chain reaction assay was used to detect 7 target bacterial species. The most prevalent species detected in Brazilian samples were Porphyromonas endodontalis (79% of the cases) , Treponema denticola (79%), and Dialister pneumosintes (76%). The most prevalent species found in South Korean samples were Fusobacterium nucleatum (38% of the cases) , Tannerella forsythia (26%), and Treponema maltophilum (24%). Overall, P endodontalis, D pneumosintes, Filifactor alocis, T denticola, and T forsythia were significantly more detected in Brazilian samples than in South Korean samples ( P < .05). Findings indicated that the prevalence of some species in infections of endodontic origin may significantly differ from one geographic location to another.