WO2019191039A1

WO2019191039A1 - Virus-specific diagnosis of hepatitis infection using isothermal amplification in resource-poor settings

Info

Publication number: WO2019191039A1
Application number: PCT/US2019/023996
Authority: WO
Inventors: Kathie BERGHORN; Julie SAHLER
Original assignee: Carrera Bioscience Inc.
Priority date: 2018-03-27
Filing date: 2019-03-26
Publication date: 2019-10-03

Abstract

The present invention relates to mobile methods to diagnose and differentiate hepatitis viral infections in a subject and to methods to treat said subject. In one embodiment, a method for differentiating a hepatitis disease comprises of obtaining a fluid sample from a subject; running said sample in a LAMP assay; measuring the reaction for the binding of viruses HAV, HBV, HCV, HDV and HEV RNAs; determining the viruses presence; and treating the subject for the hepatitis virus disease. In another embodiment, a method for treating a subject for a hepatitis virus infection comprises of obtaining a fluid sample from a subject; running a mobile LAMP assay on said sample; measuring the amount of the hepatitis virus present; and treating said subject for said hepatitis virus infection with a therapeutically effect amount of an anti-hepatitis agent.

Description

VIRUS-SPECIFIC DIAGNOSIS OF HEPATITIS INFECTION USING ISOTHERMAL AMPLIFICATION IN RESOURCE-POOR SETTINGS

BACKGROUND OF THE INVENTION

[0001] Viral hepatitis is the leading cause of liver cancer and the most common reason for liver transplantation More than 4.4 million Americans live with chronic hepatitis. To date, five viruses. hepatitis A virus (HAV), hepatitis B virus (HBV) hepatitis C virus (HCV), hepatitis delta virus (HDV) and hepatitis E virus (HEV) have been etiologically associated with viral hepatitis. The hepatitis viruses vary widely in their natural history, genome composition, and mode of transmission. Since viral hepatitis infections caused by the five viruses are clinically indistinguishable, disparate testing algorithms are being employed to determine the etiology of infection. Nucleic acid testing (NAT) remains the gold standard for detecting the viruses in blood and other body fluids. For multiplex detection of the hepatitis viruses, the CDC and several companies have developed RT-PCR assays to detect one or more of HAV RNA, HBV DNA, HCV

RNA, HDV RNA and HEV RNA in blood samples. For greater utility in resource-poor settings, multiplex technologies not dependent on thermal cycling are highly desirable and needed.

[0002] One of the reasons hepatitis viruses, in particular hepatitis C, can spread so easily is because people often do not know they have it. According to the CDC, 70-80 percent of people have no symptoms. But some can have fever, fatigue, loss of appetite or jaundice (yellowing of the skin or eyes). Right now, there are no vaccines to prevent hepatitis C. But Hepatitis C is treatable and curable - at a high cost. According to the CDC, Medicare Part D spent slightly more than $7 billion on Harvoni® treatments in 2015, while Medicaid spent nearly S2.2 billion. [0003] The drawback to these revolutionary drags is that they are extraordinarily costly. Just one pill of Sovaldi® costs approximately $1 ,000.26. This brings the total cost of the twelve-week treatment to $84,000.27. Olysio® has an estimated cost of $23,600 per month of treatment.28 However, the treatment duration of Olysio® is even longer than Sovaldi® at twenty-four to forty- eight weeks. While this is very expensive, the primary problem with the pricing is not the price alone, rather it is the combination of the price and the large number of people prescribed the drug Spending in 2014 on Sovaldi represented seventy-eight percent of all Hepatitis C antiviral drug expenditures.

[0004] The high costs associated with Sovaldi® reflect a combination of the large size of the population treated, the high cost of the drag, and how it has dominated the market due to its superior effectiveness. The total health care expenditure on Sovaldi®, in just one year was $6.5 billion, making it the drug with most overall expenditures in the United States in 2014. Of this figure, Medicare’s share of this spending was reportedly $4.5 billion dollars.32 The cost to treat the entire population of 3.7 million chronically infected Hepatitis C patients in the United States with Sovaldi®, at the estimated rate of $84,000 per patient, would be $310 bill ion. For comparison, the total spending in the United States on all drugs for 2014 was $360.7 billion. At this cost, treating all chronically infected Hepatitis C patients in the United States with Sovaldi® is not affordable.

[0005] The global hepatitis therapeutics market size was estimated at US 19.6 billion in 2016, and is anticipated to grow at a CAGR of 2.7% is during the forecast period. Few prominent factors that are expected to boost the market are growing prevalence of viral hepatitis, rise in the number of autoimmune disease, and surge in healthcare expenditures across the globe. In 2015, approximately 1.34 million people died due to this condition globally. [0006] Nucleic acid testing (NAT) remains the gold standard for detecting the viruses in blood and other body fluids. For greater utility in resource-poor settings, multiplex technologies not dependent on thermal cycling are highly desirable and needed.

[0007] An assay that is useful for pathogen screening of blood and organ/tissue donors, antenatal clients, immigrants, sexually- transmitted diseases (STD) clinics and other target populations. In the field, this new technology is portable, with few manipulative steps, no need for electricity, and easy to interpret results. Additionally, it is desirable that the format has the potential to add targets for other pathogens. The technology is not to be electrically driven during the sample processing, detection and test-outcome display stage, in order to accomplish the transportability of the assay, thereby making it mobile.

[0008] The current cost of an HCV RNA test is approximately $100.00. In Africa, the cost of hepatitis treatment has decreased, but the cost of hepatitis diagnostics has not. One of the speakers from WHO said the“major testing gaps calls for rapid innovation.” Dr. Wang who also spoke at the first Plenary of the Summit stated that a Hepatitis B DNA test is not available in any countries. A speaker from John Hopkins University stated that field-based testing is needed. Currently, in New Haven, CT there is a mobile medical clinic that is trying to screen for HCV. And Jilian Sacks from the Cliihon Health Access Initiative (CHAI) indicated that there is a need for rolling out new tests in low and middle income countries

[0009] The field friendly hepatitis diagnostic provided herein would help globally with the diagnosis of hepatitis. With such a diagnostic providing data within a short time frame, treatment can then be initiated more quickly. SUMMARY OF THE INVENTION

[0010] The present invention relates to a L A VIP- based assay for hepatitis viruses HAY, 1 1 BV. HCV, HDV and HEY LAMP and RT-LAMP primer sets from both published sources and invented design efforts are evaluated using synthetic hepatitis genomic targets. Ultimately, the down-selected set or sets targeting each virus are demonstrated to be sensitive for target viral genome amplification and have little cross reactivity with selected other hepatitis genome targets in (RT-) LAMP assays. In the NIH SBIR the LAMP-based assay for BAY, HBV, HCV, HDV and HEV using a BioRanger instrument (Diagenetix), and a tested sample preparation module is provider. These assays are used to detect acute hepatitis infections, and the assay validated using the LAMP-based assay with clinical samples against the (RT-)qPCR assay. The assay is enhanced to reach the highest level of performance in the diagnosis of acute hepatitis infection using serum from patients.

[0011] The method for detecting hepatitis of the present invention comprises: obtaining a fluid sample, preferably blood or serum, from a subject, human being. Then, the sample is treated with the reagents in the LAMP assay. The primer is added to the assay. These primers are selected from primers that are indicative of HAY, HBV, HCV, HDV and HBV The primer sequences are provided in the detailed description of the invention. Once the assay is run, a determination of which hepatitis virus has infected the subject is made and appropriate therapies are provided

[0012] Further, the present invention relates to a method to enhance the differentiation of hepatitis viruses. Also, methods to treat individuals (subjects) infected by a hepatitis vims by utilizing the methods to differentiate that virus are presented. [0013] Also, the present inventions relates to methods for treating subjects infected by a hepatitis virus that is identified by the methods of differentiating said hepatitis viral infection and treating with the appropriate therapeutic agent.

[0014] Additionally, the present invention relates to the RNA sequences useful in the methods to differentiate hepatitis viruses of the present inventions. This and further methods for constructing the RN A sequences useful in the present inventions are provided hereinbelow.

DESCRIPTION OF DRAWINGS

[0015] Figure 1 shows initial data from a test. Wells 1 , 3, 5, and 7 are samples with 5ng of purified genomic Salmonella DNA and Wells 2, 4, 6, and 8 are samples with ddH20

[0016] Figure 2 shows the BioRanger instrument useful in the present invention. After the instrument is turned on and the software on the tablet is opened the tablet is connected to the instrument by Bluetooth.

[0017] Figure 3 shows the GUI software.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The rate of deaths attributable to viral hepatitis is trending upward. According to the WHO, nearly 1 4 million people die every year globally due to this disease, and approximately 400 million people are infected with at least one form of hepatitis.

[0019] This condition is generally classified into two types based on the duration for which the patient sutlers. If the condition lasts for less than six months, then it is classified as acute, when the condition persists for more than six months then it is a case of chronic condition. On the basis of disease type, the hepatitis therapeutics market is segmented into A, B, C, and others, such as D and E.

[0020] The others segment covers the market estimates for hepatitis D and E, which occur rarely compared to the other three conditions. HAV spreads through intake of contaminated water, food, or through direct contact with an infected person. According to the estimates of WHO, approximately 90% of children in developing economies have been infected with the virus before reaching 10 years of age owing to poor hygiene & sanitary conditions.

[0021] Hepatitis B Virus (HBV) infects the liver and can lead to chronic & acute liver disease As per the WHO, an estimated 240 million people have chronic hepatitis B and more than 686,000 people die every year due to complications arising from it. The virus can be transmitted through blood and other body fluids of an infected person. The market is valued at US 2.6 billion in 2016 and held the second largest revenue share owing to the high prevalence of hepatitis B.

[0022] The Hepatitis D Virus (HDV) needs the help of HBV to replicate and proliferate in the body. HDV is a Ribonucleic Acid vims. Globally, around 15 million people are co-infected with HDV & HBV and presently no effective medication is available for HDV Therefore, immunization against HBV is strongly recommended to prevent both hepatitis B and D.

[0023] As compared to hepatitis A and B, HCV leads to various serious complications. According to the WHO estimates, approximately 130 to 150 million people are infected by HCV. In addition, a significant number of HCV infected patients develop liver cancer or liver cirrhosis and nearly 700, 000 people die due to these conditions. [0024] The introduction of technologically advanced therapeutic products for the treatment of hepatitis has been a major driver for growth. Earlier hepatitis C was not curable, but with the introduction of products such as Sovaldi® and Harvoni®, in 2013 and 2014 respectively, the treatment rate significantly increased over the past few years. In 2015, these products reportedly had over US 19.1 billion in sales.

[0025] The present invention is illustrated by the following examples, that are not to be limitative, but are illustrative of the present invention.

[0026] Example 1 : Positive controls for each of the hepatitis viruses are developed. (Kodani and Winched, 2012). The hepatitis targets are also used as the targets for designing LAMP primers for the testing of clinical samples in Phase I. This assay format is not compatible with a multiplex operation that would allow for the inclusion of an internal control. Thus, to preserve the simple nature of the reaction, a positive-control is developed to be run in parallel on the same sample as the HEP-LAMP assay.

[0027] The alignments of HAY, HBV, HCV, HDV and HEV are evaluated to target a sequence similar in overall G+C content as the target, but not identical to the targeted HAY, HBV, HCV, HDV and HEV sequences. A series of LAMP primers are designed to detect the positive-control target sequence. The positive control target sequence is cloned into a plasmid containing a T7 promoter to generate RNA m vitro which serves as a synthetic target for the HEP-LAMP assay.

HAV

[0028] The S’-UTR region (181 -680 nt) of HAV strain KRM031 (GenBank # AB300206) is used tor gene synthesis to prepare HAY RNA templates. This strain is chosen because it is of genotype IA, the most common HAV isolates. The HAY 5’-UTR is cloned into the pET22 vector (5’ -EcoRI/3’ -Sacl). The vector contains a T7 promoter for robust in vitro transcription alter plasmid linearization with the downstream Hindlll site

These primers target a region and are based in the 5’ configuration;

HBV

[0029] While the S gene is the most conserved region among the whole genome, the plasmid pAM6 (ATCC 45020) that contains foil length HBV genome (AF46204L1 ) in pBR322 vector serves as the target template in Phase I. The primer are as follows:

HCV

[0030] As with HAY, sequence analysis show's highly conserved 5’UTR among 7 HCV genotypes, but no significant homology between HCV 5'-UTR and that of HAY, HDV or HEV. For the genome template, the l-5GQnt (5’ UTR, 1 -341) region of HAY genotype lb (GenBank# G1J451224.1) is synthesized and cloned into pET22 vector at BamHI/EeoRI sites. The 5’-

UTR/core gene (1B119,247-433nt) is used

HDV

[0031 ] The UTR region flanking the hepatitis D antigen (HDAg) is used as targets for LAMP assay development, which had no cross reaction to HAV, HBV, HCV or HEV (Wang, et al., 2012)

The corresponding target region is synthesized and cloned into the BamHI/EcoRI sites of pET22.

The target HDAg gene region (1158-1340nt) is used.

HEV

RT-LAMP assays have been developed targeting the ORF3/capsid gene region ofHEV (Chen, et al., 2014, journal of Virological Methods 197, 29; Lan, et. ah, 2009, J Clin. Microbiol. 47, 2304).

This targeted region is able to detect ah HEY genotypes and show specificity against HAV, HBV, HCV, and HDV. This target template is synthesized (5131-563 lnt of AB193177.1, genotype IV) in the BamHI/EcoRI sites of pET22. The capsid protein gene (5939-6l49nt) is used.

HEV

BRIEF DESCRIPTION OF SEQUENCE LISTING

The following sequence listing (1 through 6) are the sequence listing identification for the listed HAY primers, respectively.

Sequence ID No.: 1;

Sequence ID No.: 2;

Sequence ID No.: 3;

Sequence ID No.: 4;

Sequence ID No.: 5; and

Sequence ID No.: 6.

HAV

The following sequence listing (7 through 13) are the sequence listing identification for the listed HBV primer, respectively. Sequence ID No.: 7;

Sequence ID No.: 8;

Sequence ID No.: 9;

Sequence ID No.: 10;

Sequence ID No.: 11;

Sequence ID No.: 12; and

Sequence ID No.: 13.

HBV

The following sequence listings (14 through 20) are the sequence listing identification for the linked HCV primers, respectively.

Sequence ID No.: 14

Sequence ID No.: 15

Sequence ID No.: 16

Sequence ID No.: 17

Sequence ID No.: 18

Sequence ID No.: 19; and

Sequence ID No.: 20.

HCV

The following sequence listings (21 through 26) are the sequence listing identification for the listed HDV primers, respectively. Sequence ID No.: 21;

Sequence ID No.: 22;

Sequence ID No.: 23;

Sequence ID No : 24;

Sequence ID No.: 25; and

Sequence ID No.: 26.

HDV

Sequence ID No.: 27;

Sequence ID No.: 28;

Sequence ID No.: 29;

Sequence ID No.: 30;

Sequence ID No.: 3! ; and

Sequence ID No.: 32. HEV

Primer concentrations used in the present invention are found hereinbelow.

[0033] The primer sets for each virus are found in the following charts, with the corresponding plasmid positive control for each primer set, along with the further information regarding the plasmids. Primer Concentrations

[0034] The methods of the present invention are carried out as provided hereinbelow in Table 1. As is seen in Table 1, the LAMP assay of the present invention is comprised of water, and RNA as inhibitor, dNTP, an isothernal amplifier buffer Mq50₄, a dilution stock of Syto 9, Bst polymerase and a reverse transcriptase. The LAMP assay is carried out by using the BioRanger® or Real Plex 2® devices. However, other devices may be used that are portable and mobil, and as such, the methods provided herein are not limited to those devices, but are illustrative thereof. Table 1

[0035] Table 2 provides an illustrated protocol for the methods of the present invention.

[0036] Table 3. Primers and corresponding plasmid information for each Hepatitis virus

Numbers indicate length in base pairs

[0037] Bacteria containing plasmids listed in Table 3 are grown in order to isolate fresh stocks of each plasmid. The custom-ordered Optigene“Fast isothermal Reverse Transcriptase Master Mix- Dried reagents- No Dye” (OG) in compound against our previously optimized mastermix that uses mostly New England Biolabs reagents (NEB). Syto9 is added to the Optigene master mix for a final concentration of 10 mM. Primer concentrations are consistent between both master mixes. Maximum amount of is loaded into each reaction (13.5 mL for NEB and 7.5 mL for OG).

[0038] Positive control plasmids, frozen RNA, and frozen plasma from several strains of hepatitis, are all tested to compare the two master mixes. Table 4 compares results of hepatitis positive plasma samples detected with either the Optigene (OG) or the present master mix containing mostly NEB reagents (NEB). Comparing only the column-extracted samples, Optigene master mix has comparable times of detection. However, they also have robust negative control signals. The early threshold times of negative controls cause great concern because they: 1) create a smaller dynamic range for detection of varying quantities of virus, and 2) could generate a false positive signal, which is unacceptable for diagnostic purposes. The high background of negative controls could be due to the DNA polymerase of the Optigene master mix having non-specific amplification behavior NEB master mix provides a wider dynamic range of detection, and slower amplification of negative controls, if at all. Therefore, the“NEB” master mix is the preferred mix to use with this assay production, although others are also useful.

[0039] Table 5 , NEB Comparison with Optigeee

[0040] The master mix of the HEP LAMP reaction is optimized and validated. 1 OmL of custom

RT-LAMP master mix from NEB are used. Upon arrival, the master mix is aliquoted and stored at -20°C, When comparing the new, premixed master mix to an identical master mix that was freshly prepared in the lab across several experiments and analyzed on the Realplex2 PCR machine, results from both master mixes across all experiments are very comparable. Therefore, the new pre-made master mix is validated across all viruses (Table 5).

[0041] Occasionally, one of the duplicates does not amplify. This maybe because either the reagents or nucleic acids are not adequately interacting at the bottom of the well. 96 well plates are not centrifuged prior to the assay in the RealPlex2 instrument. Previously, tubes had been spun prior to assays on the BioRanger. Therefore a final“quick spin” step of the prepared tube or plates is added to the reagent preparation SOP, prior to commencing the LAMP assays. [0042] Table 6. Comparison of freshly prepared master mix (Old MM) vs. NEB custom master mix (New MM). Duplicate samples of plasma or plasmid for five hepatitis viruses are simultaneously prepared and analyzed on the Realplex2 machine using the indicated master mix preparation.

Example 2:

[0043] Next, lyophilize aliquots of the master mix for longer stability and durability in the field are made.

[0044] The HEP-LAMP assay has been validated to determine its perform ance against a standard

(RT-)qPCR assay. The validation takes place using a panel of serum or plasma samples characterized for HAY, HBV, HCV, HDV and HEY loads.

[0045] Analysis of hepatitis positive plasma samples from the World Health Organization using the HEP-LAMP protocol (Table 7) is performed. These plasma samples are used to determine limit of detection (lU/mL) of the LAMP assays, with a comparison of the LAMP assay, with respective qPCR assays.

[0046] The compared HBV LAMP assay is compared to its equivalent qPCR assay. Table 7 shows this comparison of various genotypes of HBV positive plasma samples. Overall, the LAMP assay detects genotypes A, B, and C. However, no amplification is seen for genotype D. The qPCR assay (a verage of duplicates) detects all genotypes. However·, readings for plasma samples B4 and C2 are too similar to the negative control readings to be considered positive signals.

[0047] Table 7. Comparison of HBV LAMP and qPCR assays. Various HBV genotypes (gt; left column) or negative control (NC) plasma are used in LAMP or qPCR assays.

[0048] From these data, as evaluation of the limit of detection of the HBV assay. To this end, the plasma samples A2 and B2 from Table 7 are then diluted with the plasma as 10-fold serial dilutions into negative plasma. Nucleic acid samples as used are isolated, as before, and LAMP and qPCR assays are performed (Table 8).

[0049] Table 8, Comparison of HBV LAMP and qPCR assays. HBV positive plasma is diluted in negative plasma before processing for LAMP or qPCR reactions. [0050] The HBV LAMP assay detects both samples through 10⁴ IU/mL concentrations and could

detect the B2 through 10² IU/mL If qPCR ct values over 30 are discarded, as the no-template control for qPCR is 34.02, then the qPCR assay also detects both samples through 10⁴ lU/mL, and the A2 plasma through 10³ lU/mL. Of note, the LAMP assay is ran as single replicates due to constraint of the instrument, and qPCR is run in duplicates.

[0051] Deciding the cut off threshold of any assay is a delicate balance if too stringent, the user may reject positive singles. If too lenient, the user may read a false positive result. Both errors are unacceptable for clinical practice. Advantageously, the LAMP reactions produce very late amplifications if at all, for negative controls.

[0052] HAY positive plasma sample (Seracare) that is commercially estimated to have a concentration between 4043-28,000 lU/mL is used to compare the qPCR and LAMP assays. Nucleic acids from neat and diluted plasma samples are isolated with the Zymo Research Quick Viral RNA/DNA column isolation kit Elutants are divided into replicates to be run on qPCR or LAMP assays with their respective primers Cycle thresholds and time to thresholds are indicated in Table 9.

[0053] Table 9. Comparison of Hepatitis A LAMP and qPCR assays. Cycle threshold (qPCR) and time to threshold (LAMP) are indicated in the table. Triplicates are averaged for the qPCR assay and duplicates are averaged for the LAMP assay. ND indicates that sample signals are not detected, and {*) are indicated tha t one of the replicates was not detected.

[0054] HCV positive plasma samples from the Seracare Linearity Panel are used to compare the qPCR and LAMP assays. Nucleic acids from each sample are isolated with the Zymo Research Quick Viral RNA/DNA column isolation kit. Eluates are divided into replicates to he run on qPCR or LAMP assays with their respective primers.

[0055] Estimated concentrations (provided by Seracare datasheet), cycle thresholds, and time to thresholds are indicated in Table 10. Whereas, qPCR HCV assays appear to be more sensitive, the determination of the threshold line between positive and negative samples is less clear than with the LAMP assay.

[0056] Table 10. Comparison of Hepatitis C LAMP and qPCR assays. Cycle threshold (qPCR) and time to threshold (LAMP ) are indicated in the table. Duplicates are averaged for the qPCR assay and single replicates are used on the LAMP assay. ND indicates that sample signals are not detected.

[0057] The HDV positive plasma sample used by the World Health Organization as the International Standard was used to compare the qPCR and [.AMP assays. Nucleic acids from neat and diluted plasma samples were isolated with the Zymo Research Quick Viral RNA/DN A column isolation kit. Eiutants were divided into replicates to be run on qPCR or LAMP assays with their respective primers. Estimated concentrations, cycle thresholds, and tune to thresholds are indicated in Table 11, In this comparison, the [.AMP assay was superior to the qPCR assay, as the qPCR positive and negative controls were too close in cycle threshold to differentiate.

[0058] Table 11. Comparison of Hepatitis D LAMP and qPCR assays, Cycle threshold (qPCR) and time to threshold (LAMP) are indicated in the table. Duplicates are averaged for the qPCR assay and the LAMP assay ND indicates that sample signals are not detected.

[0059] The LAMP assays have a lower limit of detection in the 10E5 range or better, which matches the LGD testing with positive control plasmids described previously. Deciding the cut off threshold of any assay is a delicate balance. If too stringent, the user may reject positive singles. If too lenient, the user may read a false positive result. Both errors are unacceptable for clinical practice. Advantageously, the LAMP reactions produce very late amplifications, if at all, for negative umbras. Conversely, the qPCR assay has positive signal for negative controls very dose to some of the sample signals.

[0060] Several HEV genotypes are tested from the WHO panel of positive plasma samples, simultaneously compares qPCR to the HEY LAMP assay using the same extracted nucleic acid sample prep. Table 12 show the results of both assays. None of the samples were detected by qPCR, as all of their cycle threshold values are higher than the negative control water sample. The HEY LAMP assay was able to detect one sample: genotype 3b with an estimated of concentration >15,000 IU/mL. Must of the samples were not detected, likely due to then low concentration in plasma. As many of the other LAMP assays showed, the limit of detection is around 1E5 KJ/mL, making the HEV assay slightly more sensitive. Interestingly, genotype 4c was not detected by either assay, despite having a higher estimated concentration. It is possible that the estimation is incorrect, or that these LAMP primers are not as sensitive for that sub-genotype

[0061] Table 12, Comparison of HEV qPCR amt LAMP assays across genotypes, LAMP assay is performed on each sample as single replicates, whereas qPCR assay values indicate an average of duplicates. N.D. stands for not detectable. Water is not tested in the LAMP assay.

[0062] Many plasma samples and genotypes purchased from the World Health Organization are tested. All five hepatitis viruses are detected in their respective assays, with limits of detection around 1 E4 lU/mL.

[0063] Another area of investigation is the viral load for acute infection of the 5 different hepatitis viruses. After much research it has been determined what the acute viral loads for the 5 hepatitis viruses are. These results are shown in Tablc 13. Of interest is the number of references that stated for HBY. DNA is not a measure of acute infection.

[0064] Table 13. Estimated Viral Loads m Acute Hepatitis Infections

Claims

WHAT IS CLAIMED IS:

1. A method for differentiating a hepatitis disease, said method comprising:

a. obtaining a fluid sample from a subject;

b. running said sample in a LAMP assay;

c. measuring the reaction for the binding of viruses H AV, HBV, HCV. HDV and HE V RNAs:

d. determining the viruses presence; and

e. treating the subject for the hepatitis virus disease

2. The method according to claim 1, wherein the fluid sample is serum.

3. The method according to claim 2 wherein the method is mobile.

4. The method according to claim 1, wherein the subject is a human being.

5. A method according to claim 2, wherein the method uses the RNA selected from HAY.

6. The method according to claim 2, wherein the method uses the RNA selected from HBV.

7. The method according to claim 2, wherein the method uses the RNA selected from HCV.

8. The method according to claim 2, wherein the method uses the RNA selected from HDV.

9. The method according to claim 1 , wherein the method uses the RNA selected from HEV.

10. The method according to claim 5, wherein the RNA is produced in plasmid HAY with primer pET22 vector.

1 1. The method according to claim 6, wherein the RNA is produced in plasmid HBV pAM6 with primer 101.

12. The method according to claim 7, wherein the RNA is produced in plasmid HCV with primer 1D44.

13. The method according to claim 2 wherein the RNA is produced in plasmid pET22 with Bam Hl/ECORJ sites having the UTR region flanking the hepatitis B antigen.

14. The method according to claim 8, wherein the RNA is produced in plasmid HDV1 with primer Wang.

15. The method according to claim 9, wherein the RNA is produced in plasmid HEV₂ with primer ID2.

16. A method for treating a subject for a hepatitis vims infection, said method comprising: a. obtaining a fluid sample from a subject;

b. running a mobile LAMP assay on said sample;

c. measuring the amount of the hepatitis virus present:

d. treating said subject for said hepatitis vims infection with a therapeutically effect amount of an anti-hepatitis agent.

17. The method of treating a subject for a hepatitis virus infection according to claim 16, wherein the vims is HAY, HBV, HCV, HDV or HEV.

18. The method of treating a subject infected with HAV, HBV, HCV, HDV or HEV according to claim 16, wherein said subject is treated with a therapeutically effective amount of Harvoni®, Sovaldi® or combinations thereof.

19. The method according to claim 5, wherein the HAV primer is selected from the group A-

20. The method according to claim 6 wherein the HBV primer is selected from the group

21. The method according to claim 7, wherein the HCV primer is selected from the group

22. The method according to claim 8, wherein the HAV primer is selected from the group

23. The method according to claim 9, wherein the HEV primer Is selected from the group