Parasite Nucleic Acids Research Articles

International review of blood donation nucleic acid amplification testing.

Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion-transmitted infections. This project reviewed NAT for screening blood donations globally. A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion-transmitted Infectious Diseases (ISBT WP-TTID), was distributed through ISBT WP-TTID members. Data were analysed using descriptive statistics. Forty-three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT-positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT-positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT-positivity rate was higher in first-time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT.

Evaluation of genome skimming to detect and characterise human and livestock helminths

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.

Molecular and Antifilarial IgG4 Detection Using the miniPCR-Duplex Lateral Flow Dipstick and BmSxp-ELISA in Myanmar Immigrant Communities

Lymphatic filariasis (LF) is an important tropical disease that affects over a billion people in more than 80 countries and approximately 40 million people are currently suffering from severe disfigurement and disability. A diagnostic tool is the principal impact factor to determine the infection status of lymphatic filariasis. The purpose of the present study was to investigate nucleic acid of Wuchereria bancrofti as well as antifilarial IgG4 in a Myanmar immigrant community living along the Moei River, a natural border between Mae Sot, Tak province Thailand and Myawaddy, Myanmar which is an endemic area of bancroftian filariasis. Blood was collected from 300 Myanmar immigrants in Mae Sot district, Tak Province. The nucleic acid of W. bancrofti was assessed in the study population using our recent published miniPCR-Duplex Lateral Flow dipstick (DLFD) platform as well as the standard PCR technique. The antifilarial IgG4 was detected in the study population using the developed ELISA which used BmSxp protein as antigen. The miniPCR-DLFD method delivered results comparable to the standard PCR technique and it enables convenient and rapid visual detection of the parasite nucleic acid. Furthermore, the ELISA using BmSxp antigen demonstrated a sensitivity, specificity, and positive and negative predictive values of 98.1%, 98.9%, 96.3%, and 99.4% respectively. The W. bancrofti nucleic acid and antifilarial IgG4 were detected in 1.6% (5/300), and 2% (6/300) of the study population, accordingly. The results of this study also revealed important epidemiological data about LF on the Thai–Myanmar border.

Evaluating the diagnostic test accuracy of molecular xenomonitoring methods for characterising the community burden of Onchocerciasis.

BackgroundMolecular xenomonitoring (MX), the detection of parasite nucleic acid in the vector population, is recommended for onchocerciasis surveillance in elimination settings. However, the sensitivity of MX for detecting onchocerciasis-positive communities has not previously been evaluated. MX may have additional applications for control programmes but its utility is restricted by a limited understanding of the relationship between MX results and human prevalence.MethodsWe conducted a systematic review of studies reporting the prevalence of Onchocerca volvulus DNA in wild-caught Simulium spp. flies (MX rate) and corresponding prevalence of microfilaria (mf) in humans. We evaluated the sensitivity of MX for detecting onchocerciasis-positive communities and describe the characteristics of studies with reduced sensitivity. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate.ResultsWe identified 15 relevant studies, with 13 studies comprising 34 study communities included in the quantitative analyses. Most communities were at advanced stages towards elimination and had no or extremely low human prevalence. MX detected positive flies in every study area with >1% mf prevalence, with the exception of one study conducted in the Venezuelan Amazonian focus. We identified a significant relationship between the two measurements, with mf prevalence accounting for half of the variation in MX rate (R2 0.50, p<0.001).ConclusionMX is sensitive to communities with ongoing onchocerciasis transmission. It has potential to predict human mf prevalence, but further data is required to understand this relationship, particularly from MX surveys conducted earlier in control programmes before transmission has been interrupted.

Choline and PEG dually modified artemether nano delivery system targeting intra-erythrocytic Plasmodium and its pharmacodynamics in vivo

Objective The choline derivative (CD) and polyethylene-glycol (PEG) dually modified artemether (ARM) nanostructured lipid carriers (CD-PEG-ARM-NLC) have been designed to prolong the circulation of ARM in blood, as well as to develop targeting for new permeability pathways (NPPs) and erythrocyte choline carriers (ECCs) that are expressed on the Plasmodium-infected erythrocyte membrane. Significance The CD-PEG-ARM-NLC constructed in this study was found to be able to target endoerythrocytic Plasmodium by increasing the drug concentration and residence time in the infected erythrocytic microenvironment and minimizing toxicity and side effects. Methods CD-PEG-ARM-NLC was prepared using high-pressure homogenization followed by physicochemical characterization. The targeting ability of CD-PEG-NLC to infected erythrocytes probed by coumarin-6 was investigated by using fluorescence microscopy imaging. The SYBR Green I assay for parasite nucleic acid was adapted in order to assess the efficacy of inhibition against parasite growth in vitro. The antimalarial activity of ARM-loaded NLCs was evaluated by a Pearson four-day suppressive test in Pyy265BY-bearing mice. Results In vitro imaging indicated that the intracellular delivery of CD-PEG-ARM-NLC was efficiently taken up by the infected erythrocytes via ECCs and NPPs, which could be inhibited by addition of furosemide (an inhibitor of NPPs) and excessive choline (native substrate of ECCs). Moreover, in vitro and in vivo studies that evaluated antimalarial activity suggested that CD-PEG-ARM-NLC exhibited higher antimalarial activity in comparison to ARM-NLC and PEG-ARM-NLC. Conclusion These findings suggested that choline and PEG dually modified NLC could be promising preparations for the production of hydrophobic antimalarial drugs, particularly for ARM.

Efficiency of molecular detection of Toxoplasma gondii contrasting to serological method.

Toxoplasmosis infection of the mother during pregnancy is linked to transplacental transmission to the fetus. Toxoplasma gondii infection is difficult to diagnose and necessitates a comprehensive examination that includes laboratory and clinical testing. This study used RT-PCR using B1 gene based on paired blood samples and clinical data, for the detection of Toxoplasma infections in miscarriage women, this technique was compared to enzyme-linked immunosorbent assay (ELISA). There are a total of 125 miscarriage women at Al-Muthanna Hospital. Anti-Toxoplasma antibodies, IgG (latent infection) and IgM (recently acquired infection), were positive in 50/125 cases, with 25.6% IgG, 8.8% IgM, and 5.6% for IgM and IgG. In addition, women who have had two miscarriages have a high seropositivity rate. Furthermore, the development of molecular technologies to amplify parasite nucleic acids has enhanced toxoplasmosis diagnosis. The cycle threshold values Ct, which indicate the target gene's quantity, were calculated. With DNA from 500 T. gondii tachyzoites, a Ct of 25-28 was usually obtained. Present results show that the assay's reproducibility was found in 50 which considered (40%) from total miscarriage women of ELISA positive samples while 28/125 samples (22.4%) will positively in molecular detection and 56% from serological test was positive in molecular test. A move forward was the invention of diagnostic methods that used ELISA IgG and IgM followed by an RT-PCR methods in women were the most effective in detecting recent and reactive Toxoplasma gondii infection.

Type I Interferons and Malaria: A Double-Edge Sword Against a Complex Parasitic Disease.

Type I interferons (IFN-Is) are important cytokines playing critical roles in various infections, autoimmune diseases, and cancer. Studies have also shown that IFN-Is exhibit ‘conflicting’ roles in malaria parasite infections. Malaria parasites have a complex life cycle with multiple developing stages in two hosts. Both the liver and blood stages of malaria parasites in a vertebrate host stimulate IFN-I responses. IFN-Is have been shown to inhibit liver and blood stage development, to suppress T cell activation and adaptive immune response, and to promote production of proinflammatory cytokines and chemokines in animal models. Different parasite species or strains trigger distinct IFN-I responses. For example, a Plasmodium yoelii strain can stimulate a strong IFN-I response during early infection, whereas its isogenetic strain does not. Host genetic background also greatly influences IFN-I production during malaria infections. Consequently, the effects of IFN-Is on parasitemia and disease symptoms are highly variable depending on the combination of parasite and host species or strains. Toll-like receptor (TLR) 7, TLR9, melanoma differentiation-associated protein 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) coupled with stimulator of interferon genes (STING) are the major receptors for recognizing parasite nucleic acids (RNA/DNA) to trigger IFN-I responses. IFN-I levels in vivo are tightly regulated, and various novel molecules have been identified to regulate IFN-I responses during malaria infections. Here we review the major findings and progress in ligand recognition, signaling pathways, functions, and regulation of IFN-I responses during malaria infections.

Lysercell M enhances the detection of stage-specific Plasmodium-infected red blood cells in the automated hematology analyzer XN-31 prototype

The automated hematology analyzers XN-30 (for research) and XN-31 prototype (for diagnosis support) can easily and rapidly detect Plasmodium-infected red blood cells (iRBCs) and distinguish the developmental stages of the parasite in approximately 1min. Two dedicated reagents, Lysercell M and Fluorocell M, are available with the analyzers. Lysercell M plays an indispensable role in enhancing the fluorescence intensity of the nucleic acid staining dye in Fluorocell M and altering cell morphology. These effects of Lysercell M have been empirically determined but insufficiently analyzed. In this study, the properties of Lysercell M were analyzed using two flow cytometers and a fluorescence microscope. First, the fluorescence intensity emitted by iRBCs treated with Lysercell M or phosphate-buffered saline (PBS) was evaluated. Second, the size of RBCs treated with Lysercell M or PBS was measured. Finally, the morphology of individual parasites was observed after reconstruction of an M scattergram, a cytogram of the XN-31 prototype system, using an imaging flow cytometer. These analyses showed that treatment of iRBCs with Lysercell M increased the fluorescence intensity of stained parasite nucleic acids by approximately 10-fold and reduced the size of iRBCs in a stage-specific manner, facilitating the identification and quantification of ring form, trophozoite, and schizont stage iRBCs. These properties suggest that Lysercell M is useful for rapidly detecting iRBCs and accurately distinguishing the parasite developmental stages, thereby contributing to the usability of the XN-30 and XN-31 prototype analyzers.

Combined Immunofluorescence (IFA) and Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Babesiosis in Patients from the USA, Europe and Australia.

Apicomplexan parasites of the genus Babesia cause babesiosis in humans and animals worldwide. Human babesiosis is a predominantly zoonotic disease transmitted by hard ticks that is of increasing health concern in the USA and many other countries. Microscopic examination of stained blood smears, detection of serum antibodies by immunoassays and identification of parasite nucleic acid in blood by qPCR and fluorescence in situ hybridization (FISH) are some methods available for diagnosing babesiosis. This study investigated the use of a Babesia genus-specific FISH test for detecting Babesia parasites in blood smears and immunofluorescence assay (IFA) for detecting serum antibodies to Babesia duncani and Babesia microti, two common species that cause human babesiosis in the USA. The findings with clinical samples originating from USA, Australia, Europe and elsewhere demonstrate that the parallel use of Babesia genus-specific FISH and IFA tests for B. duncani and B. microti provides more useful diagnostic information in babesiosis and that B. duncani infections are more widespread globally than presently recognized.

A Fluorescence in Situ Hybridization (FISH) Test for Diagnosing Babesiosis.

Apicomplexan parasites of the genus Babesia cause babesiosis in humans and animals. The microscopic examination of stained blood smears, detection of serum antibodies by immunoassays, and PCR-based identification of parasite nucleic acid in blood are common laboratory methods for diagnosing babesiosis. The present study evaluated a commercially available Babesia genus-specific fluorescence in situ hybridization (FISH) test for detecting Babesia parasites in blood smears. The FISH test detected Babesia duncani and Babesia microti, two common species that cause human infections in the USA, and other Babesia species of human and veterinary importance in less than two hours. The Babesia genus-specific FISH test supplements other existing laboratory methods for diagnosing babesiosis and may be particularly useful in resource-limited laboratories.

Malaria Parasite Density in Individuals with Different Rapid Diagnostic Test Results and Concentrations of HRP2 Antigen.

Low-density malaria infections are a source of human morbidity in endemic settings and potentially contribute to ongoing malaria transmission. Conventional rapid diagnostic tests (RDTs) were designed to detect clinically relevant parasite and antigen levels, but it is largely unknown what proportion of parasite (and antigen positive) infections are missed by conventional RDTs. Furthermore, RDTs can also provide false positives from lingering histidine-rich protein 2 (HRP2) antigenemia from a past infection. We analyzed 207 samples from Angolan outpatients with a bead-based HRP2 antigen assay and by qRT-PCR for the presence of parasite nucleic acids. Among patients HRP2 positive but negative by conventional RDT, the rate of quantitative reverse transcription-PCR (qRT-PCR) positivity was 45% (95% CI: 35-56%), with a median parasitemia of 3.4 parasites/µL (interquartile range: 0.14-4.8). Only 15% (7-26%) of HRP2-negative samples were found to have parasite nucleic acids. A substantial proportion of persons with blood HRP2 antigen concentrations not detected by the conventional RDT were found to have evidence of active infection, but at low parasite density levels.

Superior real‐time polymerase chain reaction detection of Babesia microti parasites in whole blood utilizing high‐copy BMN antigens as amplification targets

Babesiosis is a zoonotic disease transmitted to humans by the bite of infected ticks and caused by apicomplexan parasites, most commonly Babesia microti. Additionally, blood and blood products collected from asymptomatically infected blood donors may cause transfusion-transmitted infections in recipients. Highly sensitive molecular assays that detect parasite nucleic acid are needed for laboratory diagnosis and to identify and defer clinically silent but parasitemic blood donors. Here we report the development and analytical and clinical characterization of a real-time polymerase chain reaction (RT-PCR)-based assay for the detection of B.microti genomic DNA in whole blood. We evaluate the detection of Babesia parasites using two separate targets, the traditional18S ribosomal subunit gene (Bm18S) and members of the abundant BMN family of seroreactive antigens (BmBMN). Analytical sensitivity determination using a probit analysis demonstrated an analytical sensitivity of 30.9 parasites/mL for 18S amplification and 10.0 parasites/mL for BMN amplification The BMN primer set also demonstrates superior sensitivity for serial dilution panels prepared from clinically diagnosed Babesia-infected blood samples, generally detecting 10-fold more dilute nucleic acid. Cumulatively, our data demonstrate that RT-PCR detection of the BMN family of seroreactive antigens reflects a sensitive and superior assay for the detection of B.microti in whole blood samples.

Investigating zoonotic infection barriers to ape Plasmodium parasites using faecal DNA analysis

African apes are endemically infected with numerous Plasmodium spp. including close relatives of human Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Although these ape parasites are not believed to pose a zoonotic threat, their ability to colonise humans has not been fully explored. In particular, it remains unknown whether ape parasites are able to initiate exo-erythrocytic replication in human hepatocytes following the bite of an infective mosquito. Since animal studies have shown that liver stage infection can result in the excretion of parasite nucleic acids into the bile, we screened faecal samples from 504 rural Cameroonians for Plasmodium DNA. Using pan-Laverania as well as P. malariae- and P. vivax-specific primer sets, we amplified human P. falciparum (n = 14), P. malariae (n = 1), and P. ovale wallikeri (n = 1) mitochondrial sequences from faecal DNA of 15 individuals. However, despite using an intensified PCR screening approach we failed to detect ape Laverania, ape P. vivax or ape P. malariae parasites in these same subjects. One faecal sample from a hunter-gatherer contained a sequence closely related to the porcupine parasite Plasmodium atheruri. Since this same faecal sample also contained porcupine mitochondrial DNA, but a matching blood sample was Plasmodium-negative, it is likely that this hunter-gatherer consumed Plasmodium-infected bushmeat. Faecal Plasmodium detection was not secondary to intestinal bleeding and/or infection with gastrointestinal parasites, but indicative of blood parasitaemia. Quantitative PCR identified 26-fold more parasite DNA in the blood of faecal Plasmodium-positive than faecal Plasmodium-negative individuals (P = 0.01). However, among blood-positive individuals only 10% - 20% had detectable Plasmodium sequences in their stool. Thus, faecal screening of rural Cameroonians failed to uncover abortive ape Plasmodium infections, but detected infection with human parasites, albeit with reduced sensitivity compared with blood analysis.

Comparison of methods for detecting asymptomatic malaria infections in the China\u2013Myanmar border area

BackgroundSensitive methods for detecting asymptomatic malaria infections are essential for identifying potential transmission reservoirs and obtaining an accurate assessment of malaria epidemiology in low-endemicity areas aiming to eliminate malaria. PCR techniques to detect parasite nucleic acids (DNA or RNA) are among the most commonly used molecular methods. However, most of these methods are of low throughput and cannot be used for large-scale molecular epidemiological studies. A recently developed capture and ligation probe-PCR (CLIP-PCR) is claimed to have the sensitivity of molecular techniques and the high throughput capacity needed for screening purposes. This study aimed to compare several molecular methods for detecting asymptomatic and submicroscopic Plasmodium infections in healthy residents of a malaria-hypoendemic region in Southeast Asia, where malaria elimination is in sight.MethodThis study compared three molecular detection methods side-by-side, namely nested PCR targeting the rRNA genes, nested RT-PCR to detect parasite rRNA, and CLIP-PCR to detect parasite rRNA in 1005 healthy individuals in northeastern Myanmar. For nested PCR and RT-PCR, parasite DNA and total RNA were extracted from ~100 µL of blood, whereas RNA used for CLIP-PCR was from a 3 mm disk of dried blood filter paper. The sensitivity and specificity of these methods were compared with those of conventional light microscopy. In addition, RT-PCR and quantitative RT-PCR (qRT-PCR) targeting the Pvs25 gene in Plasmodium vivax were used to assess gametocyte prevalence in the samples.ResultsLight microscopy detected Plasmodium infections in only 1.19% of the residents harbouring the parasites. CLIP-PCR had slightly better performance and detected Plasmodium infections in 1.89% of the population. Further improvement was achieved by nested PCR to detect parasite DNA, which detected P. vivax and Plasmodium falciparum infections in 2.39% of the residents. The nested RT-PCR targeting rRNA, however, detected as many as 187 (18.61%) individuals having Plasmodium infections with P. vivax being the predominant species (176 P. vivax, 5 P. falciparum and 6 P. falciparum/P. vivax mixed infections). Of the 210 Plasmodium-positive samples detected by all molecular methods, 115 were Pvs25-positive by qRT-PCR, indicating that a large proportion of asymptomatic individuals were gametocyte carriers.ConclusionNested RT-PCR based on the detection of asexual-stage parasite rRNA was the most sensitive, with a more than sixfold higher sensitivity than the other two molecular methods of parasite detection. CLIP-PCR has an increased throughput, but its sensitivity in this study was much lower than those of other molecular methods, which may be partially due to the smaller amount of RNA input used.

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification.

Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject’s blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200–2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

A cautionary note on fecal sampling and molecular epidemiology in predatory wild great apes.

Fecal samples are an important source of information on parasites (viruses, prokaryotes, or eukaryotes) infecting wild great apes. Molecular analysis of fecal samples has already been used for deciphering the origins of major human pathogens such as HIV-1 or Plasmodium falciparum. However, for apes that hunt (chimpanzees and bonobos), detection of parasite nucleic acids may reflect either true infection of the host of interest or ingestion of an infected prey, for example, another non-human primate. To determine the potential magnitude of this issue, we estimated the prevalence of prey DNA in fecal samples obtained from two wild chimpanzee communities. We observed values >15%, which are higher than or close to the fecal detection rates of many great ape parasites. Contamination of fecal samples with parasite DNA from dietary origin may therefore occasionally impact non-invasive epidemiological studies. This problem can be addressed (at least partially) by monitoring the presence of prey DNA.

Diagnosis of toxoplasmosis and typing of Toxoplasma gondii.

Toxoplasmosis, caused by the obligate intracellular protozoan Toxoplasma gondii, is an important zoonosis with medical and veterinary importance worldwide. The disease is mainly contracted by ingesting undercooked or raw meat containing viable tissue cysts, or by ingesting food or water contaminated with oocysts. The diagnosis and genetic characterization of T. gondii infection is crucial for the surveillance, prevention and control of toxoplasmosis. Traditional approaches for the diagnosis of toxoplasmosis include etiological, immunological and imaging techniques. Diagnosis of toxoplasmosis has been improved by the emergence of molecular technologies to amplify parasite nucleic acids. Among these, polymerase chain reaction (PCR)-based molecular techniques have been useful for the genetic characterization of T. gondii. Serotyping methods based on polymorphic polypeptides have the potential to become the choice for typing T. gondii in humans and animals. In this review, we summarize conventional non-DNA-based diagnostic methods, and the DNA-based molecular techniques for the diagnosis and genetic characterization of T. gondii. These techniques have provided foundations for further development of more effective and accurate detection of T. gondii infection. These advances will contribute to an improved understanding of the epidemiology, prevention and control of toxoplasmosis.

UPDATE IN DIAGNOSIS AND TREATMENT OF CRYPTOSPORIDIOSIS AN UPDATE REVIEW

Cryptosporidiosis represents a major public health problem which transmitted by contamination of food or water by sporulated Cryptosporidial oocyst. Causing self- limited diarrhea in immunocompetent person and chronic and life threatening diarrhea among immunocompromised individuals. It can be diagnosed by concentration and detection of its Oocyst in different environmental samples and water by microscopic and immunological examination such as enzyme immunoassay (ELISA) for parasite antigens and nucleic acid amplification assay as well as use of molecular techniques such as Polymerase Chain Reaction (PCR). Treatment is face challenges, Macrolides, Paramomycin, Nitazoxanide and Mirazid. All these drugs have partial efficacy in reducing disease severity in immunocompetent individuals. Nitazoxanide has partial efficacy in immunocompromised individuals. Resolution of Cryptosporidiosis can be maintained with effective Highly Active Antiretroviral Therapy (HAART).

Update in Diagnosis and Treatment of Cryptosporidiosis an Update Review

Cryptosporidiosis represents a major public health problem which transmitted by contamination of food or water by sporulated Cryptosporidial oocyst. Causing self- limited diarrhea in immunocompetent person and chronic and life threatening diarrhea among immunocompromised individuals. It can be diagnosed by concentration and detection of its Oocyst in different environmental samples and water by microscopic and immunological examination such as enzyme immunoassay (ELISA) for parasite antigens and nucleic acid amplification assay as well as use of molecular techniques such as Polymerase Chain Reaction (PCR). Treatment is face challenges, Macrolides, Paramomycin, Nitazoxanide and Mirazid. All these drugs have partial efficacy in reducing disease severity in immunocompetent individuals. Nitazoxanide has partial efficacy in immunocompromised individuals. Resolution of Cryptosporidiosis can be maintained with effective Highly Active Antiretroviral Therapy (HAART).

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited.