Trypanosome SL-RNA detection in blood and cerebrospinal fluid to demonstrate active gambiense human African trypanosomiasis infection.

BACKGROUND: Spliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF. METHODOLOGY/PRINCIPAL FINDINGS: Blood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood (78/97 positive, 80.4%, p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values. CONCLUSIONS/SIGNIFICANCE: Detection of SL-RNA in blood and CSF allows sensitive demonstration of active gambiense HAT infection, even if trypanosomes remain undetectable in blood or lymph. As this condition often occurs in treatment failures, SL-RNA detection in blood and CSF for early detection of relapses after treatment deserves further investigation. TRIAL REGISTRATION: This study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier: NCT03112655).

Detection of Babesia RNA and DNA in whole blood samples from US blood donations.

BACKGROUND: Human babesiosis is a zoonotic infection caused by an intraerythrocytic parasite. The highest incidence of babesiosis is in the United States, although cases have been reported in other parts of the world. Due to concerns of transfusion-transmitted babesiosis, the US Food and Drug Administration (FDA) recommended year-round regional testing for Babesia by nucleic acid testing or use of an FDA-approved device for pathogen reduction. A new molecular test, cobas Babesia (Roche Molecular Systems, Inc.), was evaluated for the detection of the four species that cause human disease, Babesia microti, Babesia duncani, Babesia divergens, and Babesia venatorum. STUDY DESIGN AND METHODS: Analytical performance was evaluated followed by clinical studies on whole blood samples from US blood donations collected in a special tube containing a chaotropic reagent that lyses the red cells and preserves nucleic acid. Sensitivity and specificity of the test in individual samples (individual donation testing [IDT]) and in pools of six donations were determined. RESULTS: Based on analytical studies, the claimed limit of detection of cobas Babesia for B. microti is 6.1 infected red blood cells (iRBC)/mL (95% confidence interval [CI]: 5.0, 7.9); B. duncani was 50.2 iRBC/mL (95% CI: 44.2, 58.8); B. divergens was 26.1 (95% CI: 22.3, 31.8); and B. venatorum was 40.0 iRBC/mL (95% CI: 34.1, 48.7). The clinical specificity for IDT was 99.999% (95% CI: 99.996, 100) and 100% (95% CI: 99.987, 100) for pools of six donations. CONCLUSION: cobas Babesia enables donor screening for Babesia species with high sensitivity and specificity.

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain.

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3-98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7-100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered.

Stage-specific Plasmodium falciparum immune responses in afebrile adults and children living in the Greater Accra Region of Ghana.

BACKGROUND: Asymptomatic carriage of Plasmodium falciparum is widespread in adults and children living in malaria-endemic countries. This study identified the prevalence of malaria parasites and the corresponding levels of naturally acquired anti-parasite antibody levels in afebrile adults living in two communities in the Greater Accra Region of Ghana. METHODS: Two cross-sectional studies conducted in January and February 2016 and repeated in July and August 2016 recruited subjects aged between 6 and 75 years from high parasite prevalence (Obom) and low parasite prevalence (Asutsuare) communities. Whole blood (5 ml) was collected from each volunteer, plasma was aliquoted and frozen until needed. An aliquot (10 µl) of the blood was used to prepare thick and thin blood smears, 100 µl was preserved in Trizol and the rest was separated into plasma and blood cells and each stored at - 20 °C until needed. Anti-MSP3 and Pfs230 antibody levels were measured using ELISA. RESULTS: Asexual parasite and gametocyte prevalence were higher in Obom than Asutsuare. Antibody (IgG, IgG1, IgG3, IgM) responses against the asexual parasite antigen MSP3 and gametocyte antigen Pfs230 were higher in Obom during the course of the study except for IgM responses against Pfs230, which was higher in Asutsuare than in Obom during the rainy season. Antibody responses in Asutsuare were more significantly associated with age than the responses measured in Obom. CONCLUSION: The pattern of antibody responses measured in people living in the high and low malaria transmission setting was similar. All antibody responses measured against the asexual antigen MSP3 increased, however, IgG and IgG1 responses against gametocyte antigen Pfs230 decreased in moving from the dry to the peak season in both sites. Whilst asexual and gametocyte prevalence was similar between the seasons in the low transmission setting, in the high transmission setting asexual parasite prevalence increased but gametocyte prevalence decreased in the rainy season relative to the dry season.

qRT-PCR versus IFA-based Quantification of Male and Female Gametocytes in Low-Density Plasmodium falciparum Infections and Their Relevance for Transmission.

BACKGROUND: Accurate quantification of female and male gametocytes and sex ratios in asymptomatic low-density malaria infections are important for assessing their transmission potential. Gametocytes often escape detection even by molecular methods, therefore ultralow gametocyte densities were quantified in large blood volumes. METHODS: Female and male gametocytes were quantified in 161 PCR-positive Plasmodium falciparum infections from a cross-sectional survey in Papua New Guinea. Ten-fold concentrated RNA from 800 µL blood was analyzed using female-specific pfs25 and male-specific pfmget or mssp qRT-PCR. Gametocyte sex ratios from qRT-PCR were compared with those from immunofluorescence assays (IFA). RESULTS: Gametocytes were identified in 58% (93/161) P. falciparum-positive individuals. Mean gametocyte densities were frequently below 1 female and 1 male gametocyte/µL by qRT-PCR. The mean proportion of males was 0.39 (95% confidence interval, 0.33-0.44) by pfs25/pfmget qRT-PCR; this correlated well with IFA results (Pearsons r2 = 0.91; P < .001). A Poisson model fitted to our data predicted 16% P. falciparum-positive individuals that are likely to transmit, assuming at least 1 female and 1 male gametocyte per 2.5 µL mosquito bloodmeal. CONCLUSIONS: Based on model estimates of female and male gametocytes per 2.5 µL blood, P. falciparum-positive individuals detected exclusively by ultrasensitive diagnostics are negligible for human-to-mosquito transmission.Estimating the transmission potential of ultralow-density malaria infections informs interventions. Almost all infections with ≥1 female and male gametocyte per 2.5 µL mosquito bloodmeal, and thus with highest likelihood of contributing to human-to-mosquito transmission, were detectable by standard molecular diagnostics.

Genetic Diversity of Theileria equi From Horses In Different Regions of Brazil Based On the 18S rRNA Gene.

Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (∼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.

Minimal infectious dose and dynamics of Babesia microti parasitemia in a murine model.

BACKGROUND: Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. STUDY DESIGN AND METHODS: Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. RESULTS: In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. CONCLUSION: The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

Direct Detection of Unamplified Pathogen RNA in Blood Lysate using an Integrated Lab-in-a-Stick Device and Ultrabright SERS Nanorattles.

Direct detection of genetic biomarkers in body fluid lysate without target amplification will revolutionize nucleic acid-based diagnostics. However, the low concentration of target sequences makes this goal challenging. We report a method for direct detection of pathogen RNA in blood lysate using a bioassay using surface-enhanced Raman spectroscopy (SERS)-based detection integrated in a "lab-in-a-stick" portable device. Two levels of signal enhancement were employed to achieve the sensitivity required for direct detection. Each target sequence was tagged with an ultrabright SERS-encoded nanorattle with ultrahigh SERS signals, and these tagged target sequences were concentrated into a focused spot for detection using hybridization sandwiches with magnetic microbeads. Furthermore, the washing process was automated by integration into a "lab-in-a-stick" portable device. We could directly detect synthetic target with a limit of detection of 200 fM. More importantly, we detected plasmodium falciparum malaria parasite RNA directly in infected red blood cells lysate. To our knowledge, this is the first report of SERS-based direct detection of pathogen nucleic acid in blood lysate without nucleic acid extraction or target amplification. The results show the potential of our integrated bioassay for field use and point-of-care diagnostics.

Serological detection and molecular characterization of piroplasmids in equids in Brazil.

Equine piroplasmosis is a disease caused by the hemoparasites Babesia caballi and Theileria equi and is considered to be the most important parasitic infection affecting Equidae. The objective of the present study was to carry out an epidemiological molecular and serological survey for the presence of these two protozoal organisms in equids from the northwestern region of the State of Rio Grande do Sul (RS), south Brazil. For this purpose, blood samples were collected from 90 equids in the city of Passo Fundo, RS, Brazil. Those were animals used for sport activities, outdoor recreational riding, and work including cattle herding and mounted patrol. Anti-T. equi and anti-B. caballi IgG antibodies were detected in the sera of those animals by commercial ELISA kits. The molecular diagnosis of equine piroplasmosis due to T. equi or B. caballi (or both) consisted in the amplification of the 18S rRNA gene by nested PCR followed by sequencing of the amplified PCR product and sequence comparison and phylogenetic analysis of the isolates; 17 (18.9%) and 5 (5.55%) out of the 90 serum samples tested in this study were positive for T. equi and B. caballi, respectively. Piroplasmid 18S rRNA gene fragments were detected by PCR in 24.4% (22/90) of the samples analysed and shared 99-100% identity with sequences of T. equi by BLASTn. Samples for the phylogenetic analysis were divided into 2 groups. In group A, there was close phylogenetic relationship between 4 sequences and sequences previously reported along the US-Mexico border, in South Africa, and in Brazil. There was a phylogenetic proximity between 5 samples from group B and samples tested by other authors in the US and Spain. Variation of the 18S rRNA gene allowed the identification of 9 new T. equi genotypes in the geographical region studied.

Molecular Detection of Hepatozoon canis in Dogs of Ardabil Province, Northwest of Iran.

Hepatozoon species are protozoan parasites that infect some animals such as birds, reptiles, amphibians, and carnivores. Previous studies performed on canine hepatozoonosis in Iran have never used molecular techniques for diagnosis of this disease. The main objective of the present study was to detect Hepatozoon canis in the blood of dogs using polymerase chain reaction (PCR) method and sequencing. A total of 104 blood samples were collected from dogs of Meshginshahr County (Ardabil Province), and DNA was extracted from blood samples by dint of DNG-plus Extraction Kit. Then, 18S rRNA gene was amplified by using the conventional PCR methods. PCR products yielded an amplicon of the approximate length of 897 bp for all the positive samples. Twenty-four out of the 104 (23.07%) samples were found to be positive for H. canis. This rate of infection is relatively high among dogs in Ardabil Province. Sequence analysis confirmed the molecular identity of 99% of the samples by comparison with GenBank profiles. This is the first report of molecular detection of H. canis from Iran.

Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate.

Strand-specific RNA sequencing in Plasmodium falciparum malaria identifies developmentally regulated long non-coding RNA and circular RNA.

BACKGROUND: The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive and precise gene regulation to allow invasion and hijacking of host cells, transmission, and immune escape. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. Yet it is becoming increasingly clear that long non-coding RNAs (lncRNAs) could represent a missing regulatory layer across a broad range of organisms. RESULTS: To investigate the regulatory capacity of lncRNA in P. falciparum, we harvested fifteen samples from two time-courses. Our sample set profiled 56 h of P. falciparum blood stage development. We then developed and validated strand-specific, non-polyA-selected RNA sequencing methods, and pursued the first assembly of P. falciparum strand-specific transcript structures from RNA sequencing data. This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing. We validated the complete splicing structure of three lncRNAs with compelling properties. Non-polyA-selected deep sequencing also enabled the prediction of hundreds of intriguing P. falciparum circular RNAs, six of which we validated experimentally. CONCLUSIONS: We found that a subset of lncRNAs, including all subtelomeric lncRNAs, strongly peaked in expression during invasion. By contrast, antisense transcript levels significantly dropped during invasion. As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference. We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.

Cost-effectiveness of Babesia microti antibody and nucleic acid blood donation screening using results from prospective investigational studies.

BACKGROUND: Babesia microti causes transfusion-transmitted babesiosis (TTB); currently, blood donor screening assays are unlicensed but used investigationally. STUDY DESIGN AND METHODS: We developed a decision tree model assessing the comparative- and cost-effectiveness of B. microti blood donation screening strategies in endemic areas compared to the status quo (question regarding a history of babesiosis), including testing by: (1) universal antibody (Ab), (2) universal polymerase chain reaction (PCR), (3) universal Ab/PCR, and (4) recipient risk-targeted Ab/PCR. The model predicted the number of TTB cases, complicated TTB cases, cases averted, and quality-adjusted life years (QALYs). Economic outcomes included each strategy's per-donation cost, waste (number of infection-free units incorrectly discarded), and waste index (number wasted units/number true positives). Sensitivity analyses examined uncertainty in transmission probabilities, prevalence rates, and other key model inputs. RESULTS: Universal PCR in four endemic states would prevent 24 to 31 TTB cases/100,000 units transfused (pht) at an incremental cost-effectiveness ratio (ICER) of $26,000 to $44,000/QALY (transmission probability dependent) and waste index of zero. Universal Ab/PCR would prevent 33 to 42 TTB cases pht at an ICER of $54,000 to $83,000/QALY and waste index of 0.05. The questionnaire is most wasteful (99.62 units wasted pht; 208.62 waste index), followed by the risk-targeted strategy (76.27 units wasted pht; 0.68 waste index). The model predicted zero cases of TTB or complicated TTB with universal Ab/PCR (versus [33, 42] and [13, 18] pht, respectively [no screening]). Results are highly sensitive to transmission probabilities. CONCLUSIONS: Universal PCR in endemic states is an effective blood donation screening strategy at a threshold of $50,000/QALY. Using a higher cost-effectiveness ratio, universal Ab/PCR is the most effective strategy.

Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.

Trypanosomatids transcribe their genes in large polycistronic clusters that are further processed into mature mRNA molecules by trans-splicing. During this maturation process, a conserved spliced leader RNA (SL-RNA) sequence of 39 bp is physically linked to the 5' end of the pre-mRNA molecules. Trypanosomatid infections cause a series of devastating diseases in man (sleeping sickness, leishmaniasis, Chagas disease) and animals (nagana, surra, dourine). Here, we investigated the SL-RNA molecule for its diagnostic potential using reverse transcription followed by real-time PCR. As a model, we used Trypanosoma brucei gambiense, which causes sleeping sickness in west and central Africa. We showed that the copy number of the SL-RNA molecule in one single parasitic cell is at least 8600. We observed a lower detection limit of the SL-RNA assay in spiked blood samples of 100 trypanosomes per milliliter of blood. We also proved that we can detect the trypanosome's SL-RNA in the blood of sleeping sickness patients with a sensitivity of 92% (95% CI, 78%-97%) and a specificity of 96% (95% CI, 86%-99%). The SL-RNA is thus an attractive new molecular target for next-generation diagnostics in diseases caused by trypanosomatids.

Revealing natural antisense transcripts from Plasmodium vivax isolates: evidence of genome regulation in complicated malaria.

Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation.

Mosaic VSGs and the scale of Trypanosoma brucei antigenic variation.

A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG) coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct 'mosaic' VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection.

[Toxoplasmosis in HIV infection: invasion reactivation criteria].

Contemporary representation of toxoplasmosis reactivation criteria in HIV infection is generalized. Significance of the issue is justified: toxoplasmosis is a leading neurological pathology in AIDS with a high lethality percentage due to complexity of clinical confirmation and difficulties of laboratory confirmation of the start of reactivation. Clinical, instrumental, immunologic, molecular genetic invasion reactivation criteria are discussed in the article and analysis of their effectiveness is performed; their most feasible combinations are justified. Further system analysis of the cerebral toxoplasmosis reactivation criteria specified in the article in combination with search of new pathogen dissemination markers will allow to obtain important information that has both fundamental interest and important practical significance.

Polymerase chain reaction-based tests for pan-species and species-specific detection of human Plasmodium parasites.

BACKGROUND: There is still a need to improve the sensitivity of polymerase chain reaction (PCR) tests for malaria to detect submicroscopic asexual stage Plasmodium infections during the early phase and chronic, asymptomatic phase of infection when the parasite burden is very low. STUDY DESIGN AND METHODS: The inhibitory effect of hemoglobin (Hb) on PCR limits the volume of blood that can be used in the PCR-based detection of intraerythrocytic Plasmodium parasites. We lysed red blood cells with saponin to reduce the Hb concentration in extracted nucleic acid and, as a result, significantly increased the volume of blood that can be tested by PCR. The analytical sensitivity of the PCR was determined using whole blood spiked with ring-stage Plasmodium falciparum parasites, and its clinical sensitivity by testing blood film-positive and blood film-negative samples from individuals living in an endemic area in Ghana. RESULTS: We have developed a pan-Plasmodium PCR that detects all five human Plasmodium species with the highest analytical sensitivity of two P. falciparum parasites/mL of whole blood and species-specific PCR tests that distinguished between the five human Plasmodium species. Pan-Plasmodium PCR detected 78 of 78 (100%) blood film-positive and 19 of 101 (18.81%) blood film-negative samples from asymptomatic individuals living in Ghana. Pan-Plasmodium PCR was equally sensitive with samples collected as anticoagulated whole blood and clotted blood and in blood collected by finger stick into capillaries. CONCLUSION: We have developed PCR tests with the highest reported sensitivity to date for pan-Plasmodium diagnosis and species-specific diagnosis and detected blood film-negative asymptomatic infections in individuals living in malaria-endemic countries.