This work was made possible by funding from The Wellcome Trust. JM (080964) and SCW (080948) were supported by Wellcome Trust fellowships and DT by a Malawi-Liverpool-Wellcome Trust studentship.

1. Sample Collection
Platelets can be easily activated through temperature, agitation or storage and therefore they should be handled with care. The use of vacutainers to collect blood for platelet preparation is unavoidable in most clinical situations, but should be carefully considered as suction can potentially cause platelet activation. Due to the clinical protocol used at our research hospital, our samples are carefully collected in sodium citrate vacutainers. We have not experienced any problems with premature platelet activation in this or our previous study 12. We collect blood for platelet preparation from blood group O+ individuals to minimize nonspecific blood group antigen aggregation. Donors have also not taken medication in the previous 7-10 days as some drugs are known to influence platelet function.
1.1 Preparation of platelet-rich plasma (PRP)
<ol>
	<li>Collect approximately 5 ml of whole blood from a malaria naive host or 2.5 ml of blood from CM or severe malaria (SM) patients in a sodium citrate vacutainer (BD product number 36276).</li>
	<li>Centrifuge the whole blood at 250 x g for 10 min at room temperature. Do not centrifuge at 4 &deg;C as low temperatures may cause the platelets to aggregate.</li>
	<li>Carefully transfer the cloudy supernatant in a clean 15 ml tube. Platelets are easily activated by temperature variations and physical shocks and should therefore be handled with care. Avoid shaking or any jerky movements of the tube.</li>
	<li>Use a Neubauer's haematocytometer to count and adjust the platelet suspension to &gt;300 x 103 platelets/&mu;l for healthy donors and &lt;150 x 103 platelets/&mu;l from CM and SM patients using 1X phosphate buffer (pH 7.2-7.4; PBS). Make sure to choose a high dilution factor to ease the platelet count and ensure its accuracy.</li>
</ol>
Note: Malaria patients have a reduced platelet count compared to the healthy individuals 16,17,18. Therefore platelet concentrations for CM and UM are adjusted according to the average platelet counts for patients within each malaria diagnostic group. The P. falciparum clumping phenotype is common in CM isolates and displays a strong binding affinity therefore the reduced platelet concentrations does not affect clump size or frequency since platelet concentration is standardized for all CM cases.
1.2 Preparation of platelet-poor plasma (PPP)
<ol>
	<li>To obtain PPP, centrifuge a portion of the PRP obtained above at 1,500 x g for 10 min. The majority of platelets are pelleted at the bottom of the tube.</li>
	<li>Both PRP and PPP can be stored at 4 &deg;C for up to two weeks. Ideally, use fresh platelets for the clumping assay. After 8-10 days most of the platelets are likely to be inactive and probably aggregated.</li>
</ol>
2. Parasite Culture
<ol>
	<li>Wash the pelleted pRBC three times with 5-10 ml RPMI 1640 by centrifugation at 370 x g for 5 min. 
	Note: in this protocol all cultures started from approximately 1 ml packed cell volume (PCV) and maintained at 5% haematocrit. Cultures can be started with any PCV of pRBC and adjusted accordingly with uninfected RBC and media to reach desired haematocrit.</li>
	<li>Place the pRBC in a culture flask and supplement with standard malaria culture medium of RPMI 1640 supplemented with 25 mM HEPES, 5% Albumax II or 10% serum and 40 &mu;g/ml gentamycin to achieve a 5% haematocrit.</li>
	<li>Permeate culture flasks with a mixture of 92.5% nitrogen, 2.5% oxygen and 5% carbon dioxide before sealing and incubating. Alternatively, the air-tight candle jar method of Trager-Jensen can be used.</li>
	<li>For pRBC obtained directly from patients, incubate their culture flask for 24-36 hr in a 37 &deg;C incubator in 5% CO2 to get mature parasites. 
	NOTE: Most laboratory and culture-adapted lines are very easy to maintain in culture under standard conditions. There are simple techniques described below that can be used to synchronise different parasite stages to retain growth rate.</li>
	<li>Prepare a thin blood smear as described below and examine parasite maturation under a light microscope.</li>
</ol>
3. Thin Blood Film Slide Preparation
<ol>
	<li>Place approximately 10-15 &mu;l of blood on one end of a frosted glass slide resting on a flat surface.</li>
	<li>Touch the drop of blood with the edge of a second slide until the blood is evenly spread across the edge of the second slide.</li>
	<li>While holding the second slide at a 45&deg; angle, quickly but gently, without exerting too much pressure on the first slide, slide the blood across the first slide to make a thin film of blood that is evenly spread. Air dry.</li>
	<li>Dip the slide in methanol for 10 sec. Air dry.</li>
	<li>Dip the slide in 2% Giemsa stain for at least 10 min. Rinse extensively until the water runs clear. Air dry.</li>
	<li>Examine slide using a 90-100x oil emulsion lens on a light microscope</li>
</ol>
4. Purification and Synchronization of Asexual Stages
The purification of mature P. falciparum pRBC is a required step for the clumping assay. Indeed, only mature parasites are capable of binding to platelet receptors as it is at this life cycle stage that the relevant ligands are expressed and protrude from the surface of infected erythrocytes. There are several methods to purify and synchronize asexual stages of P. falciparum.: Late asexual stages can be enriched by Percoll gradient 19; magnetic cell sorting 20 or using the gelatin sedimentation 21 protocol described here. Sorbitol-synchronization selects for ring stages 22, after which the rings are cultured for 24-26 hr to obtain mature stages (with no need to perform gelatin flotation).
4.1 Plasmagel Flotation
Plasmagel flotation uses gelatin-like solution to select for lower weight knobbed erythrocytes to remain in solution while the dense ring forms and rosettes sink to the bottom. This technique gives up to 90% purity of the mature stage-infected erythrocytes and can be used for both field and laboratory isolates. However, as previously mentioned, plasmagel flotation removes rosetting pRBC as well as immature stages which could result in a reduced clumping frequency in those samples with a high rosetting frequency. However, the absence of the rosetting pRBC after plasmagel flotation would not affect the clumping assay because rosetting is an interaction of pRBC and uninfected erythrocytes while clumping is pRBCs binding mediated by platelets. The ligand involved in these two traits is different; with mainly complement receptor CR-1 on uninfected erythrocytes mediating rosetting.
<ol>
	<li>Prewarm Gelofusine solution and serum/Albumax II-free medium to 37 &deg;C in a water bath.</li>
	<li>Transfer culture to a clean sterile 15 ml tube and pellet cells by centrifugation at room temperature at 370 x g for 5 min.</li>
	<li>Carefully aspirate supernatant so as not to disturb the pellet and resuspend cells in an equal volume of Gelofusine and protein-free medium.</li>
	<li>Transfer mixture to a sterile 15 ml tube and allow to stand for 15-20 min in a 37 &deg;C incubator or until a clear demarcation can be seen between an upper and lower level.</li>
	<li>Carefully transfer the upper layer to a fresh sterile 15 ml tube and add 10 ml of fresh RPMI.</li>
	<li>Centrifuge at 370 x g for 5 min and carefully discard supernatant.</li>
	<li>Assess parasitaemia and developmental stage by thin blood smear and examine under a light microscope as described in section 4.</li>
</ol>
NOTE: Mature pRBC range between 60-90% of infected erythrocytes depending on the initial parasitaemia of the culture. For high percentage of mature parasites, use cultures with an initial parasitaemia of &ge;10%.
5. Clumping Assay
<ol>
	<li>Use a Neubauer's haematocytometer to count and adjust the pRBC suspension obtained after plasmagel flotation to 1 x 108 pRBC/&mu;l.</li>
	<li>In a fresh 1.5 ml tube, resuspend pRBC at 5% hematocrit, acridine orange at 20 &mu;g/ml final concentration and PRP at 10% of the total volume. 
	Instead of acridine orange, parasite cultures can be stained with 25 &mu;g/ml ethidium bromide for 5 min before use. 
	E.g. For a 200 &mu;l reaction mix, add 10 &mu;l mature pRBC, 20 &mu;l of 300 x 103 platelets/&mu;l for PRP from healthy donors or 150 x 103 platelets/&mu;l from SM patients and 170 &mu;l of protein-free medium. 
	NOTE: The ratio of platelets:pRBC in the final reactions is 20:1. This is important as is allows the maximum clumping frequency of a sample to be determined. If fewer platelets are added in controls then not all clumping pRBCs might have the chance to clump.</li>
	<li>In the same way prepare the following controls; uninfected red blood cells and PRP, and pRBC with PPP to ascertain the role of platelets in the pRBCs clumping.</li>
	<li>Transfer the suspension to a 1.5-2.0 ml conical screw-cap vial.</li>
	<li>Place the vial under gentle agitation by rolling at 10-12 rpm at room temperature.</li>
	<li>Check for clump formation by a wet-slide prepared by pipetting 10 &mu;l of pRBC &plusmn; platelet mix on a glass slide, cover with a glass slip and examine under fluorescence.</li>
	<li>Sampling can be done at 15-20 min intervals for a total of 120 min. A clump is considered as an aggregate of three or more infected erythrocytes.</li>
</ol>

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No conflicts of interest declared.

We have described a platelet-mediated clumping assay used for studying pRBC in clinical isolates directly in the field. Platelet-mediated clumping, a characteristic behavior in P. falciparum clinical isolates, has been identified as a distinct adhesive phenotype, frequent in CD36-binding isolates 12,23-24. We have used this assay to identify three main points: 1) a strong in vitro clumping phenotype of P. falciparum isolated from Malawian children that is associated with disease severity and diagnosis 2) novel receptor P-selectin mediaties clumping on platelets together with CD36, 3) the degree of thrombocytopenia present in patients with CM is sufficient to limit the further formation of pRBC clumps in vitro 12. The involvement of platelets in clump formation is demonstrated by examining a wet-slide preparation as described in section 6. Platelets can be isolated from PRP by centrifugation at high speeds in order to minimize blood group antigenic reactions; however, we used whole PRP in order to minimize premature platelet activation from excessive handling and from the high-speed centrifugation. Platelet activity can be measured by platelet aggregation test using weak platelet agonists, epinephrine or adenosine diphosphate (ADP) 25 as described in 26.
There are several aspects of the assay that have previously been poorly characterized, one of them being the importance of selecting PRP sources and the effect of blood antigen groups on the outcome of the assay. We prepared PRP from individuals who were blood group O+ and had not taken any medication in the last 7-10 days before blood is drawn as some pharmaceuticals can affect platelet behavior.
Other factors that may affect clumping success include pRBC haematocrit, parasitaemia levels and length of the clumping assay. Using high haematocrit and platelet count it would be difficult to tell whether the cluster is truly a clump or whether too many cells are merely sitting together because they are densely packed 15. Clumping also generally increases with longer incubation times. These are all useful guidelines that should be considered when designing clumping studies.
We find that samples from different clinical groups can be analyzed in parallel using PRP from the same donor if it is possible to do so, but in limited resource settings such as field sites the transfusion pool is generally limited. It is therefore difficult to have a single O+ donor to standardize the assays. We therefore identified several O+ donors to ensure blood group compatibility wherever possible. Using multiple donors is also helpful in cases of large samples sizes so that burden of blood donation does not fall on only one individual.
Sampling points at 15-20 min are much more feasible if a single person is conducting the assays, allowing for wet-slide preparations and performing assay kinetics without sampling times overlapping. Most of the microscopic data is visual and somewhat objective; therefore, it is important that cell counting is verified by more than one person. In that case, handling one sample at a time can allow complete analysis of three to four samples a day depending on personal efficiency.
Platelet-mediated clumping can be performed using both clinical and laboratory isolates. For laboratory lines, it is recommended that CD36 binding parasites are used to maximize clumping frequencies. The assay can be coupled with other agglutination assays such as rosetting or used in clumping inhibition assays that would enable the study of receptors on the platelets that may mediate pRBC binding, a poorly understood and examined aspect of this phenotype.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>21870-092</td>
 <td></td>
 </tr>
 <tr>
 <td>Acridine orange</td>
 <td>Invitrogen</td>
 <td>A3568</td>
 <td>10 mg/ml</td>
 </tr>
 <tr>
 <td>Gelofusine</td>
 <td>B Braun</td>
 <td>Gelofusine</td>
 <td></td>
 </tr>
 <tr>
 <td>1 M HEPES</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamycin</td>
 <td>Invitrogen</td>
 <td>15750045</td>
 <td>50 mg/ml</td>
 </tr>
 <tr>
 <td>Albumax</td>
 <td>GIBCO</td>
 <td>1102-037</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium citrate vacutainer</td>
 <td>BD</td>
 <td>362760</td>
 <td>4 ml capacity</td>
 </tr>
 <tr>
 <td>Inverted Microscope</td>
 <td>Leica</td>
 <td>MD16000 B</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescent microscope</td>
 <td>Leica</td>
 <td>EL6000</td>
 <td>Contains blue and green light fluorescent filters for acridine orange and ethidium bromide </td>
 </tr>
 </tbody>
</table>

a simple protocol for platelet-mediated clumping of plasmodium falciparum-infected erythrocytes in a resource poor setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

An example of a platelet-mediated clump assay using approximately 70% mature stages of P. falciparum after enrichment by plasmagel flotation is shown in Figure 2. A clump is an aggregate of three or more infected erythrocytes. Staining with acridine orange or ethidium bromide can be visualized by fluorescence microscopy. Acridine orange is spectrally similar to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green), while ethidium bromide has an excitation maximum at 493 nm and an emission maximum at 620 nm (similar to rhodamine dyes, red). Clumps are easily spotted under the microscope as under normal light they appear as cell aggregates and under fluorescence as spots of green or red when stained with acridine orange or ethidium bromide dyes, respectively, as shown in Figure 2 (30 min time point using acridine orange). The larger the clumps, the larger the cell aggregate appears under normal light and the larger the spots of green or red under fluoresce for the respective dyes. Since the dyes target DNA and therefore stain nuclei, platelets and uninfected RBC are not detected under fluorescent microscopy due their lack of nuclei.
Random formation of small pRBC clumps of 4.7&plusmn;1.6 pRBCs/clump occur after 30 min. The clump size significantly increases to an average of &gt;15 pRBC/clumps after incubation for 120 min with some clumps containing numerous pRBC/clump that could not be visually counted. The frequency of the clumping phenotype is calculated as the number of infected cells present in clumps/1,000 infected cells, counted in duplicate assays.
<img alt="Figure 1" fo:content-width="3.5in" fo:src="/files/ftp_upload/4316/4316fig1highres.jpg" src="/files/ftp_upload/4316/4316fig1.jpg" /> 
 Figure 1. Work flow for clumping experiment. Platelet rich and poor plasma (A) and a P. falciparum-infected erythrocyte culture (B) are prepared and then combined for the clumping assay (C).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4316/4316fig2highres.jpg" src="/files/ftp_upload/4316/4316fig2.jpg" /> 
 Figure 2. Clumping of erythrocytes infected by P. falciparum laboratory isolate. Clumps formed by HB3 at time 0, 30 and 120 min in platelet-rich plasma (A) and poor clump formation in platelet poor plasma (B).
Assay Limitations
There are limitations that specifically affect platelet function or influence outcome and most of these have already been highlighted by Arman and Rowe 15. Here we mention some of the potential problems that might be encountered at different stages of the assay:
<ol>
	<li>Adapting clinical isolates to in vitro culture can be a challenge as sometimes longer culture periods are required in order to achieve high enough parasitaemia to allow formation of clumps. With high antigenic variation rates in P. falciparum, the adhesive phenotype of the culture-adapted parasite might not reflect the original infecting parasite population after prolonged times in culture.</li>
	<li>In order to avoid non-specific agglutination due to blood group antigens, platelet donors should be matched for blood group antigen with clinical isolates used in the same assay, or the universal donor blood antigen group O- used. However, individuals with blood group O- are rare in African sites such as Malawi and therefore platelets commonly used are obtained from blood group O+ donors and have to be matched for blood group.</li>
	<li>Counting clumps on a wet slide preparation is cumbersome and challenging for image capture as the cells are in constant motion. Usually, pRBC accumulate on the edges of the cover slip or they tend to stick together forming "false" clumps that can be easily confused with real clumps. In order to reduce cell motion, allow the cells to sediment for 15 min at room temperature prior to analysis.</li>
	<li>The clumping assay is time-sensitive and therefore only allows one sample to be analyzed per person at a time for optimal accuracy. Sampling time points for two different samples can easily overlap, resulting mismatched sampling time points between samples if handling more than one sample at the same time. In such circumstances, sophisticated quantitative software could calculate clump sizes or alternatively cell-specific antibodies could be used to analyze the composition of the clumps. While these techniques could greatly improve the significance of the data, in resource-poor settings where this assay is likely to be used, such techniques and equipment are not readily available.</li>
</ol>

Watch this Scientific Journal Video about A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting at JoVE.com

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

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18.6K Views.  Malawi-Liverpool-Wellcome Trust Clinical Research Programme. This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Video: A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.