Low Levels Of Parasitemia Research Articles

Single-Cell Optical Absorbance Characterization With High-Throughput Microfluidic Microscopy

Morphological changes in cells associated with disease states are often assessed using clinical microscopy. However, the changes in chemical composition of cells can also be used to detect disease conditions. Optical absorption measurements carried out on single cells using inexpensive sources, detectors can help assess the chemical composition of cells; thereby enable detection of diseases. In this article, we present a novel technique capable of simultaneously detecting changes in morphology and chemical composition of cells. The presented technique enables characterization of optical absorbance-based methods against microscopy for detection of disease states. Using the technique, we have been able to achieve a throughput of about 1000 cells per second. We demonstrate the proof-of-principle by detecting malaria in a given blood sample. The presented technique is capable of detecting very lower levels of parasitemia within time scales comparable to antigen-based rapid diagnostic tests.

History of malaria treatment as a predictor of subsequent subclinical parasitaemia: a cross-sectional survey and malaria case records from three villages in Pailin, western Cambodia.

BackgroundTreatment of the sub-clinical reservoir of malaria, which may maintain transmission, could be an important component of elimination strategies. The reliable detection of asymptomatic infections with low levels of parasitaemia requires high-volume quantitative polymerase chain reaction (uPCR), which is impractical to conduct on a large scale. It is unknown to what extent sub-clinical parasitaemias originate from recent or older clinical episodes. This study explored the association between clinical history of malaria and subsequent sub-clinical parasitaemia.MethodsIn June 2013 a cross-sectional survey was conducted in three villages in Pailin, western Cambodia. Demographic and epidemiological data and blood samples were collected. Blood was tested for malaria by high-volume qPCR. Positive samples were analysed by nested PCR to determine the Plasmodium species. To identify previous episodes of malaria, case records were collected from village malaria workers and local health facilities and linked to study participants.ResultsAmong 1343 participants, 40/122 (32.8 %) with a history of clinical malaria were parasitaemic during the cross-sectional survey, compared to 172/1221 (14.1 %) without this history (p < 0.001). Among the 212 parasitaemic participants in the survey, 40 (18.9 %) had a history of clinical malaria, compared to 87 out of 1131 (7.7 %) parasite-negative participants; p < 0.001, adjusted OR 3.3 (95 % CI; 2.1–5.1). A history of Plasmodium vivax was associated with sub-clinical P. vivax parasitaemia in the survey (p < 0.001), but this association was not seen with Plasmodium falciparum (p = 0.253); only three participants had both P. falciparum parasites in the survey and a clinical history of P. falciparum.ConclusionsA clinical episode of vivax malaria was associated with subsequent sub-clinical parasitaemia. Treatment of P. vivax with artemisinin-based combination therapy without primaquine often resulted in recurrent episodes. Targeting individuals with a history of clinical malaria will be insufficient to eliminate the sub-clinical reservoir as they constitute a minority of parasitaemias.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

Application of magnetic cytosmear for the estimation of Plasmodium falciparum gametocyte density and detection of asexual stages in asymptomatic children.

BackgroundConventional malaria parasite detection methods, such as rapid diagnostic tests (RDT) and light microscopy (LM), are not sensitive enough to detect low level parasites and identification of gametocytes in the peripheral blood. A modified and sensitive laboratory prototype, Magnetic Deposition Microscopy (MDM) was developed to increase the detection of sub-microscopic parasitaemia and estimation of gametocytes density in asymptomatic school children.MethodsBlood samples were collected from 303 asymptomatic school children from seven villages in Bagamoyo district in Tanzania. Participants were screened for presence of malaria parasites in the field using RDT and MDM whereas further examination of malaria parasites was done in the laboratory by LM. LM and MDM readings were used to calculate densities and estimate prevalence of asexual and sexual stages of the parasite.ResultsPlasmodium falciparum parasites (asexual and sexual stages) were detected in 23 (7.6 %), 52 (17.2 %), and 59 (19.5 %) out of 303 samples by LM, RDT and MDM respectively. Gametocytes were detected in 4 (1.3 %) and 12 (4.0 %) out of the same numbers of samples by LM, and MDM, respectively. Likewise, in vitro results conducted on two laboratory strains of P. falciparum, 3D7 and NF54 to assess MDM sensitivity on gametocytes detection and its application on concentrating gametocytes indicated that gametocytes were enriched by MDM by 10-fold higher than LM. Late stages of the parasite strains, 3D7 and NF54 were enriched by MDM by a factor of 20.5 and 35.6, respectively. MDM was more specific than LM and RDT by 87.5 % (95 %, CI 71.2–89.6 %) and 89.0 % (95 % CI 82.9–91.4) respectively. It was also found that MDM sensitivity was 62.5 % (95 % CI 49.5–71.8) when compared with RDT while with LM was 36.5 % (95 % CI 32.2–60.5).ConclusionsThese findings provide strong evidence that MDM enhanced detection of sub-microscopic P. falciparum infections and estimation of gametocyte density compared to current malaria diagnostic tools. In addition, MDM is superior to LM in detecting sub-microscopic gametocytaemia. Therefore, MDM is a potential tool for low-level parasitaemia identification and quantification with possible application in malaria transmission research.

Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels.

The malaria SYBR green assay, which is used to profilein vitrodrug susceptibility ofPlasmodium falciparum, is a reliable drug screening and surveillance tool. Malaria field surveillance efforts provide isolates with various low levels of parasitemia. To be advantageous, malaria drug sensitivity assays should perform reproducibly among various starting parasitemia levels rather than at one fixed initial value. We examined the SYBR green assay standardized procedure developed by the Worldwide Antimalarial Resistance Network (WWARN) for its sensitivity and ability to accurately determine the drug concentration that inhibits parasite growth by 50% (IC50) in samples with a range of initial parasitemia levels. The initial sensitivity determination of the WWARN procedure yielded a detection limit of 0.019% parasitemia.P. falciparumlaboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC50s were comparable for laboratory strains with between 0.0375% and 0.6% parasitemia and for field isolates with between 0.075% and 0.6% parasitemia for all drugs tested. Furthermore, assay quality (Z') analysis indicated that the WWARN procedure displays high robustness, allowing for drug testing of malaria field samples within the derived range of initial parasitemia. The use of the WWARN procedure should allow for the inclusion of more malaria field samples in malaria drug sensitivity screens that would have otherwise been excluded due to low initial parasitemia levels.

Consistent prevalence of asymptomatic infections in malaria endemic populations in Colombia over time.

BackgroundMalaria control programmes rely on confirmation of parasite presence in patients’ blood prior to treatment administration. Plasmodium parasites are detected mostly by microscopy or rapid diagnostic test (RDT). Although these methods contribute significantly to malaria control/elimination, they are not suitable for detecting the significant proportion of asymptomatic subjects harbouring low levels of parasitaemia, which endure untreated as potential reservoirs for transmission. Malaria prevalence was assessed in endemic regions of Colombia over a 4-year follow-up.MethodsA series of cross-sectional surveys were conducted between 2011 and 2014 in low to moderate malaria transmission sentinel sites (SS) of Tumaco, Buenaventura and Tierralta municipalities of Colombia. A census was performed and a random sample of houses was selected from each SS prior to each survey. Inhabitants were asked to answer a questionnaire on clinical, epidemiological and demographic aspects, and to provide a blood sample for malaria diagnosis using microscopy and quantitative real time polymerase chain reaction (qPCR).ResultsA total of 3059 blood samples were obtained from all SS, 58.5 % of which were from women and displayed a malaria prevalence ranging from 4 % (95 % CI 3–5 %) to 10 % (95 % CI 8–12 %) in the 4 years’ study period. Almost all malaria cases (n = 220, 97 %) were sub-microscopic and only detectable by qPCR; 90 % of the cases were asymptomatic at the time of blood collection. While Buenaventura and Tierralta had a decreasing tendency during the follow-up, Tumaco had a rise in 2013 and then a decrease in 2014. Plasmodium vivax accounted for the majority (66–100 %) of cases in Tierralta and Buenaventura and for 25–50 % of the cases in Tumaco.ConclusionsThis study demonstrates an important prevalence of asymptomatic malaria cases not detectable by microscopy, which therefore remain untreated representing a parasite pool for malaria transmission. This demands the introduction of alternative strategies for diagnosis and treatment, especially for areas of low transmission to reduce it to appropriate levels for malaria pre-elimination efforts to start.

Canine Rangeliosis: A Rare Case of Hyperparasitemia in the Acute Phase

Background: R. vitalii causes a remerging tick-borne disease known as rangeliosis. The parasite is not always identifable in blood smears, especially in the chronic phase of the disease. Low parasitemia levels have been observed in cases of rangeliosis caused by natural infection, even in acute situations, while hyperparasitemia has been reported only in acuteexperimental infection. This paper describes an unusual case of acute natural R. vitalii infection with hyperparasitemia.Case: The dog (a 12-year-old male German Shepherd) had presented apathy, dyschezia and hyporexia for three days prior to seeing the veterinarian, whose examination revealed discrete pale mucous membranes, soft bloody stools, hyperthermia, splenomegaly and lethargy. Numerous intra-erythrocytic forms, as well as free-living parasites compatible with R. vitalii and/or Babesia sp, were also found in the CBC (complete blood count), and the parasite load was estimated at 12 parasites/feld – x1000. After diagnosing hemoparasitosis from the blood smear, therapy was started immediately. After 18 days of treatment, the animal returned to the veterinary hospital showing visibly improved health. The dog’s mucous membranes showed normal coloration. A new CBC showed no intra-erythrocytic parasite in the blood smear. Some of the blood drawn during the animal’s frst examination was sent for DNA extraction. Two specifc TaqMan real-time PCR-based assays were performed to test for R. vitalii, and Ehrlichia canis. The sample was also tested for Babesia (Babesia canis and Babesia gibsoni), but tested positive only for R. vitalii.Discussion: Clinical signs related to the disease depend on its acute, subclinical, and chronic evolution, which may be reflected in clinical and pathological conditions. Our dog presented mild clinical signs of the disease, such as apathy, lethargy, hyporexia, hyperthermia, discrete pale mucous membranes, splenomegaly, dyschezia and soft bloody stools.In acute experimental cases, parasitemia increases progressively after inoculation, when R. vitalii becomes detectable in erythrocytes and leukocytes. In the chronic form of the disease, it is particularly rare to identify free-living forms of R. vitalii in the bloodstream or of the parasite in erythrocytes, and they are identifed in only in few cases of natural infection. The blood smear of our canine patient contained numerous parasitized cells, showing different shapes and sizes of the parasite, as well as a variable number of microorganisms parasitizing each cell. The number of parasitized erythrocytes was comparatively higher than that of leukocytes. This case report reveals that acute natural canine rangeliosis with hyper parasitemia is possible, indicating that the acute phase of the disease does not occur only in experimental cases. Although the animal showed nonspecifc clinical signs in the acute phase, the blood smear and PCR enabled the detection of the parasite. This leads us to suggest that, to ensure a better diagnosis, treatment and prognosis of rangeliosis, veterinarians should also determine whether the disease is in the acute or chronic stage.Keywords: Rangelia vitalii, natural acute phase, high parasitemia, dogs.

Field Evaluation of Malaria Microscopy, Rapid Malaria Tests and Loop-Mediated Isothermal Amplification in a Rural Hospital in South Western Ethiopia

BackgroundIn up to one third of the hospitals in some rural areas of Africa, laboratory services in malaria diagnosis are limited to microscopy by thin film, as no capability to perform thick film exists (gold standard in terms of sensitivity for malaria diagnosis). A new rapid molecular malaria diagnostic test called Loop-mediated isothermal DNA amplification (LAMP) has been recently validated in clinical trials showing exceptional sensitivity and specificity features. It could be a reliable diagnostic tool to be implemented without special equipment or training.ObjectiveThe objective of this proof of concept study was to confirm the feasibility of using LAMP technique for diagnosis of malaria in a rural Ethiopian hospital with limited resources.Methodology/Principal FindingsThis study was carried out in Gambo General Hospital, West Arsi Province (Ethiopia), from November 1st to December 31st 2013. A total of 162 patients with a non-focal febrile syndrome were investigated. The diagnostic capability (sensitivity, specificity, positive predictive and negative predictive values) of rapid malaria tests and microscopy by thin film was evaluated in comparison with LAMP. Eleven (6.79%) out of the 162 patients with fever and suspected malaria, tested positive for LAMP, 3 (1.85%) for rapid malaria tests and none of the eleven cases was detected by thin film microscopy.Conclusions/SignificanceLAMP can be performed in basic rural laboratories without the need for specialized infrastructure and it may set a reliable tool for malaria control to detect a low level parasitemia.

Optofluidic single-cell absorption flow analyzer for point-of-care diagnosis of malaria.

In this work, an optofluidic flow analyzer, which can be used to perform malaria diagnosis at the point-of-care is demonstrated. The presented technique is based on quantitative optical absorption measurements carried out on a single cell level for a given population of Human Red Blood Cells (RBCs). By measuring the optical absorption of each RBC, the decrease in the Hemoglobin (Hb) concentration in the cytoplasm of the cell due to the invasion of malarial parasite is detected. Cells are assessed on a single cell basis, as they pass through a microfluidic channel. The proposed technique has been implemented with inexpensive off-the-shelf components like laser diode, photo-detector and a micro-controller. The ability of the optofluidic flow analyzer to asses about 308,049 cells within 3 minutes has been demonstrated. The presented technique is capable of detecting very low parasitemia levels with high sensitivity.

Cytauxzoonosis in cats: ABCD guidelines on prevention and management.

Cytauxzoon species are apicomplexan haemoparasites, which may cause severe disease in domestic cats, as well as lions and tigers. For many years, cytauxzoonosis in domestic cats was only reported in North and South America, but in recent years the infection has also been seen in Europe (Spain, France and Italy). Cytauxzoon felis is the main species; it occurs as numerous different strains or genotypes and is transmitted via ticks. Therefore, the disease shows a seasonal incidence from spring to early autumn and affects primarily cats with outdoor access in areas where tick vectors are prevalent. Domestic cats may experience subclinical infection and may also act as reservoirs. Cytauxzoonosis caused by C felis in the USA is an acute or peracute severe febrile disease with non-specific signs. Haemolytic anaemia occurs frequently; in some cats neurological signs may occur in late stages. The Cytauxzoon species identified in Europe differ from C felis that causes disease in the USA and are probably less virulent. The majority of infected cats have been healthy; in some cases anaemia was found, but disease as it occurs in the USA has not been reported to date. Diagnosis is usually obtained by Cytauxzoon detection in blood smears and/or fine-needle aspirates from the liver, spleen and lymph nodes. PCR assays are able to detect low levels of parasitaemia and may be used for confirmation. Currently a combination of the antiprotozoal drugs atovaquone and azithromycin is the treatment of choice. Concurrent supportive and critical care treatment is extremely important to improve the prognosis. Cats that survive the infection may become chronic carriers for life. Cats with outdoor access in endemic areas should receive effective tick treatment.

Enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection.

Despite significant advancements over the years, there remains an urgent need for low cost diagnostic approaches that allow for rapid, reliable and sensitive detection of malaria parasites in clinical samples. Our previous work has shown that magnetic resonance relaxometry (MRR) is a potentially highly sensitive tool for malaria diagnosis. A key challenge for making MRR based malaria diagnostics suitable for clinical testing is the fact that MRR baseline fluctuation exists between individuals, making it difficult to detect low level parasitemia. To overcome this problem, it is important to establish the MRR baseline of each individual while having the ability to reliably determine any changes that are caused by the infection of malaria parasite. Here we show that an approach that combines the use of microfluidic cell enrichment with a saponin lysis before MRR detection can overcome these challenges and provide the basis for a highly sensitive and reliable diagnostic approach of malaria parasites. Importantly, as little as 0.0005% of ring stage parasites can be detected reliably, making this ideally suited for the detection of malaria parasites in peripheral blood obtained from patients. The approaches used here are envisaged to provide a new malaria diagnosis solution in the near future.

Submicroscopic and asymptomatic Plasmodium falciparum and Plasmodium vivax infections are common in western Thailand - molecular and serological evidence.

BackgroundMalaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels.MethodsThree-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals.ResultsOf 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others.ConclusionThese findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0611-9) contains supplementary material, which is available to authorized users.

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n=105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2=0.694, P=0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry.

Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine's mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite's life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.

Comparative pathogenicity in Swiss mice of Trypanosoma cruzi IV from northern Brazil and Trypanosoma cruzi II from southern Brazil

The geographical heterogeneity of Chagas disease (ChD) is mainly caused by genetic variability of the etiological agent Trypanosoma cruzi. Our hypothesis was that the pathogenicity for mice may vary with the genetic lineage (or Discrete Typing Unit – DTU) of the parasite. To test this hypothesis, parasitological and histopathological evaluations were performed in mice inoculated with strains belonging to the DTU T. cruzi IV (TcIV) from the State of Amazonas (northern Brazil), or the DTU T. cruzi II (TcII) from the State of Paraná (southern Brazil). Groups of 10 Swiss mice were inoculated with eight strains of TcIV obtained from acute cases (7) from two outbreaks of orally acquired ChD, and from the triatomine Rhodnius robustus (1) from Amazonas; and three strains of TcII obtained from chronic patients in Paraná. We evaluated the pre-patent period, patent period, maximum peak of parasitemia, day of maximum peak of parasitemia, area under the parasitemia curve, inflammatory process, and tissue parasitism in the acute phase. TcIV was less virulent than TcII, and showed significantly (p < 0.005) lower parasitemia levels. Although the levels of tissue parasitism did not differ statistically, mice infected with TcIV displayed significantly (p < 0.001) fewer inflammatory processes than mice infected with TcII. This supported the working hypothesis, since TcIV from Amazonas was less pathogenic than TcII from Paraná; and agreed with the lower severity of human cases of ChD in the Amazon region.

Performance of a new gelled nested PCR test for the diagnosis of imported malaria: comparison with microscopy, rapid diagnostic test, and real-time PCR

Microscopy and rapid diagnostic tests (RDTs) are the techniques commonly used for malaria diagnosis but they are usually insensitive at very low levels of parasitemia. Nested PCR is commonly used as a reference technique in the diagnosis of malaria due to its high sensitivity and specificity. However, it is a cumbersome assay only available in reference centers. We evaluated a new nested PCR-based assay, BIOMALAR kit (Biotools B&M Labs, Madrid, Spain) which employs ready-to-use gelled reagents and allows the identification of the main four species of Plasmodium. Blood samples were obtained from patients with clinical suspicion of malaria. A total of 94 subjects were studied. Fifty-two (55.3%) of them were malaria-infected subjects corresponding to 48 cases of Plasmodium falciparum, 1 Plasmodium malariae, 2 Plasmodium vivax, and 1 Plasmodium ovale. The performance of the BIOMALAR test was compared with microscopy, rapid diagnostic test (RDT) (BinaxNOW® Malaria) and real-time quantitative PCR (qPCR). The BIOMALAR test showed a sensitivity of 98.1% (95% confidence interval [CI], 89.7-100), superior to microscopy (82.7% [95% CI, 69.7-91.8]) and RDT (94.2% [95% CI, 84.1-98.8]) and similar to qPCR (100% [95% CI, 93.2-100]). In terms of specificity, the BIOMALAR assay showed the same value as microscopy and qPCR (100% [95% CI, 93.2-100]). Nine subjects were submicroscopic carriers of malaria. The BIOMALAR test identified almost all of them (8/9) in comparison with RDT (6/9) and microscopy (0/9). In conclusion, the BIOMALAR is a PCR-based assay easy to use with an excellent performance and especially useful for diagnosis submicroscopic malaria.

Implications of low-density microfilariae carriers in Anopheles transmission areas: molecular forms of Anopheles gambiae and Anopheles funestus populations in perspective

BackgroundPrevious studies have shown a general reduction in annual transmission potential (ATP) of Anopheles species after mass drug administration (MDA) in lymphatic filariasis endemic communities. Whereas results obtained from a monitoring programme after three years of MDA revealed a decrease in ATP of Anopheles funestus this was not the same for An. gambiae s.s. in Ghana. In this study, the ability of these vectors in transmitting Wuchereria bancrofti in nine lymphatic filariasis endemic communities in Gomoa District of Ghana after four rounds of MDA with ivermectin and albendazole was investigated.MethodsAfter mass screening of inhabitants in these communities, twelve consenting volunteers with different intensities of microfilariae (mf) slept under partly opened mosquito nets as sources of mf blood meal. Hourly collection of mosquitoes and finger-pricked blood were taken from 21.00 to 06.00 hours the following day. For each hour, half of the mosquitoes collected were immediately killed and dissected for mf. The remaining half were maintained up to 13 days for parasite maturation. Parasitaemia and infection rates in the mosquitoes were determined by microscopy. The mosquitoes were identified by microscopy and molecular techniques.ResultsA total of 1,083 participants were screened and the overall parasite prevalence was 1.6% with mf intensities ranging from 0 to 59 per 100 μl and geometric mean intensity of 1.1 mf per ml of blood. Of the 564 mosquitoes collected, 350 (62.1%) were Anopheles spp., from which 310 (88.6%) were An. funestus and 32 (9.1%) An. gambiae. Six anopheline mosquitoes (1.7%) were found infected with L1, but no larva was observed in any of the mosquitoes maintained up to 13 days. Molecular studies showed all An. gambiae s.l. to be An. gambiae s.s., of which 21 (70%) were of the M molecular form.ConclusionAt low-level parasitaemia after 4 rounds of MDA, there was no recovery of infective stage larvae of W. bancrofti in An. funestus s.l. as well as M and S forms of An. gambiae.

A longitudinal study of Babesia microti infection in seropositive blood donors

Babesia infection is caused by intraerythrocytic tick-borne parasites. Cases of transfusion-transmitted babesiosis have been increasingly recognized. To date, no Babesia test has been licensed for screening US blood donors. We conducted a longitudinal study to assess the course and markers of Babesia infection among seropositive donors identified in a seroprevalence study. Eligible donors had B. microti indirect fluorescent antibody (IFA) titers of 64 or greater. Enrollees were monitored up to 3 years, by IFA and three methods for evidence of parasitemia: B. microti nested polymerase chain reaction (PCR) analysis (at two laboratories), hamster inoculation, and blood-smear examination. Among 115 eligible donors, 84 (73%) enrolled. Eighteen enrollees (21%) had evidence of parasitemia for 30 total specimens (17% of 181), which were collected in 9 different months and tested positive by various approaches: PCR (25 specimens/16 persons), hamster inoculation (13 specimens/8 persons), and blood smear (one specimen positive by all three approaches). Overall, 14 persons had one or more specimen with positive PCR results at both laboratories (12 persons) and/or had parasitologically confirmed infection (eight persons). Three of nine persons who had more than one specimen with evidence of parasitemia had nonconsecutive positives. Several enrollees likely had been infected at least 1 year when their last positive specimen was collected. The final three specimens for seven persons tested negative by all study methods, including IFA. Seropositive blood donors can have protracted low-level parasitemia that is variably and intermittently detected by parasitologic and molecular methods. Donor-screening algorithms should include serologic testing and not solely rely on molecular testing.

Editorial Commentary: Xenodiagnosis for Posttreatment Lyme Disease Syndrome: Resolving the Conundrum or Adding to It?

Editorial Commentary: Xenodiagnosis for Posttreatment Lyme Disease Syndrome: Resolving the Conundrum or Adding to It?