02 - Hav 2006
02 - Hav 2006
02 - Hav 2006
VIRAL HEPATITIS
Zahid Hussain, Bhudev C Das, Syed A Husain, Sunil K Polipalli, Tanzeel Ahmed, Nargis Begum, Subhash Medhi,
Alice Verghese, Mohammad Raish, Apiradee Theamboonlers, Yong Poovorawan, Premashis Kar
Zahid Hussain, Sunil K Polipalli, Tanzeel Ahmed, Nargis P = 0.44). The CD4+/CD8+ ratio in AHA patients (1.17
Begum, Subhash Medhi, Premashis Kar, PCR Hepatitis ± 0.11; P = 0.22) and s-AH (0.83 ± 0.12; P = 0.0002)
Laboratory, Department of Medicine, Maulana Azad Medical were lower than seen in normal healthy controls (1.52).
College, New Delhi 110002, India Self-limited cases had peak viral load at the beginning of
Bhudev C Das, Division of Molecular Oncology, Institute of analysis while in s-AH patients this occurred at the 15
th
Cytology and Preventive Oncology, (ICMR), Noida, Sector 39, th
or 30 d. In acute and severe groups, one patient each
Uttar Pradesh, India
Zahid Hussain, Syed A Husain, Tanzeel Ahmed, Nargis belonged to genotype IA, with the remaining 8 cases
Begum, Mohammad Raish, Human Genetics Laboratory, belonging to genotype IIIA. The only fulminant hepatic
Department of Biosciences, Jamia Millia Islamia, New Delhi failure case belonged to genotype IA. HAV viral load and
110025, India ALT values collected during the entire course of the self-
Alice Verghese, Advance Center for AIDS, National Institute of limited infection were directly correlated but this was not
Communicable Diseases, New Delhi 110041, India the case for s-AH patients.
Apiradee Theamboonlers, Yong Poovorawan, Viral Hepatitis
Research Unit, Department of Pediatric, Faculty of Medicine, CONCLUSION: Based on a small-scale study, the
Chulalolongkorn University and Hospital, Rama IV Road, persistently higher viral load of s-AH might be due
Bangkok 10330, Thailand
to diminished cellular immunity and hemolysis. The
Correspondence to: Dr. Premashis Kar, Room No. 111,
duration of viremia was dependent on the host, as the
Department of Medicine, Maulana Azad Medical College, New
Delhi 110002, India. [email protected] viral genotype had no apparent role in clinical outcome
Telephone: +91-11-23230132 Fax: +91-11-23230132 of AVH and s-AH cases.
Received: 2006-03-11 Accepted: 2006-04-21
© 2006 The WJG Press. All rights reserved.
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4684 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol August 7, 2006 Volume 12 Number 29
To date, however, there is limited knowledge of viral containing EDTA and was employed for CD4+ (T helper)
load, or the length of viral persistence both in the blood and CD8 + (T suppressor) cell counts within 24 h of
circulation and in fecal excretion. It has been reported collection, using a Fluorescence Activated Cell Sorter
that a relapse may occur 30-90 d after the initial onset of (FACS) (Becton Dickenson Electronics Laboratory,
the disease[10-12] and virus has been detected in the stool Mountain View, California). This system quantifies CD4+,
of patients[13]. Recently, outbreaks of HAV have occurred CD8 + and CD3 + T lymphocytes as absolute numbers
among hemophiliacs receiving organic solvent and of lymphocytes per μL (mm3) of blood, and the CD4+/
detergent-treated factor Ⅷ, a fact that stresses the potential CD8+ T lymphocyte ratio. Samples from healthy controls
usefulness of a reliable and widely applicable technique for and patients were also run for cell counts using the
quantifying viral load in blood samples[14-18]. The level and manufacturer’s protocol and reagents.
the length of HAV viremia involve the additional risk of
the carrier becoming an infectious source of hepatitis A[19]. Primers, probe and standard for real-time amplification
We undertook further examination of hepatitis A HAV RNA was extracted using the QIAamp TM viral
viremia during the course of infection to understand RNA extraction kit (Qiagen, Germany). Viral RNA was
whether viral load was correlated with cell-mediated amplified using primers derived from the most constant
immunity. The pathogenetic mechanisms underlying region, the 5’ non-coding region (5’ NCR) [25,26] . The
hepatocellular injur y in acute hepatitis are poorly primers used were, forward primer HAV1 (22: 5’-TTT
understood [20]. There is general agreement that HAV CCG GAG CCC CTC TTG-3’), as wild type (M14707)
infection does not evolve to chronic hepatitis in man[21], reverse primers HAV2 (85: 5’-AAA GGG AAA TTT AGC
and immune mechanisms have been suspected of playing a CTA TAG CC-3’) and HAV3 (85: 5’-AAA GGG AAA
major role in eliminating virus-infected liver cells[22-24]. The ATT TAG CCT ATA GCC-3’), and HAV-probe (58: 5’
aim of this study was to undertake analysis of HAV viral -FAM-ACT TGA TAC CTC ACC GCC GTT TGC CT-
load, alanine aminotransferase, and viral genotypes with TAMRA-3’) and RNA standard representing the 5’NCR
the duration of viremia, and to correlate these parameters region was constructed according to Costa-Mattioli et al[27].
with populations of CD4 + /CD8 + lymphocytes that
controls cell-mediated immunity. Fluorogenic quantitative Real-Time PCR and direct
sequencing
RT-PCR was carried out with a HAV quantification kit
MATERIALS AND METHODS (Roche Diagnostics GmbH, Germany) according to the
Patients and blood samples manufacturer’s instructions. The total volume of the
Patients attending the Medical Out Patient Department of reaction mixture was 25 μL (15 μL of mastermix with 10
Lok Nayak Hospital, New Delhi, with the characteristic μL of the RNA template) in 0.2 mL tubes. The capillaries
symptoms of acute viral hepatitis such as jaundice, fever, were sealed, centrifuged, and transferred to the Rotor
general malaise, fatigue, nausea, vomiting, anorexia and Gene 3000 real-time PCR machine (Corbett Research,
right upper quadrant discomfort, were screened for the Sydney, Australia). Reverse-transcription was done for
study. Ten mL of blood were collected by venipuncture 15 min at 50℃ followed by 5 min denaturation at 95℃.
from those patients, who gave consent for five different The corresponding cDNA's were amplified by PCR (20
visits. The study was approved by the ethical committee of s at 95℃, 30 s at 50℃ acquiring FAM, and 20 s at 72℃)
Maulana Azad Medical College, as per the Declaration of over 45 cycles, and an 87 bp fragment was obtained. The
Helsinki (1995). Consecutive blood samples were collected CT values from the clinical samples were plotted on the
from 10 acute hepatitis A patients on the 0th, 7th, 15th, 30th standard curve, and the number of copies was calculated
and 45th d between July 2004 and June 2005. No sample automatically.
was collected before the onset of symptoms. The ‘0th’ d PCR amplification from part of the VP1/2A region of
was defined as the first day when the patient presented HAV genome was directly sequenced for genotyping[28-30].
after the onset of jaundice. Ten healthy subjects who had Sequencing was done with an ABI Prism 310 Genetic
no evidence of liver disease or dysfunction were taken as Analyzer (ABI, Foster City, CA). Sequencing analysis
control. was performed using ClustalW and the phylogenetic
inference by version 1.81 of the PHYLIP software
Serological tests package (Professor J. Felsenstein, Department of Genetics,
Laboratory examination of alanine aminotransferase University of Washington, Washington, DC).
(ALT), aspartate aminotransferase (AST), prothrombin
time (PT), and total bilirubin levels were carried out by Statistical analysis
standard methods. IgM anti-HAV were detected by ELISA All data were analyzed by two tailed tests, and a P value
(HAVAB-MEIA); Abbott Laboratories, North Chicago, less than 0.05 was considered significant. We used chi-
IL), IgM anti-HEV (Qiagen, Hilden, Germany), HBsAg square test and student’s t-test as appropriate.
(Qiagen, Hilden, Germany) and anti-HCV (Bio-Rad, San
Francisco, CA, USA) were measured according to the
manufacturer’s protocol. RESULTS
Comparison of clinical features between AVH and s-AH
FACS analysis of T-lymphocyte profile groups
One milliliter whole blood was collected into a vial The average age of all patients was 20.8 ± 15.5 years
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Hussain Z et al. Duration of hepatitis A viremia in relation to different parameters 4685
Table 1 Clinical, biochemical and immunological Comparison of viral load between AVH and s-AH groups
characterization of different groups (AVH, s-AH, and normal In the acute case, Ind-301, the viral load on the initial day
control) was 4.5 × 105, and decreased further to > 103 copies/mL.
As shown in Figure 1, Ind-303 and Ind-306 followed
1 2 3
Characteristic AVH s-AH Normal Control almost similar trends with viral loads of 2.6 × 105 and
No. of cases 07 03 10
1.2 × 10 5 respectively at 0 th d while < 10 2 copies/mL
Sex (M/F) 3/4 3/0 7/3
still persisted at the end of follow up. The viral load of
Age 0.4 ± 17.9 27.7 ± 2.08 26.2 ± 3.6
(mean ± SD)
Ind-304 was 1.0 × 105 at start and complete elimination
ALT 1070.9 ± 894.3 1713.9 ± 886.3 23.6 ± 7.2 of the virus with zero copies/mL was seen at the end
(mean ± SD; IU/L) of follow up, as shown in Figure 1. Patients Ind-308 and
AST 621.3 ± 242.8 1614 ± 234.7 23.8 ± 5.8 Ind-310 displayed a peak viral load at 0th d like the cases
(mean ± SD; IU/L) described above, while Ind-309 peaked on the 15th d, but
T. Bilirubin 9.8 ± 3.8 24.5 ± 3.0 0.74 ± 0.43 values then decreased to less than 100 copies/mL in all
(mean ± SD; mg/dL)
three. In the severe cases Ind-302 and Ind-305, the viral
Prothrombin Time 14.3 ± 1.1 21.0 ± 2.0 12.5 ± 0.5
(Seconds)
load on the initial day of follow up was 1.2 × 105 and 1.1
Mean CD4+/CD8+ 1.17 ± 0.11 0.83 ± 0.12 1.52 ± 0.11 × 105 respectively, and reached a peak on the 30th d, quite
different from the acute case shown in Figure 1. At the
ALT: AVH vs s-AH P = 0.29 (not significant); AVH vs NC P = 0.0014 (highly end of follow up, the viral load was significantly higher (>
significant); s-AH vs NC P = 0.001 (highly significant); AST: AVH vs s-AH 1.0 × 105) as compared to AVH. In-patient Ind-307, viral
P = 0.0034 (highly significant); AVH vs NC P = 0.00014 (highly significant);
s-AH vs NC P = 0.00044 (highly significant); T. Bil: AVH vs s-AH P = 0.076
load reached the peak (4.6 × 105 ) at 15th d and decreased
(significant); AVH vs NC P = 0.00017 (highly significant); s-AH vs NC P = subsequently to 4.3 × 104 copies/mL at the end of the
0.0023 (highly significant); PT: AVH vs s-AH P = 0.11 (not significant); AVH follow up.
vs NC P = 0.44 (not significant); s-AH vs NC P = 0.02 (significant); Mean
CD4+/CD8+: AVH vs s-AH P = 0.46 (not significant); AVH vs NC P = 0.22 (not
significant); s-AH vs NC P = 0.0002 (highly significant). Note: AVH1 (Acute Comparison of genotype (s) between AVH and s-AH
viral hepatitis); s-AH2 (Severe acute hepatitis); NC3 (Normal Control). groups
In Table 2, the maximal viral load was compared with
genotyping and geographical distribution. As shown
and ranged from 3 to 59 years. The average age and sex in phylogenetic tree (Figure 2), the patients Ind-301
ratio of two groups of patients is shown in Table 1. 10 (DQ179131) and Ind-302 (DQ179132) were categorized
of 11 patients could be followed completely, of which as genotype IA, while the remainder Ind-303 (DQ179133),
3 progressed to severe acute hepatitis (s-AH), which Ind-304 (DQ179134), Ind-305 (DQ179135), Ind-306
is defined on the basis of a prothrombin time (PT) < (DQ179136), Ind-307 (DQ179134), Ind-308 (DQ182495),
40% of normal range. The remainder of patients had Ind-309 (DQ182496), and Ind-310 (DQ182497)) were
self-limited acute hepatitis A (AHA) and one died of classified as the ⅢA genotype. The only fulminant hepatic
fulminant hepatic failure (Grade Ⅳ encephalopathy) at failure case, Ind-274 (DQ182500), belonged to genotype
the 4th d of follow up. There was no difference between ⅠA.
the groups with respect to clinical symptoms, such as flu-
like prodromes (including arthralgia, or headache), fever,
nausea, vomiting, abdominal pain, pruritus, and diarrhea. DISCUSSION
Hepatitis A remains the most frequent form of viral
Comparison of biochemical features between AVH and hepatitis observed in a large number of countries[31,32].
s-AH groups Recent publications have demonstrated that the duration
In Figure 1, the time course of viral load and serum of the viremic phase is much longer than assumed[10,27]. A
transaminase ALT levels is presented for all patients. serum HAV viral load assay could therefore be helpful in
ALT values for both groups followed a decreasing trend the management of severe hepatitis A. Real-Time reverse
towards normal from the initial to final day of follow up. transcription (Rotor Gene 3000, Corbett Research, Sydney,
The mean liver function profile of s-AH patients was Australia), was used for the quantitative detection of the
higher compared to the AVH cases as shown in Table 1. HAV genome in human sera in individuals who displayed
The mean prothrombin time (PT) of s-AH patients was varying disease courses[27,33,34]. The fluorescence signal due
(21.0 ± 2.0; P = 0.02) significantly higher than that of to the cleavage of the fluorogenic probe is generated only
acute cases (14.3 ± 1.1; P = 0.44). if the target sequence for the probe is amplified by the
PCR. Therefore, no signals are generated by non-specific
Comparison of immunological profiles between AVH and amplification.
s-AH groups The alanine aminotransferase level of AVH cases on
The mean immunological (CD4+/ CD8+) ratio in patients the initial day was significantly higher (≥ 103) and the
with acute viral hepatitis A was (1.17 ± 0.1) higher than that decreased upon subsequent follow up, which corresponds
in the severe acute cases (0.83 ± 0.12). As shown in Table to earlier findings that demonstrate a direct correlation
1, the CD4+/CD8+ ratio in normal controls (NC) (1.52 ± of viral load with serum ALT[27,35]. In our study, the mean
0.11) ratio was almost twice as high as in s-AH (P = 0.0002). prothrombin time in AVH was not higher while severe
There was no significant decrease in the immunological cases showed significant elevations compared to normal
ratio of AVH cases compared to normal controls. controls. This could be due to anemia (hemolysis) as this
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4686 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol August 7, 2006 Volume 12 Number 29
Acute hepatitis A
Table 2 Comparison of patient’s genotypes to viral load and
Ind-301; 22/M
600 000 10 000 geographical distribution
ALT (IU/L)
copies/mL
HAV viral
400 000
5000 Patients Genotype Maximal viral load Geographical
200 000 Copies/mL distribution
0 0 Ind-301 ⅠA 4.5 × 105 New Delhi
0th 7th 15th 30th 45th 1
Ind-302 ⅠA 6.0 × 105 New Delhi
Ind-303; 59/F Ind-303 ⅢA 2.6 × 105 Uttar Pradesh
300 000 4000
ALT (IU/L)
1.0 × 105
copies/mL
Ind-304 Delhi
HAV viral
ⅢA
200 000 1
Ind-305 ⅢA 5.0 × 105 Delhi
2000
100 000 Ind-306 ⅢA 1.2 × 105 New Delhi
1
0 0 Ind-307 ⅢA 4.6 × 105 Uttar Pradesh
0th 7th 15th 30th 45th Ind-308 ⅢA 4.1 × 103 New Delhi
Ind-304; 15/F Ind-309 ⅢA 3.4 × 104 Haryana
200 000 4000 9.8 × 104
ALT (IU/L)
Ind-310 New Delhi
copies/mL
ⅢA
HAV viral
50 000 1000 of follow up. The kinetics of peak viral load attainment
0 0 in s-AH was quite different from that in the acute self-
0th 7th 15th 30th 45th
limited cases since at the end of follow up high copies/mL
Severe acute hepatitis A still persisted, as shown in Figure 1. The acute results
*Ind-302; 26/M; s-AH confirm the levels recently estimated by Chudy et al[15].
1 000 000 6000 The progression of severity due to diminished cellular
ALT (IU/L)
copies/mL
HAV viral
500 000 4000 immunity and hemolysis might be directly linked to high
2000 viral persistence throughout the follow up.
0
0 Most of the patients examined during acute self-limited
0th 7th 15th 30th 45th
*Ind-305; 27/M; s-AH
illness belonged to genotype ⅢA, other than Ind-301
600 000 10 000 who belonged to ⅠA[6]. Among the severe cases, Ind-302
ALT (IU/L)
copies/mL
HAV viral
400 000
5000
belonged to ⅠA while other two belonged to ⅢA, which
200 000 means genotypic variations likely do not play a crucial
0 0 role in determination of the viral load as described earlier
0th 7th 15th 30th 45th
by Normann[35]. The only FHF case, who died at the 4th
*Ind-307; 30/M; s-AH
600 000 4000 d of follow up, belonged to genotype ⅠA. The question
ALT (IU/L)
copies/mL
HAV viral
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Hussain Z et al. Duration of hepatitis A viremia in relation to different parameters 4687
1AL20552
1AL20549
DQ179131
ⅠA
DQ179132
1AL20553
1AL20551
DQ182495
DQ179134
DQ179133
DQ182497
DQ179136 ⅢA
DQ182496
DQ179137
DQ179135
3AAJ299465
3AAJ299464
3BL20544
3BL20536 ⅢB
3BL20532
3BL20530
Ⅳ
4
Ⅵ
6L07731
2 Ⅱ
2L07693
7 Ⅶ
7L07729
1BL07728
1BL07703
1BL07702 ⅠB
1BL07701
1BL07700
0.1
Figure 2 A neighbor-joining phylogenetic tree for isolates of hepatitis A virus based on the sequencing of the VP1-P2A region. Isolates DQ179131 (Ind-301), DQ179132
(Ind-302), DQ179133 (Ind-303), DQ179134 (Ind-304), DQ179135 (Ind-305), DQ179136 (Ind-306), DQ179137 (Ind-307), DQ182495 (Ind-308), DQ182496 (Ind-309),
DQ182497 (Ind-310) were collected during the present study, in PCR Hepatitis Laboratory, MAM College and associated LNJ Hospital, New Delhi, India.
findings which showed that a long duration of viremia was of immunological markers by FACS, and Mr. Pradeep
found in patients infected with HAV genotype IA[35]. K Singhal, Professional Biotech Ltd. New Delhi, India
In conclusion, HAV viral load and alanine aminotrans- for their excellent guidance and assistance in viral load
ferase (ALT) values collected during the entire course determination.
of a self-limited acute infection were directly correlated,
but this was not found in s-AH cases. The duration of
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