Patent Application
20090258347
CD4 monitoring and HIV viral load measurement in HIV disease are the bedrock to monitoring quality-care of HIV infected patients. In HIV disease, viral load is one of the best markers of dynamic changes over time. The viral load is principal to facilitate prediction about disease progression, predict response to therapy and monitor the effects of that therapy. The viral load assays currently quantitate across a wide range of viral load levels (linear dynamic range), and have good reproducibility of 0.2 log. Quantitative measurements of plasma HIV RNA are expressed in two ways: the number of HIV-RNA copies/ml of plasma (or IU/ml), or the logarithmic equivalent (log10, where a 1-log change represents a 10-fold change). A 3-fold variation (0.5log10 copies) is accounted for by intra-assay variability and biological variability, but clinically a 10 fold (1-log10) difference is regarded as significant.
The laboratory measure of HIV plasma viral load is performed by nucleic acid amplification techniques that amplify a target region of DNA or RNA. It is an extremely sensitive and skilled laboratory tool that requires a dedicated laboratory environment with skilled staff that adhere to strict protocol to prevent carry over contamination. This methodology is also expensive and dependent on the supply of expensive kits and equipment for testing. Currently there are three FDA licensed HIV RNA assays accepted for clinical management—reverse transcriptase PCR Roche Amplicor HIV-1 Monitor™ Test, bioMerieux NucliSens® HIV-1 QT Assay, and Versant® HIV-1 RNA 3.0 Assay (bDNA). All three assays are high throughput, the Amplicor and the NASBA assays amplify the target HIV-RNA into measurable amounts of nucleic acid product (target amplification), whereas the bDNA amplifies the signal obtained from a captured HIV-RNA target (signal amplification).
The cost of a single viral load test (Roche Amplicor) ranges from about US$50-US$100. This is either unaffordable or unavailable (not feasible from an implementation perspective for high throughput testing) in the developing world, especially for patient follow-up. Several alternative cost effective methodologies are being investigated that use different platforms. For example, the p24 antigen quantitation ELISA assay (Perkin-Elmer Life and Analytical Sciences, Turku, Finland) is becoming increasingly popular as an inexpensive alternative that measures viral replication in vivo by quantitating the major viral core protein-p24 The measure of viral reverse transciptase activity recovered from plasma and measured in an ELISA format by the ExaVir™ enzyme immunoassay (Cavidi Tech-AB, Uppsala, Sweden) has also been developed as an alternative cost effective assay.
Several other factors have been shown to correlate with disease progression, and form the basis of other approaches to laboratory diagnostic monitoring tools under exploration. A few examples of these factors are:
In spite of all these alternative approaches, the viral load remains the most important and clinically useful measure for monitoring. Nevertheless, there is still a need for a viral load monitoring assay or alternative disease monitoring assay or test that is affordable, reliable, simple and robust to increase the accessibility to viral load measurement in the developing world.
According to a first embodiment of the invention, there is provided an assay for diagnosing and/or monitoring a viral infection or disease, the assay including the steps of:
The sample of leucocytes may be from a blood sample of a patient (which includes a cord blood sample), in which case the assay may also include the step of lysing the red blood cells so as to obtain the leucocyte sample. Alternatively, cultured cells may form the leucocyte sample.
Typically, the monocyte, granulocyte and lymphocyte sub-populations are all identified in the assay.
The fluorescence intensity of each sub-population may be determined from the mean or median fluorescence intensity or from marker or region limits of the respective sub-population.
Typical ratios that may be calculated by comparing the fluorescence intensity of one to sub-population to the fluorescence intensity of another sub-population are: monocytes:granulocytes, monocytes:lymphocytes and granulocytes:lymphocytes.
The ratio of the mean fluorescence intensity of the monocyte population to the mean fluorescence intensity of the granulocyte population or lymphocyte population may be an indicator of the cellular viral reservoir in the patient.
The viral infection may be HIV. Similarly, the disease may be AIDS. For example, when monitoring HIV/AIDS infection, the monocyte:granulocyte ratio will be greater than one and is expected to increase with increase of the virus reservoir. However, the ratio of these two sub-populations or the ratio of a different combination of two of the leucocyte sub-populations may vary when monitoring a different disease, such as tuberculosis.
The assay may also be used to monitor co-infection of the patient with another disease, for example, another viral, parasitic or bacterial infection For example, if the ratio of the mean fluorescence intensity of the monocyte population to the mean fluorescence intensity of the granulocyte population is less than the ratio of the mean fluorescence intensity of the monocyte population to the mean fluorescence intensity of the lymphocyte population, this may be an indicator of a co-infection, such as Mycobacteium tuberculosis infection. This relationship may similarly be shown by the mean fluorescence intensity of the granulocyte to lymphocyte population being either <1 (showing lymphocyte activity/disease) or >1 (showing granulocyte activity/disease).
The dye is preferably a compound which stains RNA or both DNA and RNA. The dye may be selected from the group consisting of thiazole orange, SYTO dyes, LDS-751 and acridine orange.
May be performed using a flow cytometer, haematology analyser or other suitable instrumentation that measures fluorescence, such as a fluorimeter.
The assay may also include a step for obtaining a CD4 count. In particular, an antibody that fluoresces in a different fluorescent channel to the dye may be added to the sample so that the CD4 count can be obtained. Other antibody markers may also be used, for example cell activation markers such as CD38 or specific sub-population markers such as CD14 and CD16 or p24.
According to a second embodiment of the invention, there is provided a method of diagnosing and/or monitoring the cellular viral reservoir (load) of a patient with HIV or other bacterial infection, the method including the step of comparing the mean fluorescence intensity of the patient's monocytes that have been stained with a fluorescent dye to the mean fluorescence intensity of the patient's granulocytes and/or lymphocytes that have also been stained with a fluorescent dye.
This comparison may be used as a marker of the viral load of the patient, and hence as a marker of disease infection or progression and related infections, as well as being used to indicate the patient's response to therapy.
According to a third embodiment of the invention, there is provided a kit for performing the assay described above, the kit including a cell membrane-permeable dye which stains RNA or both DNA and RNA, typically but not necessarily with a single fluorescence.
The kit may further include a set of computer readable instructions for performing the assay or at least a portion of the assay, and in particular, for:
The computer readable instructions may further interpret the ratio or ratios obtained above. For example, the computer readable instructions may indicate to a user whether the patient has a low, medium or high virus reservoir or has a co-infection.
The fluorescence intensities of each sub-population may be the mean or median fluorescence intensity or may be a region or marker limit of that sub-population.
The kit may further include an antibody for determining the CD4 count (or other cell marker) of the sample.
The kit may further include one or more reagents selected from the group consisting of a red cell lysing agent, a stabilizer, a fixative, control cells, media and bead reagents.
The kit may further include means for dispensing the red cell lysing agent, dye, antibody reagents and/or other reagents used in the assay.
The kit may further include other sets of cell membrane markers or intracellular markers for phenotyping, such as CD38, CD14/CD16 or p24.
According to a further embodiment of the invention, there is provided a machine readable medium comprising instructions for diagnosing or monitoring a viral infection or disease according to the method of the invention, which when executed by a machine, cause the machine to perform all or at least some of the steps of the assay described above.
The machine readable medium may be configured for use in conjunction with a flow cytometer and/or haematology analyser.
The machine readable medium may include instructions for performing analysis methods selected from the group consisting of impedance, light scatter and fluorescence.
The invention provides an assay for diagnosing and/or monitoring a viral infection or disease, such as HIV/AIDS.
It has long been documented that the death of CD4 T-cells is an untoward outcome of the viral replicative cycle in these cells. Emerging in the literature is the premise that CD4 T-cells are innocent bystanders and the CD4+ macrophages have a more significant and direct role to play in HIV/AIDS pathogenesis.
Macrophages have been shown to be the principal reservoir of HIV and SHIV (simian immunodeficiency virus/HIV-1 chimera) and sustain high virus loads after the depletion of the CD4 T-cells. The macrophages are infected during the acute infection and the number infected gradually increases over time and become a major contributor to total body virus burden during the symptomatic phase of the disease. Long-term infections of HIV in monocytes have also been shown in patients receiving HAART [1].
It is being recognised that assessing viral load should not be restricted to the plasma RNA, and changes in HIV DNA and RNA copy numbers in peripheral blood mononuclear cells should be given equal focus. In particular, HIV-1 mRNA expression in peripheral blood cells has been shown to predict disease progression independently of the CD4 count [2].
The applicant thus set out to investigate whether it would be possible to monitor viral load by quantifying the cellular nucleic acid in leucocytes. HIV/AIDS was chosen for testing as a suitable example of a viral infection and disease, as there is a pressing need for an affordable and reliable viral monitoring assay for this disease.
It has now been found that monocytes of HIV positive patients contain an increased amount of nucleic acids, and this increase correlates to the plasma viral load. Furthermore, the applicant has found that by quantifying the cellular (whole cell) nucleic acid (RNA or both DNA and RNA) using a fluorescent dye, and comparing the amount of nucleic acids in the monocytes with the amount of nucleic acids in the granulocytes (neutrophils) and in the lymphocytes, it is possible to monitor the cellular viral reservoir load. More particularly, the applicant has shown that the index (ratio) of monocyte, lymphocyte and granulocyte mean fluorescent intensities (MFI) can be used as a marker of HIV/AIDS disease progression and related infections.
As the increased nucleic acid concentration in the monocytes is probably a measure of virus reservoir (or cellular response to infection), the mean fluorescence ratio or index (MFI) calculated according to the invention has been termed the HIV reservoir monitoring index (HIVrmi).
Mycobacterium tuberculosis is the etiological agent for tuberculosis infection. This bacterium is a facultative parasite capable of surviving and multiplying in phagocytes. During primary infection, M.tuberculosis enters and survives in alveolar macrophages, and disseminates from the lung by a heterogeneous group of tissue macrophages. It has also been shown that neutrophils play a role in TB infection as the ‘Trojan horse’ by hiding mycobacteria from the immune system. In addition, neutrophil function has been shown to be impaired in HIV/TB infection, resulting in increased susceptibility to secondary infections. The identification of certain groups of patients from TB cohorts with increased neutrophil fluorescence in the HIVrmi assay provides an additional application of cellular reservoir identification using HVrmi. The hypothesis that the HIVRMI increased neutrophil fluorescence is a measure of intracellular M.tuberculosis infection (or cellular response to infection) is being investigated.
The other infections may also be parasitic infections, such as bilharzia or worms.
Flow cytometry is a platform well-used for measuring antigen expression and cell enumeration. Several studies using this platform have found correlates to HIV disease progression. The flow cytometry platform has also been used to detect and quantitate viruses directly, including HIV, and was therefore decided to be a particularly suitable platform for performing the assay of the invention. It will be apparent to a person skilled in the art, however, that the assay may also be performed on a haematology analyser or by fluorimetry without requiring undue experimentation.
Nucleic acid binding dyes are well described in flow cytometry for discriminating non-nucleated from nucleated cell events in assays that measure cell viability and ploidy analysis. The direct measure of nucleic acid specific dyes on intact cells has, however, been mostly applied to study apoptosis and necrosis, and is relatively uninvestigated for the direct measurement of viral DNA or RNA for viral load measurement.
Suitable dyes for use in the assay should have the following properties:
Some of the commercially available vital probes (permeate) that have been described for use in flow cytometry and that have these properties are thiazole orange, SYTO group dyes (from Molecular Probes), LDS-751, acridine orange and the combination of Hoechst 33342 and pyronin Y (some SYTO dyes, like SYTO RNA Select, which are also cell membrane-permeable but only stain RNA, may also show the same increased fluorescence).
Acridine orange can be used as a vital stain without fixation of the cells, but requires two different excitation sources to visualize DNA and RNA at the same time. The absorption of acridine orange is in the range between 440 nm and 480 nm (blue), and the emission is in the range between 520 nm (green for DNA) and 650 nm (orange for RNA). The combination of Hoechst 33342 and pyronin Y can be used for DNA and RNA content in intact cells, but requires two light sources.
The above examples do not include DNA/RNA binding dyes that are currently used for microscopy, DNA/RNA amplification, and detection molecular methods that have not yet been cited for use in flow cytometry. Although the most popular flow cytometry configurations use 488 nm lasers light sources, there are also other light sources at different wavelengths that would be compatible with different dyes.
Thiazole orange is an asymmetric cyanine that consists of two aromatic rings connected by a bond and is sufficiently soluble in a phosphate buffer or distilled water solution to make appropriate dilutions for long term storage, with negligible fluorescence in solution. The interaction of thiazole orange with nucleic acids is through complex intercalation (insertion of planar compounds between adjacent base pairs) which is dependant on the state of the nucleic acid (single or double stranded) and has higher affinity for A-T rich sequences. Once bound to nucleic acid the thiazole orange aromatic rings become restricted and reduce their rotation, which is believed to cause the intense fluorescence [3]. Thiazole orange is used in flow cytometry to identify Plasmodium parasitized red blood cells, stain RNA in reticulocytes and measure the percentage reticulated platelets within whole blood. Quantities of thiazole orange used for nucleic acid detection are generally in the order of 10−6 to 10−7 M free dye and 10−5 M in applications for flow cytometry.
Thiazole orange is a suitable dye for use in this invention, because it is membrane permeate, it is suitable with standard ‘lyse no wash’ protocols and it has an emission and excitation spectrum similar to FITC (fluoroscein isothiocyanate). It can also be used with standard blue laser light (488 nm) flow cytometers. The commercial cost of thiazole orange is approximately ZAR778.00 (−$80) for 1 gram. Dilutions of thiazole orange to the concentrations required in this assay would result in about 600 tests costing only 1 cent (ZAR0.01). Such minimal expense makes this dye a good candidate for affordable HIV/AIDS monitoring in the developing world.
The assay is typically performed as follows:
A sample of peripheral whole blood in EDTA is prepared and the red cells are lysed. A cell-permeable dye is then added to the remaining white cell suspension and the dye binds to the DNA and RNA within the cells. The bound dye fluoresces, making it possible for the cells in suspension to be analysed for fluorescence and side angle light scatter by flow cytometry (488 nm laser instrument detecting thiazole orange in channel FL1).
Three white cell populations (granulocytes, monocytes, lymphocytes) are identified using a dual scattergram (SSC vs FL1), although it would also be possible to identify only the monocyte population and one of the granulocyte and lymphocyte populations.
The mean fluorescent (FL1) intensity (MFI) in each gated cell type is recorded, and the ratio of monocyte mean fluorescent (FL1) intensity (MFI) to granulocyte MFI the ratio of the monocyte MFI to lymphocyte MFI, and the ratio of granulocyte mean fluorescent (FL1) intensity (MFI) to lymphocyte mean fluorescent (FL1) intensity (MFI) is calculated.
A CD4 count can be determined in the same tube at the same time, by adding an antibody that fluoresces in a different channel to the dye used for the cellular nucleic acids.
This assay is best performed on fresh (<24hrs) blood, since aged blood shows a general increase, throughout all the leucocytes, in thiazole orange mean fluorescent intensity (MFI).
Preliminary investigation into the exact cause of the increased mean fluorescent intensity (MFI) in monocytes indicates that the thiazole orange measures RNA in the cytoplasm. It is also probable that because the dye is used in small molar concentrations, it is just sufficient to enter cells and stain cytoplasmic nucleic acid such as RNA. Increases in DNA due to cell replication in the nucleus may not be able to be measured at these low dye concentrations and therefore not interfere with the MFI measurement. The hypothesis that this increased RNA is viral and/or upregulated mRNA (cellular response to infection) is being further validated, but the role of monocytes in HIV further strengthens this hypothesis.
HIV-1 replication has been shown to continue in patients receiving ARV with suppressed plasma vireamia. Sites of replication are found In cellular reservoirs including monocytes. In particular a specific subgroup of monocytes with the phenotype CD14low/CD16high have been shown to be more susceptible to HIV infection, and to contribute to those monocytes that differentiate into macrophages to traffic the virus through tissue. A preliminary study has shown that the percentage of these CD14low/CD16high monocytes correlates with increasing HIVRMI (highest index value: monocytes to granulocytes or monocytes to lymphocytes in the presence of probable TB co-infection), and further validates HIVRMI as a measure of cellular HIV reservoir. The graph in
The HIVRMI assay was primarily investigated as a monitoring tool for HIV adult patients on ARV. A single HIVRMI result may not be useful for direct conversion (prediction) to a plasma viral load value without knowledge of patient treatment status. The HIVRMI does appear useful for longitudinal monitoring as an early indicator of virus production/cell activity for disease progression and response to therapy.
The HIVRMI, however, was also (secondarily) investigated as a qualitative assay for use in diagnosis of HIV. This became apparent when the HIVRMI values measured in a paediatric cohort (infants age 30-50 days old) where found to exceed values typical of adult monitoring values. Applying a cut-off value of HIVRMI=2.0 (
The fact that early HIV infection may be detected by the HIVRMI (as found in the paediatric cohort) may also mean that the HIVRMI assay may be useful in detecting PHI (primary or acute HIV infection) in adults that are sero-negative and in the <2 week after infection window period. This is being investigated.
It is envisaged that a kit for performing the assay described above can be provided to make it easier for the invention to be performed. The kit would include one or more of the following:
It is further envisaged that there will be provided a machine readable medium comprising instructions, which when executed by a machine, cause the machine to perform all or at least some of the steps of the invention described above. The machine readable medium may be configured for use in conjunction with a flow cytometer and/or haematology analyser, and may include instructions for performing analysis methods such as impedance, light scatter and fluorescence.
The present invention is further described by the following examples. Such examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention.
Blue plastic tubes (Beckman Coulter, cat #2523749) were labelled with individual laboratory numbers, and 50 μl AB human reagent serum (blood transfusion services) was inserted into each tube as a blocking agent.
Fresh EDTA was mixed with a sample of whole blood from each patient on a blood rocker for 3-5 minutes at room temperature. 50 μl of each EDTA and whole blood sample was added to a tube containing the AB serum, taking care to wipe excess blood off the pipette tip so as to ensure that no blood was deposited onto the sides of the tube. The blood and serum were mixed for 30 seconds and the tubes were incubated for 15 minutes at room temperature. The red cells were then lysed using Immunoprep™ reagent (Beckman Coulter) dispensed by an automated Q-Prep system (Beckman Coulter).
A 10 μM thiazole orange (Sigma/Aldrich, cat #39,006-2) solution in methanol was prepared. 1 μM was diluted in Sorenson's Phosphate Buffer, (pH adjusted to 7.2) or distilled water. A volume of 40 μl of this 1 μM diluted thiazole orange solution was added to each tube after red cell lysis and the tubes were incubated at room temperature for a further 20 minutes in the dark.
The samples were then analysed on an XL MCL (Beckman Coulter) flow cytometer, counting a minimum 25000 leucocyte events. All leucocytes were identified using heterogeneous gating (SSC vs FL1 thiazole orange) in the FL1 channel.
Three regions were set around the granulocytes, monocytes and lymphocytes, and the mean fluorescent intensity (MFI) in the FL1 channel for each region was measured. The ratios of monocyte MFI to granulocyte MFI and monocyte MFI to lymphocyte MFI and granulocytes to lymphocytes was calculated using the following formula, as an example:
ratio (or index value)=Monocyte MFI/Granulocyte MFI or Lymphocyte MFI=Reservoir Monitoring Index (RMI)
It was found that the leucocytes from an HIV negative sample share similar mean fluorescence intensity (MFI) in the FL1 channel, as shown by the single FL1 histogram in
However, the mean fluorescent intensity ratios in HIV positive patients (
The samples which were assayed as described above were also analysed using the standard Roche Amplicor Monitor version 1.5 assay to determine the log plasma viral load. A positive correlation was shown to exist between the HIVRMI and plasma viral load (VL) (Table 1 and
Explanation of Table 1:
Value Indicative of Virus Reservoir (Column 4 and 5)
The HIVRMI with the highest value (monocyte/granulocyte or monocyte/lymphocyte) is the index correlated to plasma viral load and indicative of the amount of intracellular viral reservoir or mRNA cellular response to infection.
Value Indicative of Additional Disease (such as TB) (Column 6).
Granulocyte/lymphocyte=1 shows no other background cellular activity;
Granulocyte/lymphocyte <1 shows disease with lymphocyte activity (may be early or late stage lymphocyte infection/activation);
Granulocyte/lymphocyte >1 shows disease with granulocyte activity (possible TB).
The correlation between HIVRMI and plasma PCR viral load on ARV naïve patients was initially shown to be r=0.677 p<0.0001 (R2=0.357, n=80) (Table 3).
The viability of the samples was determined, and only those samples with ≧60% viability (n=80) were included in the study. The highest HIVRMI was compared with the log viral load (Roche Amplicor) (Table 2).
Linear regression:
The HIVRMI was shown to significantly correlate with the viral load, although only 35% of the data is represented by the equation of the line in
However in the era of anti-retrovirals (ARV), this correlation has reduced to r=0.244 (R2=0.106, n=20), as shown in
Patients receiving anti-retroviral treatment (ARV) will pass through different phases of infection, and will show differences between plasma and cellular viral loads, which is why a single HIVRMI result is not useful for direct conversion to a plasma viral load value without knowledge of the patient treatment status:
Samples from some patients also showed an increase in granulocyte fluorescent intensity, resulting in the monocyte/granulocyte index being less than the monocyte/lymphocyte index (highlighted in column 5 of Table 1). These patients were found to be co-infected with tuberculosis and this relationship is thus being investigated as an additional tool (TBRMI) for identification and monitoring of co-infection. This additional index may assist in overall patient monitoring.
These differences in naïve and ARV patients highlight the strength of the HIVRMI for long term follow-up of cellular reservoirs and not circulating plasma virus that is more readily cleared by ARV. This is shown in
The HIVRMI is also applicable to disease monitoring in paediatric patients as in adults. The HIVRMI values in paeditrics, however, have been noticed to reach higher values than found with adults. Table 4 lists HIVRMI values from a paediatric and an adult cohort.
This concept of paediatrics having higher HIVRMI values was investigated to determine whether the HIVRMI assay could also be used as a qualitative assay for HIV diagnosis. DNA PCR is routinely used for HIV infant diagnosis at 6 weeks of age. When the cut-off of 2.0 for the HIVRMI was applied to a paediatric cohort also tested by DNA PCR, as shown in
Several studies have shown difficulty in determining differences in lymphocyte subsets between infected and un-infected infants using immune activation markers, due to changes occurring in the maturation of the infant immune system. This same effect may apply to the HIVRMI, and infants at earlier ages are being investigated, including cord blood.
The inventor believes that the assay according to the invention is advantageous for at least the following reasons:
While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention. It is therefore intended that the claims cover or encompasses all such modifications, alterations and/or changes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004/10087 | Dec 2004 | ZA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2005/003738 | 12/12/2005 | WO | 00 | 6/14/2007 |
